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 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.
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 β-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.
1. Substrate Preparation
2. Dendrimer Nanopatterning
3. Preparation of Control Substrates
Note: All steps were performed in a sterile tissue culture hood, and only sterile materials, solutions and techniques were used. At least, six control substrates were used (three replicas of the positive control and three replicas of the negative control).
4. Surface Characterization
5. Cell Culture
NOTE: All steps were performed in a tissue culture hood, and only sterile materials, solutions and techniques were used.
6. Cell Fixation and Immunostaining
NOTE: The following steps can be performed in non-sterile conditions.
7. Cell Imaging and Data Analysis
NOTE: For opaque Au(111) and thick microslide-based substrates, an upright microscope must be used.
8. Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR) Analysis
NOTE: To prevent RNase contamination, use disposable, sterile plastic ware and wear disposable gloves while handling reagents and RNA. Always use proper microbiological aseptic techniques and use an appropriate decontamination solution to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes.
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 (Figure 1B), while highly packed dendrimer aggregates formed at higher concentrations (Figure 1C). AFM surface characterization (Figures 1D-G, upper row) revealed that the surface distribution of RGD-Cys-D1 dendrimers can be adjusted as a function of the initial dendrimer concentration in solution. On the probability contour maps for dmin, this results in different local RGD nanoscale densities on the surface (Figures 1D-G, lower row).
In cell culture experiments, the cell-membrane receptor integrins of around 10 nm in diameter recognized the RGD sequence on the dendrimers periphery. Although eight copies of this sequence were provided per dendrimer, only one dendrimer interacted per integrin due to its size (4-5 nm measured by STM; Figure 1B). That is, each dendrimer provided a single site for integrin binding, thereby allowing a direct correlation from dendrimer distribution (as seen in the AFM images) and the RGD distribution available for cell adhesion. Moreover, no significant variations were found in the CA values obtained for the different nanopattern configurations that may influence cell adhesion29. These characteristics make dendrimer nanopatterns on biocompatible surfaces suitable substrates through which modulate and study cell behavior.
Cell adhesion to nanopatterned Au(111) was tested with fibroblasts within the first 24 h of culture (Figure 2A) and with MSCs on nanopatterned PLLA (Figure 2B). In both cases, the percentage of area stained for the FA protein paxillin (pax) increased progressively with dendrimer concentration, as did local RGD surface density (the percentage of nanopatterned area with dmin below the 70-nm threshold for efficient cell adhesion; Table 4). For an initial dendrimer concentration of 10-2% w/w and positive controls, the percentage of area stained for pax per cell decreased and the correlation with the percentage of nanopatterned area with dmin < 70 nm was lost.
The percentage of surface area occupied by dmin <70 nm (Figure 1D-G lower row and Table 4) is a good indication of local RGD surface density for nanopatterns up to 10-5% w/w initial dendrimer concentration but not of those up to 10-2% w/w, due to dendrimer aggregation. Aggregation produced highly heterogeneous samples in terms of ligand distribution, with only a small percentage of the surface with dmin values below the 70-nm threshold (Figure 1D lower row and Table 4). XPS results showed that these surfaces present a global RGD density comparable to 10-5% w/w-derived nanopatterns28. This observation indicates that maximum RGD density was achieved in the regions containing dendrimer aggregates and that the percentage of surface area occupied by dmin <70 nm is not representative of the local RGD surface density in this case.
The observation that positive controls (homogeneous coatings) with maximum local RGD surface density did not show the expected increase in cell adhesion can be attributed to a steric hindrance effect. These results indicate that, compared to the corresponding homogeneous surfaces commonly used in cell culture, RGD nanopatterns sustain cell adhesion more efficiently. This finding thus highlights the relevance of local ligand density.
Interactions with the surrounding environment in morphogenesis trigger the first cell differentiation events and propagate them until the establishment of the final complex tissue structures. To evaluate the effects of dendrimer nanopatterns on cell adhesion and differentiation, we used the chondrogenic induction of MSCs as a model. During chondrogenesis, there is active matrix remodeling, which plays an instructive role in directing cells through the different stages of cartilage formation.
