Name: preparation of thermoresponsive nanostructured surfaces for tissue engineering
Uploaded: 2016-03-01T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering at JoVE.com

This study was financially supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Program's Project for Developing Innovation Systems "Cell Sheet Tissue Engineering Center (CSTEC)" of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

1. Synthesis of Polystyrene-block-poly(N-isopropylacrylamide) by Two-step Reversible Addition-fragmentation Chain Transfer (RAFT) Radical Polymerization
<ol>
	<li>Dissolve styrene (153.6 mmol), 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT; 0.2 mmol), and 4,4&#39;-Azobis(4-cyanovaleric acid) (ACVA; 0.04 mmol) in 40 ml of 1,4-dioxane. Freeze the solution in liquid nitrogen under vacuum for 15-20 min to remove the reactive species and gradually thaw at RT. Make sure that the solution...

<ol>
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I., Borozenko, K., Badia, A., Bazuin, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+order+transition+in+nanodot-forming+diblock.">Pressure-induced order transition in nanodot-forming diblock.</a> J. Am. Chem. Soc. 133 (493), 19702-19705 (2011).</li><li>Wang, X. L., Ma, X. Y., Zang, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+behavior+of+polystyrene-b-poly(acrylicacid)+at+the+air-water+interface.">Aggregation behavior of polystyrene-b-poly(acrylicacid) at the air-water interface.</a> Soft Matter. 9 (2), 443-453 (2013).</li><li>Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+engineering.">Cell sheet engineering.</a> Mater. 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A temperature-responsive surface was fabricated by the Langmuir-Schaefer method, and the surface properties for cell adhesion/detachment and cell sheet recovery were optimized. When using this method for the fabrication of surfaces, several steps are critical. The molecular composition of the St-IPAAm molecules has a great effect on the surface structure and the stability of the surface, and by extension, on cell adhesion and detachment. In particular, the St-IPAAm molecules should have a narrow molecular weight distribu...

Nanostructured surfaces have recently attracted substantial attention due to their various potential applications, including patterning, cell culture, cleaning, and surface switching. For example, superhydrophobic surfaces inspired by the nanostructure of the lotus leaf and other responsive surfaces are capable of reacting to external stimuli1-4.
The Langmuir film is one of the most widely studied polymer coatings. A Langmuir film is formed by dropping amphiphilic molecules onto an air-water interface5-8. The film can then be transferred onto a solid surface by physical or chemical adsorption, and the molecular conformation on a solid surface can be controlled using vertical and horizontal transfer methods9-12. The density of the Langmuir film can be precisely regulated by compressing the air-water interface. Recently, this method has also proven effective for fabricating nanoscaled sea-island structures by utilizing amphiphilic block copolymers. The nanostructures are assumed to consist of a core of hydrophobic segments and a shell of hydrophilic segments13-17. In addition, the number of nanostructures on a surface is regulated by controlling the area per molecule (Am) of the block copolymer at the interface.
We have focused on an original, unique scaffold-free tissue engineering approach, cell sheet engineering, using a temperature-responsive culture surface. The developed technology has been applied to regenerative therapies for various organs18. A temperature-responsive culture surface was fabricated by grafting poly(N-isopropylacrylamide) (PIPAAm), a temperature-responsive molecule, onto a surface19-27. PIPAAm and its copolymers exhibit a lower critical solution temperature (LCST) in aqueous media at temperatures near 32 &#176;C. The culture surface also exhibited a temperature-responsive alternation between hydrophobicity and hydrophilicity. At 37 &#176;C, the PIPAAm-grafted surface became hydrophobic, and cells readily attached and proliferated on the surface as well as on conventional tissue culture polystyrene. When the temperature was lowered to 20 &#176;C, the surface became hydrophilic, and cells spontaneously detached from the surface. Therefore, cultured confluent cells on the surface could be harvested as an intact sheet by changing the temperature. These cell adhesion and detachment properties were also displayed by a surface fabricated by Langmuir film coating for laboratory demonstration26, 27. A Langmuir film of block copolymers composed of polystyrene (P(St)) and PIPAAm (St-IPAAm) was fabricated. The Langmuir film with a specific Am could be horizontally transferred to a hydrophobically modified glass substrate. In addition, cell adhesion on and detachment from the prepared surface in response to temperature were evaluated.
Here, we describe protocols for the fabrication of a nanostructured Langmuir film composed of thermo-responsive amphiphilic block copolymers on a glass substrate. Our method may provide an effective fabrication technique for organic nanofilms in various fields of surface science and may facilitate more effective control of cell adhesion on and spontaneous detachment from a surface.

