Name: dendrimer-based uneven nanopatterns to locally control surface adhesiveness: a method to direct chondrogenic differentiation
Uploaded: 2018-01-20T14:00:00
Description: Watch this Scientific Journal Video about Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation at JoVE.com

The authors acknowledge Oriol Font-Bach and Albert G. Casta&#241;o for their help in dmin quantification. They also acknowledge the Advanced Digital Microscopy Unit at the Institute for Research in Biomedicine (IRB Barcelona) to let the authors record the video in their premises. This work was supported by the Networking Biomedical Research Center (CIBER), Spain. CIBER is an initiative funded by the VI National R&amp;D&amp;i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III, with the support of the European Regional Development Fund. This work has been supported by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2014 SGR 1442). It was also funded by the projects OLIGOCODES (No. MAT2012-38573-C02) and CTQ2013-41339-P, awarded by the Spanish Ministry of Economy and Competitiveness, in addition to INTERREG V-A Spain-Portugal 2014-2020 POCTEP (0245_IBEROS_1_E). C.R.P. acknowledges financial support from the Spanish Ministry of Economy and Competitiveness grant (No. IFI15/00151).

1. Substrate Preparation
<ol>
	<li>Annealing 1.4 x 1.1 cm Au(111) on Mica Substrates.
	<ol>
		<li>Place the Au(111) substrate on a glass-ceramic hob and anneal it with a butane flame for 3 min. Allow the substrate to cool under an argon atmosphere. Repeat this step for each Au(111) substrate. 
		NOTE: Au(111) substrates should be used immediately after annealing.</li>
	</ol>
	</li>
	<li>Preparation of Poly(L-Lactic Acid) (PLLA)-Coated Glass Substrates.
	<ol>
		<li>Cut and wash the...

<ol>
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During the development of the described protocol, a number of critical steps should be considered. The first refers to nanopattern characterization with scanning probe microscopy techniques. To visualize the nanopatterns, the surface where patterning is produced must have a roughness value below the mean diameter of the dendrimers, which is around 4&#8211;5 nm as measured by STM (Figure 1B). Also, it should be taken into account that high resolution STM imaging is restricted to conductive substrates, in ...

Here we describe a simple and versatile dendrimer-based nanopatterning procedure to obtain cell culture surfaces that allow the control of local adhesiveness at the nanoscale. Nanoscale details of ECM organization have been reported,1,2,3 and the nanopatterning of cell adhesion surfaces has provided deep insights into the cellular requirements related to adhesion4,5. Experiments using micellar lithography-based nanopatterns revealed a threshold value of around 70 nm for RGD peptide nanospacing, cell adhesion being significantly delayed above this value6,7,8,9. These studies also highlighted the greater influence of local than global ligand density on cell adhesion9,10,11.
During morphogenesis, cell interactions with the surrounding environment trigger the first differentiation events, which continue until final complex tissue structures have been formed. Within this framework, nanopatterned surfaces have been used to tackle the influence of the initial cell-surface interactions on morphogenesis. Lithography-based RGD nanopatterns with a lateral spacing of 68 nm in &#946;-type Ti-40Nb alloys help to maintain the undifferentiated phenotype of non-committed stem cells12, while RGD nanospacings of between 95 and 150 nm enhance the differentiation of mesenchymal stem cells (MSCs) towards adipogenic/osteogenic13,14,15 and chondrogenic fates16. Also, self-assembling macromolecules modified with signaling components have been shown to direct cell adhesion and differentiation by providing nanoscale architectural regulation of the signaling cues17. In this regard, the deposition of dendrimers with cell-interacting moieties in their outer sphere18,19,20 onto surfaces has been used to study cell adhesion21,22, morphology23,24, and migration events25,26. Nevertheless, the lack of surface characterization in these studies makes it difficult to establish any correlation between dendrimer surface configuration and cell response.
Dendrimer nanopatterns with liquid-like order and defined spacing can be obtained when dendrimers adsorb to low-charged surfaces from solutions with low ionic strengths.27 On the basis of this property, here we present a method to obtain large-scale uneven nanopatterns of RGD-functionalized dendrimers on low-charged surfaces that permit the nanoscale control of local RGD surface density. Water contact angle (CA), X-ray photoelectron spectroscopy (XPS) and scanning probe microscopy techniques (STM and AFM nanopatterns) show that local ligand densities can be adjusted modifying the initial dendrimer concentration in solution. The local RGD surface density is quantified from AFM images by probability contour maps of minimum interparticle distances and then correlated with cell experiments. Compared with other nanopatterning techniques4, dendrimer-based nanopatterning is straightforward and can be easily scaled up to large surface areas, thus being fully compatible with cell culture applications. Nanopatterns are used as bioactive substrates to evaluate the effect of the local RGD surface density on cell adhesion28 and on the chondrogenic induction of adult human MSCs29. Our results show that RGD dendrimer-based nanopatterns sustain cell growth and that cell adhesion is reinforced by high local RGD surface densities. In the differentiation experiments, intermediate adhesiveness of cells to the substrates favored MSC condensation and early chondrogenic differentiation. Due to the ease with which dendrimer peripheral groups can be modified, the method described here can be further extended to other ECM ligands that exert concentration-dependent effects on cells.