MSCs underwent chondrogenesis on the nanopatterns (Figure 3). Chondrogenic differentiation begins with a cell condensation step, which involves cell recruitment to form dense condensates, the establishment of cell-to-cell communication, and concomitant changes in cell morphology33. Figure 3A shows that cell condensation occurred in all the substrates and that condensates increased in area with increasing dendrimer concentrations up to 2.5 x 10-8% w/w The condensate area then decreased again for a dendrimer concentration of 10-2% w/w and for the positive control. The cell condensation step in chondrogenesis occurs through active cell movement rather than through an increase in cell proliferation34. This cell movement is favored by flexible adhesion with the substrate: stable adhesions should be formed to permit traction forces to move the cell body, and at the same time, these adhesions should be weak enough to allow cell release from the substrate during movement. Cell condensation is favored by an increase in local RGD surface density up to 2.5×10-8% w/w-derived nanopatterns, with a good balance between cell adhesion and cell movement. For 10-2 and the positive control, the local RDG surface density was too high, thereby impairing the condensation event.
Chondrogenic differentiation proceeds from prechondrogenic condensates: cells in condensates become more rounded and start the synthesis of cartilage specific markers such as the ECM protein COL2A1 and the transcription factor SOX933,34,35. Accordingly, substrates with an intermediate adhesiveness, favoring the formation of cell condensates, showed higher COL2A1 staining (Figure 3B) and higher levels of SOX9 mRNA expression (Figure 3C).
Our results highlight the influence of cell-cell matrix interactions during the early stages of chondrogenic differentiation. Such interactions can be easily addressed through dendrimer-based nanopatterns.
Step | Time (s) | Speed (rpm) | Acceleration (rpm/s) |
1 | 5 | 500 | 300 |
2 | 30 | 3,000 | 1,500 |
Table 1: Spinner program steps used to coat the glass slides with the prepared PLLA solution. A first homogenization step was used at 500 rpm with an acceleration of 300 rpm/s for 5 s. followed by a last step at 3,000 rpm with an acceleration of 1,500 rpm/s for 30 s.
Solution | RGD-Cys-D1 (mg/mL) | RGD-Cys-D1 (mg) | MQ water (µL) |
A | 0.77 | 5 | 6,494 |
RGD-Cys-D1 (% w/w) | Solution A (µL) | ||
B | 10-2 | 779 | 5,220 |
C | 10-5 | 0.78 | 6,000 |
Solution C (µL) | |||
D | 2,5 x 10-8 | 15.0 | 5,985 |
E | 10-8 | 6.0 | 5,994 |
F | 4 x 10-9 | 2.4 | 5,998 |
Table 2: Details of the preparation of the RGD-Cys-D1 solutions used. A stock solution of RGD-Cys-D1 dendrimers was prepared at a final concentration of 0.77 mg/mL (solution A), from which solutions B and C were prepared at 10-2 and 10-5% w/w concentrations. Solution C with a 10-5% w/w concentration of RGD-Cys-D1 dendrimer was used to prepare solutions D, E and F at final concentrations of 2.5 x 10-8, 10-8 and 4 x 10-9% w/w, respectively.
Name | Volume (µL) | Blocking buffer (mL) | |
Primary mix | Rabbit mAb to pax | 65 | 12.870 |
Mouse mAb to COL2A1 | 32.5 | ||
Secondary mix | Alexa Fluor 488 goat anti-mouse | 13 | 12.961 |
Alexa Fluor 568 goat anti-rabbit | 13 | ||
Hoechst solution | 13 |
Table 3: Antibody solution preparation. The primary antibody mixture contained monoclonal antibody against pax produced in rabbit diluted 1:200 in the blocking solution with monoclonal antibody against COL2A1 produced in mouse diluted 1:400 in the blocking solution. The secondary antibody mixture contained the cell nuclei stain Hoechst and the secondary antibodies produced in goat against mouse and rabbit respectively (labeled with Alexa Fluor 488 and 568 fluorophores, respectively).