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">N-isopropylacrylamide</td>
			<td style="width:193px;">Kohjin</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Azobis(4-cyanovaleric acid)</td>
			<td style="width:193px;">Wako Pure Chemicals</td>
			<td style="width:119px;">016-19332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Styrene</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:119px;">S4972</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1,3,5-trioxane</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:119px;">T81108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1,4-Dioxane</td>
			<td style="width:193px;">Wako Pure Chemicals</td>
			<td style="width:119px;">045-24491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:193px;">Sigma&#160;</td>
			<td style="width:119px;">D6429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:193px;">Nakarai</td>
			<td style="width:119px;">11482-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Streptomycin</td>
			<td style="width:193px;">GIBCO BRL</td>
			<td style="width:119px;">15140-163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin</td>
			<td style="width:193px;">GIBCO BRL</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin-EDTA</td>
			<td style="width:193px;">Sigma</td>
			<td style="width:119px;">T4174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FBS</td>
			<td style="width:193px;">Japan Bioserum</td>
			<td style="width:119px;">JBS-11501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">BAECs</td>
			<td style="width:193px;">Health Science Reserch Resources Bank</td>
			<td style="width:119px;">JCRB0099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Cover Glasses</td>
			<td style="width:193px;">Matsunami Glass Industry</td>
			<td style="width:119px;">C024501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AFM NanoScope V</td>
			<td style="width:193px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">1H NMR INOVA 400</td>
			<td style="width:193px;">Varian, Palo Alto</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">ATR/FT-IR NICOLET 6700</td>
			<td style="width:193px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">GPC HLC-8320GPC</td>
			<td style="width:193px;">Tosoh</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">TSKgel Super AW2500, AW3000, AW4000</td>
			<td style="width:193px;">Tosoh</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Langmuir-Blodgett Deposition Troughs&#160;</td>
			<td style="width:193px;">KSV Instruments</td>
			<td style="width:119px;">KN 2002</td>
			<td>KSV NIWA Midium trough</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Nikon ECLIPSE TE2000-U</td>
			<td style="width:193px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of thermoresponsive nanostructured surfaces for tissue engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Block copolymers composed of polystyrene and poly(N-isopropylacrylamide) (St-IPAAms) with specific molecular weights were synthesized by RAFT radical polymerization. ECT was prepared as a chain-transfer agent as described in Moad et al.28. Two St-IPAAm molecules of different PIPAAm chain lengths were synthesized, and the obtained block polymers were characterized by 1H nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The mol...

Watch this Scientific Journal Video about Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering at JoVE.com