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Gold (111) on mica. 1.4x1.1 cm&#160;</td>
			<td style="width:169px;">Spi Supplies</td>
			<td style="width:307px;">466PS-AB</td>
			<td style="width:405px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Glass micro slides, plain</td>
			<td style="width:169px;">Corning</td>
			<td style="width:307px;">2947-75x25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Deionized water</td>
			<td style="width:169px;">Millipore</td>
			<td style="width:307px;"></td>
			<td>18M&#937; cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Ethanol 96%</td>
			<td style="width:169px;">PanReac</td>
			<td style="width:307px;">131085.1212</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">L-Lactide/DL-Lactide copolymer</td>
			<td style="width:169px;">Corbion</td>
			<td style="width:307px;"></td>
			<td>95/05 molar ratio</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">1,4 - dioxane</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">296309-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Silicone oil, high temperature</td>
			<td style="width:169px;">Acros Organics</td>
			<td style="width:307px;">174665000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Spinner</td>
			<td style="width:169px;">Laurell</td>
			<td style="width:307px;">WS-650MZ-23NPP/Lite</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:504px;">Tissue culture laminar flow hood</td>
			<td style="width:169px;">Telstar</td>
			<td style="width:307px;">Bio II Advance</td>
			<td>Class II biological safety cabinet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Filter unit</td>
			<td style="width:169px;">Millex-GP</td>
			<td style="width:307px;">SLGP033RB</td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Syringe 10 mL</td>
			<td style="width:169px;">Discardit</td>
			<td style="width:307px;">309110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Atomic Force microscope</td>
			<td style="width:169px;">Veeco Instruments</td>
			<td style="width:307px;">Dimension 3000 AFM instrument</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:504px;">Silicon AFM probes</td>
			<td style="width:169px;">Budget Sensors</td>
			<td style="width:307px;">Tap300AI-G</td>
			<td>Resonant Freq. 300 kHz, k = 40 N/m</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Scanning tunneling microscope</td>
			<td style="width:169px;">Molecular Imaging</td>
			<td style="width:307px;">PicoSPM microscope</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Pt0.8:Ir0.2 wire</td>
			<td style="width:169px;">Advent</td>
			<td style="width:307px;">PT671012</td>
			<td>Diameter 0.25 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">WSxM 4.0 software</td>
			<td style="width:169px;">Nanotec electronica</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Optical contact angle (CA) system</td>
			<td style="width:169px;">Dataphysics</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">SCA20 software</td>
			<td style="width:169px;">Dataphysics</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">X-ray photoelectron spectrometer</td>
			<td style="width:169px;">Physical Electronics</td>
			<td style="width:307px;">Perkin-Elmer PHI 5500 Multitechnique System</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Fibronectin from bovine plasma</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">F1141-1MG</td>
			<td>1.0 mL solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Dulbecco&#39;s Phosphate Buffer Saline (DPBS)</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:307px;">21600-10</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Mouse embryo fibroblasts</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC CRL-1658</td>
			<td>NIH/3T3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Dulbecco&#39;s modified eagle medium (DMEM) liquid high glucose</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:307px;">11960044</td>
			<td>liquid high glucose, no glutamine, 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:307px;">16000044</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">L-Glutamine</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">25030</td>
