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

Summary

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.

Abstract

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.

Introduction

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 adhesion^4,5. Experiments using micellar lithography-based nanopatterns revealed a threshold value of around 70 nm for RGD peptide nanospacing, cell adhesion being sig....

Protocol

1. Substrate Preparation

1. Annealing 1.4 x 1.1 cm Au(111) on Mica Substrates.
   1. 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.
2. Preparation of Poly(L-Lactic Acid) (PLLA)-Coated Glass Substrates.
   1. Cut and wash the glass slides.
      1. Cut microscopy slides into 18 slides of 1.25 cm x 1.25 cm with a diamond-tip cutter. Make a small indentation on the lowe....

Results

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 (

Discussion

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 4–5 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 .......

Materials

List of materials used in this article

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