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

The overall goal of this Langmuir-Schaefer method with block co-polymers is to fabricate cell sheets on various conditions of the temperature-responsive surface, by controlling strength of cell adhesion and attachments. This method can help answer key questions in the biomaterials field, such as biocompatible materials, cell culture surface, and bioseparation. The main advantage of this technique is that the interaction between temperature-responsive surface and cells can be controlled, and the modifying conditions for cell sheet recovery can be optimized.Though this method can provide insight into interaction between cell and surface, it can also be applied to other system such as bioseparation and biofilm. To begin, dissolve styrene, ECT, and ACVA in 40 milliliters of 1, 4-Dioxane. Freeze the solution in liquid nitrogen under vacuum for 15 to 20 minutes to remove the reactive species.After gradually thawing the solution at room temperature, repeat this freeze, pump, thaw, degassing cycle three times. Obtain the polystyrene as a macro-RAFT agent by polymerization at 70 degrees Celsius for 15 hours in an oil bath. Precipitate the polystyrene macro-RAFT agent with 800 milliliters of ether, and dry in vacuo.Next, dissolve IPAAm monomer, polystyrene macro-RAFT agent, and ACVA in four milliliters of 1, 4-Dioxane. Remove the oxygen in the solution using freeze, pump, thaw degassing cycles as before. Perform a polymerization at 70 degrees Celsius for 15 hours in an oil bath after degassing.Finally, obtain the synthesized St-IPAAm molecule in the same manner as the polystyrene macro-RAFT agent. Wash glass substrates with an excess of acetone and ethanol, and sonicate for five minutes to remove surface contaminants. Dry the substrates in an oven at 65 degrees Celsius for 30 minutes.Then, use oxygen plasma to activate the surfaces of the substrates at room temperature. Immerse the substrates in toluene containing 1%hexatrimethoxysilane overnight at room temperature to sylanise the substrate. Then, wash the sylanised substrates in toluene and immerse in acetone for 30 minutes to remove the unreacted agents.Anneal substrates for two hours at 110 degrees Celsius to thoroughly immobilize the surface. Finally, cut the sylanised substrates by a glass cutter to 25 millimeters by 24 millimeters to fit the cell culture dishes. Place the Langmuir film instrument in a cabinet to prevent the accumulation of dust.Wash the Langmuir trough in barriers with distilled water and ethanol to remove the contaminants. Dry the trough and barriers by wiping with a lintless towel. Then fill the trough with approximately 110 milliliters of distilled water, and set the barriers on both sides of the trough.Next, heat a platinum Wilhelmy plate for monitoring the surface tension, with a gas burner until the plate turns red. Then wash the plate with distilled water to remove the contaminants. Suspend the Wilhelmy plate on a wire attached to the surface pressure measurement instrument.Zero the surface pressure measurement instrument according to the manufacturer's protocol. Compress the air, water interface on the trough by the barriers on both sides of the trough until the interface reaches approximately 50 square centimeters without any drops of polymer. Aspirate any small contaminants until the surface pressure is nearly zero millinewtons per meter.Reposition the barriers on both sides, and add distilled water to compensate for the decrease of distilled water. Next, dissolve five milligrams of the synethesized St-IPAAm molecule in five milliliters of a development solution of chloroform. Then, gently drop 27 microliters of St-IPAAm dissolved in chloroform, onto the trough using a microsyringe or micropipette.After waiting for five minutes to allow complete evaporation of chloroform, move both barriers horizontally to compress the St-IPAAm molecule at the surface. Maintain compression rate of the barriers at 0.5 millimeters per second until the target area of 50 square centimeters is reached. Measure the surface pressure Pi-A isotherms with the platinum Wilhelmy plate attached to the surface pressure measurement instrument during compression, according to the manufacturer's protocol.After reaching the target area size, maintain the surface for five minutes to allow the St-IPAAm molecules to relax. The molecules do not reach equilibrium immediately after compression. Then, transfer the Langmuir film to a hydrophobically-modified glass substrate using a transfer apparatus for five minutes to robustly absorb the film.Fix the hydrophobic glass substrate in parallel on the device. Connect the device to an alignment stage and move perpendicularly. Lift the substrate horizontally with the transfer apparatus and dry for one day in a dessicator.To prepare cell suspensions, culture bovine carotid artery endothelial cells, or BAECs, to 1/3 confluence at 37 degrees Celsius in 5%CO2 and 95%air, on tissue culture polystyrene with DMEM, containing FBS and Penicillin. After confluence is reached, treat BAECs with three milliliters of 0.25%trypsin EDTA for three minutes at 37 degrees Celsius in 5%CO2 and 95%air. Deactivate the trypsin EDTA by adding 10 milliliters of the DMEM containing 10%FBS.And collect the cell suspension in a 50-milliliter conical tube. After centrifuging at 120 times g for five minutes, aspirate the supernatent and resuspend the cells in 10 milliliters of the DMEM. Seed the recovered cells on the St-IPAAm surfaces at a concentration of 10, 000 cells per square centimeter, counted by a disposable hemocytometer.Observe the cells on the surfaces by a microscope equipped with an incubator at 37 degrees Celsius with 5%CO2 and 95%air. Next, sterilize the St-IPAAm surfaces by an ultraviolet light equipped to a clean bench. Record timelapse images of adherent BAECs for approximately 24.5 hours at 37 degrees Celsius by a phase-contrast microscope with 10x magnification.After BAEC adhesion, record detatchment of the BAECs from the St-IPAAm surface at 20 degrees Celsius for approximately 3.5 hours. Following optimization of cell adhesion and detachment on the Langmuir film transferred surface, fabricate the cell sheets by first culturing BAECs as before. Seed a total of 100, 000 cells per square centimeter on St-IPAAm surfaces.And incubate for three days at 37 degrees Celsius in 5%CO2. Confluent BAECs spontaneously detached at 20 degrees Celsius. Preparation of a temperature-responsive Langmuir film transferred surface is shown here.The synthesized St-IPAAm chloroform solution was gently dropped onto an air, water interface. Two barriers were used to compress the St-IPAAm molecules on the interface until a target area of 50 square centimeters was reached. The surface pressure was measured during compression to detect any defects in the Langmuir film.After compression, a hydrophobically-modified cover glass substrate was horizontally placed on the interface with an alignment stage. The substrate was lifted horizontally and dried for one day. After this step, St-IPAAm surfaces were ready for use in cell culture experiments.Representative results of atomic force microsopy topographic images of St-IPAAm surfaces are shown here. Nanostructures were observed on both of the St-IPAAm surfaces. The size and shape of the nanostructures were strongly dependant on AM, and the composition of St-IPAAm.Here, a macroscopic image of a recovered cell sheet on a Langmuir film transferred surface with St-IPAAm 480, and an AM of 40 square nanometers per molecule, is shown. Cells reached confluence after three days in culture on St-IPAAm 170 and St-IPAAm 480 surfaces at 37 degrees Celsius. And the cell sheet was rapidly recovered after reducing the temperature from 37 to 20 degrees Celsius.After watching this video, you should have a good understanding of how to fabricate, how to design molecular composition of polymer, fabricate and transfer Langmuir film, control cell adhesion and detachment, and recover cell sheet. After its development, this technique paves the way for researchers in the field of the biomaterials and tissue engineering to explore interactions between cells and surfaces and cell culture strategies for use in degenerative medicine. The implication of this technique extend towards therapy of cell transplantation, because this technique has potential to fabricate cell sheets of various cell types, provided from patients.

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Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

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 ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

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 ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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

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

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

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

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 ...