			<td>200 mM (100X)</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:504px;">Penicillin-streptomycin</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">15140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Sodium pyruvate</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">11360039</td>
			<td>100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">T75 culture flasks</td>
			<td style="width:169px;">Nunclon</td>
			<td style="width:307px;">156499</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Trypsin</td>
			<td style="width:169px;">Life Technologies</td>
			<td style="width:307px;">25200072</td>
			<td>0,25% EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Centrifuge</td>
			<td style="width:169px;">Hermle Labortechnik</td>
			<td style="width:307px;">Z 206 A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Non-tissue culture treated plate, 12 well</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:307px;">351143</td>
			<td>Non-adherent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Adipose-derived hMSCs</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-011</td>
			<td>Cell vial 1 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">MSC basal medium</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">MSC growth kit</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-040</td>
			<td>Low serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Chondrocyte differentiation tool</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Formalin solution</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">HT5011-15ML</td>
			<td>neutral buffered, 10%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Ammonium chloride</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">A9434-500G</td>
			<td>for molecular biology, suitable for cell culture, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Saponin</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">47036-50G-F</td>
			<td>for molecular biology, used as non-ionic surfactant, adjuvant</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:504px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">A3059-50G</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:504px;">Rabbit monoclonal [Y113] anti-paxillin antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:307px;">ab32084</td>
			<td>Diluted 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Mouse monoclonal [1F5] anti-collagen alpha-1 XX chain&#160;</td>
			<td style="width:169px;">Acris Antibodies</td>
			<td style="width:307px;">AM00212PU-N</td>
			<td>Diluted: 1:400</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibody</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">A10667</td>
			<td>2 mg/mL. Diluted 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">A11036</td>
			<td>2 mg/mL. Diluted 1:1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Hoechst 33342</td>
			<td style="width:169px;">Thermo Fisher</td>
			<td style="width:307px;">H3570 10ML</td>
			<td>10 mg/mL. Diluted 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Cover glass 24x24 mm</td>
			<td style="width:169px;">Deltalab</td>
			<td style="width:307px;">D102424</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:504px;">Fluoromount</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">F4680-25ML</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Epifluorescence Microscope</td>
			<td style="width:169px;">Nikon</td>
			<td style="width:307px;">Eclipse E1000 upright microscope</td>
			<td>with a CCD camera</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Confocal Microscope</td>
			<td style="width:169px;">Leica Microsystems</td>
			<td style="width:307px;"></td>
			<td>Leica SPE Upright Confocal Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">ImageJ 1.50g freeware</td>
			<td style="width:169px;">http://imgej.nih.gov/ij</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">MATLAB software</td>
			<td style="width:169px;">The MATHWORKS, Inc.</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">OriginPro 8.5 software&#160;</td>
			<td style="width:169px;">OriginLab Coorporation</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

