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...

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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 ...

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.  ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

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. ...

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

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 ...

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 ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...