Figure 1: RGD-Cys-D1 dendrimer nanopatterning for the nanoscale control of local RGD surface density. (A) RGD-Cys-D1 dendrimer structure containing up to eight copies of the cell adhesive RGD peptide. Representative STM images (bias = 200 mV, set point = 0.5 nA) of RGD-Cys-D1 nanopatterns on Au(111) from an initial aqueous solution of 10-8% w/w (Scale bar = 3 nm, B) and the corresponding height-distance profile obtained on the dashed region indicated in (B), and (C) of 10-2% w/w showing dendrimer aggregation (Scale bar = 20 nm). (D-G) Representative AFM images of RGD-Cys-D1 nanopatterns on PLLA obtained from the corresponding dendrimer aqueous solutions of 10-2%, 2.5 x 10-8%, 10-8% and 4 x 10-9% w/w, respectively (Scale bar = 1 µm). The corresponding minimum interparticle distance (dmin) probability contour map is shown below each AFM image, with high density RGD regions shown in dark red (dmin <70 nm). Dendrimers and dendrimer aggregates are depicted in black for clarity. Please click here to view a larger version of this figure.
Figure 2: Cell adhesion on the RGD-Cys-D1 nanopatterns. Plot of the percentage of area stained for the FA protein pax per cell for the cells in contact with the substrate (left axis) with initial dendrimer concentration. Values are compared with the local RGD surface density (percentage of surface area in the nanopatterns containing dmin values below the 70 nm threshold) (right axis) with 100 and 0 percentages assigned to the positive (+ control) and negative (- control) controls, respectively. (A) Fibroblast adhesion on dendrimer nanopatterns on Au(111) after 4.5 h of culture. (B) MSC adhesion on dendrimer nanopatterns on PLLA evaluated at day 1 of chondrogenic induction. Values are given as the mean with the standard deviation. Please click here to view a larger version of this figure.
RGD-Cys-D1 (% w/w) | Area (< dmin = 70 nm) (%) on Au(111) | Area (< dmin = 70 nm) (%) on PLLA |
10-2 | 5 ± 1 | 2 ± 2 |
10-5 | 97 ± 2 | |
2,5×10-8 | 65 ± 11 | 90 ± 2 |
10-8 | 25 ± 16 | 45 ± 7 |
4×10-9 | 18 ± 11 |
Table 4: Percentage of area with dmin values below 70 nm obtained for RGD-Cys-D1 dendrimer deposition on Au(111) and on PLLA surfaces as a function of the initial dendrimer concentrations used.
Figure 3: Chondrogenic differentiation of MSCs on the RGD-Cys-D1 nanopatterns. (A) Condensation of MSCs cultured on RGD-Cys-D1 nanopatterns on PLLA after 5 days of chondrogenic induction. Representative epifluorescence images of stained cell nuclei (Hoechst; Scale bar = 300 µm; upper row) and plot of the area of cell condensates (lower row) obtained on RGD-Cys-D1 nanopatterns from different initial dendrimer concentrations. Differentiation proceeds from cell condensation step with the expression of specific chondrogenic markers: (B) Percentage of the area of COL2A1 staining normalized with the area of the cell condensate from the corresponding confocal z-projections obtained after 5 days of chondrogenic induction. (C) Relative SOX9 mRNA expression (against negative control) after 3 days of chondrogenic induction. Values are given as the mean with the standard deviation in (B) and (C). Please click here to view a larger version of this figure.
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 this case Au(111). Any lifting of the polymer from the corners of the slide after the spin-coating can be rectified using biocompatible glue.
Dendrimer nanopatterning is a process through which to achieve controlled local cell adhesiveness at the nanoscale. Based on the self-assembly of dendrimers on the surface by adsorption, this technique does not require any complex nanopatterning equipment, thus contrasting with previously described lithography-based methods36,37,38. Dendrimer nanopatterning can be easily scaled up to large surface areas and is fully compatible with cell culture protocols.
The dendrimer nanopatterning method described here can find future applications in regenerative medicine. The control exerted by dendrimer nanopatterning on cell adhesiveness makes this technique suitable for the conditioning of biomaterials prior to implantation, thereby facilitating their integration into host tissues. Moreover, the ease with which dendrimer peripheral moieties can be modified makes dendrimer nanopatterning appropriate for other ligands that exert concentration-dependent activity on cells.
The authors have nothing to disclose.
The authors acknowledge Oriol Font-Bach and Albert G. Castaño 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&D&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).
Gold (111) on mica. 1.4×1.1 cm | Spi Supplies | 466PS-AB | |
Glass micro slides, plain | Corning | 2947-75×25 | |
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 24×24 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 |