dendrimer-based uneven nanopatterns to locally control surface adhesiveness: a method to direct chondrogenic differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

We present a nanopatterning method that allows surface adhesiveness to be addressed at the nanoscale (Figure 1). The chemical structure of RGD-Cys-D1 is shown in Figure 1A. Dendrimers were patterned on electrical conductive Au(111) surfaces for high resolution STM characterization. Low dendrimer concentrations in solution (up to 10-5% w/w) rendered isolated dendrimers of 4-5 nm in diameter (<strong class="xfig...

Watch this Scientific Journal Video about Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation at JoVE.com

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

The overall goal of this protocol is to obtain large scale dendrimer based uneven nanopatterns that permit the nanoscale control of local surface adhesiveness. The method described is applied for the study of cell adhesion and chondrogenic differentiation. Dendrimer raised nanopatterns allow to locally control surface adhesiveness at the nanoscale.First generation polynanomers modified with the cell adhesive RGD peptide are used to create nanopatterns. They are characterized by a scanning per microscopy techniques and used in cell adhesion and differentiation studies. We then analyze the results from microscopy and immunostaining techniques.The first step is to prepare the working substrates. Customized square glass slides are produced by cutting microscopy slides with a diamond tipped cutter. Make slides of 1.25 by 1.25 centimeters in size.Wash the slides with deionized water, followed by 96%ethanol and let them air dry. Prepare the 2%PLLA solution by adding 200 milligrams of PLLA to a pressure tube and add 10 milliliters of dioxane. Add a stir bar and close the tube tightly.Warm the mixture in a glycerine bath at 60 degrees centigrade for 24 hours under gentle stirring. Place the glass slides in a glass vial containing the PLLA solution on a clean, hot plate at 60 degrees centigrade. Wait at least 10 minutes to reach the necessary temperature.Spin coat the glass substrates with the PLLA solution by placing the glass slide on the spin coater stage and running the selected program. We select 3, 000 rpm for 30 seconds with an acceleration of 1, 500 rpms per second with a pre-step of 500 rpm at five seconds at 300 rpms per second to ensure an homogenous coating. Spin coated glasses can be stored in the fridge.Prepare and sonicate for 10 minutes solution A, a 0.77 milligrams per milliliter solution of RGD-Cys-D1 dendrimer in deionized water. Then prepare dendrimer solutions of 10 to the 2%and 10 to the 5%solution B and C respectively in deionized water. Sonicate solution C for 10 minutes and prepare the following dendrimer solutions, D, E, and F also in deionized water.Solution D, 2.5x10 to the 8%solution E, 10 to the 8%and solution F, 4 10 to the 9%Preserve these solutions in the fridge until use. You graduate with a UV lamp of the cell culture cabin 18 PLLA coated glasses for 13 minutes to make them sterile. Sonicate each of the dendrimer solutions for 10 minutes.Filter the dendrimer solutions through 0.22 micrometer diameter filters on the PLLA coated glass slides in a 12 well plate under the cell culture cabin laminar flow. Leave them inside the cabin at room temperature for 16 hours and wash them with sterile, deionized water. For the positive control samples, prepare a sterile fibronectin solution in PBS and incubate the replicas with the solution for one hour at room temperature in the cell culture cabin.Wash them with PBS. The remaining three PLLA coated substrates will serve as the replicas for the negative control. Nanopattern topography is analyzed with AFM operated in tapping mode in air.Mount a silicon cantilever with a spring constant of 40 newtons per meter and a resonant frequency of 300 kilohertz into the AFM tip holder. With the nanopattern substrate on the AFM stage, approach the surface until contact and choose an amplitude set point that permits imaging the dendrimer configuration without exerting excessive pressure on the surface. Register at least three representative images of 5x5 micrometers per substrate of three independent substrates per condition.Treat the acquired images with the AFM processing software. Select dendrimers on the processed AFM height images and obtain the corresponding image thresholds. Get the particle precisions and use them to obtain the minimum inter-particle distances.Let the minimum inter-particle distances in Z to the corresponding particle precisions to obtain the probability control plugs for the minimum inter-particle distances. Adjust the color scale of the plot to visualize the regions where local RGD surface density is below 70 nanometers. Quantify the area of these regions by selecting them on the image and generating a threshold.Use commercial human adipose derived from mesenchymal stem cells from early passages, preferably below five. Culture them at 37 degrees and 4.6%CO2 atmosphere in the stem cell growth medium until 70 or 