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. Синтез Polystyrene- блока -poly (N -isopropylacrylamide) двухступенчатым Реверсивный Сложение фрагментации цепи передачи (RAFT) радикальной полимеризации <ol><li> Растворите стирол (153,6 ммоль), 4-циано-4- (ethylsulfanylthiocarbonyl) sulfanylpentanoic кислоты (ЕСТ; 0,2 ммоль), и 4,4&#39;-азобис (4-cyanovaleric кислота) (ACVA; 0,04 ммоль) в 40 м...

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Температура реагирующих поверхность была изготовлена ​​методом Ленгмюра-Шеффера, и свойства поверхности для адгезии клеток / отсоединени и клеток листа восстановления были оптимизированы. При использовании этого метода для изготовления поверхностей, несколько шагов, которые имеют...

Наноструктурированные поверхности в последнее время привлекли значительное внимание из-за их различных потенциальных применений, в том числе структурирование, культуры клеток, очистки и переключения поверхности. Например, сверхгидрофобные поверхности вдохновленные наноструктуры листа лотоса и других чувствительных поверхностей способны реагировать на внешние раздражители 1-4. Пленка Ленгмюра является одним из наиболее широко изученных полимерных покрытий. Пленка Ленгмюра формируется путем сбрасывания амфифильных молекул на интерфейс воздух-вода 5-8. Затем пленка может быть передана на твердую поверхность с помощью физической или химической адсорбции, и конформации молекулы на твердой поверхности , можно контролировать с помощью вертикальных и горизонтальных методы переноса 9-12. Плотность пленки Ленгмюра можно точно регулировать путем сжатия раздела воздух-вода. В последнее время этот метод также доказал свою эффективность для изготовления наноразмерных море остров СтруктурЕ. С. за счет использования амфифильных полиэфирных блок-сополимеров. Наноструктуры предполагаются состоят из ядра гидрофобных сегментов и оболочкой из гидрофильных сегментов 13-17. Кроме того, количество наноструктуры на поверхности регулируется путем регулирования площади на молекулу (м) блок - сополимера на границе раздела фаз. Мы сосредоточились на оригинальной, уникальной каркасного свободной ткани инженерный подход, сотовый листовой инженерии, с помощью температуры реагирующих поверхности культуры. Разработанная технология была применена для регенеративной терапии для различных органов 18. Температура поверхности реагирующих культура была изготовлена ​​прививкой поли (N -isopropylacrylamide) (PIPAAm), молекула температуры реагирующих, на поверхность 19-27. PIPAAm и его сополимеры демонстрируют более низкую критическую температуру раствора (НКТР) в водной среде при температуре около 32 ° C. Поверхность культуры также выставлены температуры реагирующих alternati<html> на между гидрофобности и гидрофильности. При 37 ° С, PIPAAm привитым поверхность становилась гидрофобной, и клетки легко присоединены и пролиферируют на поверхности, а также на обычной тканевой культуры, полистирола. При понижении температуры до 20 ° С, поверхность стали гидрофильными, и клетки спонтанно отделяться от поверхности. Таким образом, культивированные сливные клетки на поверхности может быть собран в виде интактного листа путем изменения температуры. Эти клеточной адгезии и отслойка свойства также отображаются на поверхности сфабриковано пленочным покрытием Ленгмюра для лабораторной демонстрации 26, 27. Ленгмюра пленка блок - сополимеров , состоящих из полистирола (P (St)) и PIPAAm (St-IPAAm) был изготовлен. Пленка Ленгмюра с конкретнее м может быть горизонтально переданы гидрофобно модифицированной стеклянной подложке. Кроме того, оценивали клеточную адгезию на отрыв и от подготовленной поверхности в ответ на температуру. _content &quot;&gt; Здесь мы описываем протоколы для изготовления наноструктурированного Ленгмюра пленки, состоящей из термореактивных отзывчивым амфифильные блок-сополимеры на стеклянной подложке. Наш метод может обеспечить эффективный метод изготовления для органических нанопленками в различных областях науки о поверхности и может способствовать более эффективный контроль клеточной адгезии на спонтанный и отрыв от поверхности.

<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.

Блок - сополимеры , состоящие из полистирола и поли (N -isopropylacrylamide) (Сент-IPAAms) с конкретными молекулярными массами были синтезированы на плоту радикальной полимеризации. ЕСТ была подготовлена ​​в качестве агента переноса цепи , как описано в Moad и др. 28. Бы?...

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.

Приготовление Thermoresponsive наноструктурированных поверхностей для тканевой инженерии

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.

Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

Производство биологически Производные инъекций Материалы для тканевой инженерии миокарда

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

Micropatterned Поверхности по изучению Гиалуроновая кислота Взаимодействие с раковыми клетками

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

Эластомерные PGS Строительные леса в Артериальная тканевой инженерии

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

Озвучка обработка волокна Electrospun для тканевой инженерии

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

Капиллярная сила Литография для сердечной тканевой инженерии

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

Тканевая инженерия человеческого 3D<em&gt; В пробирке</em&gt; Опухоль Тестовая система

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

Инженерные 3D Cellularized Коллаген Гели для сосудистой регенерации ткани

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

Тканевая инженерия по искробезопасности васкуляризации<em&gt; В Vivo</em&gt; Tissue Engineering палата

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

Протокол для Decellularizing Cochleae мыши для внутреннего уха тканевая инженерия

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

Сайт направлен иммобилизации костных морфогенетических белка 2 для твердых поверхностей по химии нажмите

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