80%confluence is reached. Seed the cells at a cell density of 3, 000 cells per square centimeter on the substrates in a chondrogenesis inducing medium.Change the medium every three days. The condensation step can be observed from the first day in culture, and their evolution can be followed by an optical phase contrast microscope during all the culture labs. Fix the cells with 10%formalin solution at room temperature for 20 minutes, and then wash with PBS.Lock the furaldehyde groups by adding a 50 millimolar solution of ammonium chloride in PBS. Leave for 20 minutes at room temperature. Wash with PBS.Permeabilize the cells with 0.1%solution of saponin in the blocking solution of 1%albumin in PBS for 10 minutes at room temperature. Incubate with primary antibodies in the blocking solution for one hour at room temperature. Wash with PBS.Incubate with secondary antibodies in the blocking solution for on hour at room temperature, avoiding light exposure. Wash with PBS and dry. Apply microscopy mounting medium on the samples and cover them gently with cover slips.Image the immunostained samples for the focal adhesion protein paxillin after one day of chondrogenic induction, and for the early chondrogenic marker, collagen II alpha I after five days of chondrogenic induction using an upright confocal microscope. Collect sections at a representative interval, for example 0.5 or 1 micrometers. Filter the images stained for paxillin from the basal zone of cell condensates.Remove the background, smooth, and convert to eight bit files. Make them binary by setting an empirically selected threshold. Treat a minimum of three cell condensates per condition of three independent samples.Quantify the selected areas and express them as the corresponding percentage of area divided by the number of cell nuclei in the image. To quantify collagen II alpha I staining, create confocal Z projections. Filter the resulting images, remove background, smooth, and set a threshold.Quantify the sum of the projected area maximum collagen II alpha I area per sample against the area of the corresponding cell condensate. Different nanopattern configurations can be obtained modifying the initial dendrimer concentration in solution. Nanopatterns can be visualized by AFM analysis and probability contour plots for the minimum inter-particle distance constructed.They highlight the regions on the surface with the highest local RGD surface density. Dendrimer nanopatterns are used in cell adhesion studies. Adhesion increases with local RGD surface density.For the positive control and surfaces containing dendrimer aggregates, this correlation is lost. Influence of local RGD surface density was tested in chondrogenesis, both cell condensation and differentiation are favored by nanopatterns presenting an intermediate adhesiveness. In conclusion, dendrimer-based nanopattern in a three table method to address the affect of local RGD density on cell response.Nanopatterns sustain cell adhesion more efficiently at the corresponding homogenous surface commonly used in cell culture. In differentiation experiments, intermediate adhesiveness of cells to the substrate favored mesenchymal cell condensation and early chrondogenic differentiation. Due to the easy modification of dendrimer peripheral growth, the method here described can be further extended to all the excessive rheumatic layers having density effects on cells.

dendrimer-based-uneven-nanopatterns-to-locally-control-surface

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Experimental Methods to Study Human Postural Control

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied by appropriate mathematical methods are needed to understand the role of the different sub-systems involved in human postural control. This article describes a protocol for performing perturbed standing experiments, acquiring experimental data, and carrying out the subsequent mathematical analysis, with the aim of understanding the role of musculoskeletal system and central control in human upright posture. The results generated by these methods are important, because they provide insight into the healthy balance control, form the basis for understanding the etiology of impaired balance in patients and the elderly, and aid in the design of interventions to improve postural control and stability. These methods can be used to study the role of somatosensory system, intrinsic stiffness of ankle joint, and visual system in postural control, and may also be extended to investigate the role of vestibular system. The methods are to be used in the case of an ankle strategy, where the body moves primarily about the ankle joint and is considered a single-link inverted pendulum.

This article presents an experimental/analytic framework to study human postural control. The protocol provides step-by-step procedures for performing standing experiments, measuring body kinematics and kinetics signals, and analyzing the results to provide insight into the mechanisms underlying human postural control.

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...