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 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 adhesion^4,5. 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 value^6,7,8,9. These studies also highlighted the greater influence of local than global ligand density on cell adhesion^9,10,11.

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/osteogenic^13,14,15 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 sphere^18,19,20 onto surfaces has been used to study cell adhesion^21,22, morphology^23,24, and migration events^25,26. 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

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 lower side of each slide, so that the upper and lower sides can be later distinguished.
      2. Wash the slides thoroughly with deionized water followed by 96% ethanol. Allow them to air-dry.
   2. Prepare the 2% PLLA solution.
      1. Add 200 mg of PLLA to 10 mL of 1,4-dioxane in a pressure tube. Add a stir bar and close the tube tightly.
      2. Place the pressure tube into a glycerin bath on a hot plate at 60 °C under gentle stirring for 24 h, and then transfer the solution to a 15-mL glass vial.
   3. Coating the glass slides with the PLLA solution.
      1. Place the glass slides and the vial with the PLLA solution on a clean hot plate at 60 °C. Make sure to place the slides facing upward and allow them to remain for at least 10 min to reach the necessary temperature.
      2. Prepare the spin coater and set the program specified in Table 1.
      3. Place one of the slides face up on the spin coater, using a vacuum system. With a Pasteur pipette, apply 0.25 mL of the PLLA solution to the slide, making sure that the whole surface is covered. Run the coating program. Repeat this step for each slide.

2. Dendrimer Nanopatterning

1. Preparation of RGD-Functionalized Dendrimer (RGD-Cys-D1) ²⁸ Solutions.
   1. Dissolve 5 mg of the dendrimer in 6.494 mL of deionized water. This is solution A.
      NOTE: Use dendrimer stock solution within 6 months of preparation.
   2. Sonicate solution A for 10 min and prepare solutions B and C following Table 2.
   3. Sonicate solution C for 10 min and prepare solutions D, E and F following Table 2.
   4. Store solutions B, C, D, E and F at 4 °C until use. Solution A can be stored at -20 °C for later use.
2. Nanopatterning of RGD-Cys-D1 Dendrimers on the Substrates
   1. In a tissue culture hood, sterilize the substrates by irradiating them with UV light for 13 min.
      NOTE: This step is only necessary when nanopatterned substrates are going to be used as cell culture substrates. In this case, maintain the sterile conditions (tissue culture hood, sterile materials, solutions and techniques) for the following steps.
   2. Place each substrate face up in the wells of a plate, handling them carefully with tweezers.
   3. Sonicate solutions B, C, D, E and F for 10 min.
   4. Pass the RGD-Cys-D1 dendrimer solutions through a 0.22-µm diameter filter using a syringe directly in the wells containing the substrates (2 mL/well). At least three replicas per dendrimer concentration are recommended. Close and seal the plate and leave it at room temperature (RT) for 16 h.
   5. Remove and discard the solutions. Wash the substrates with sterile deionized water and dry. Store substrates at 4 °C.
      NOTE: The protocol can be paused here.

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).

1. In a tissue culture hood, sterilize the substrates by irradiating them with UV light for 13 min.
2. Control Substrates for Fibroblast Adhesion Experiment.
   1. Use flame-annealed Au(111) substrates as the negative control. Gold has a well-known protein-denaturation effect that confers anti-cell adhesive properties²⁸. Sonicate all solutions and filter prior to substrate incubation.
   2. For the positive controls, immerse flame-annealed Au(111) substrates in a solution of RGD-modified PEG thiol, Ac-Gly-Arg-Gly-Asp-Ser-Asp-NH-ethylene glycol mono-11-mercaptoundecanamide (RGD-PEG-SH)³⁰ and triethylene glycol mono-11-mercaptoundecyl ether (PEG-SH) at a 1:100 molar ratio in 96% ethanol for 16 h at RT.
   3. Wash substrates thoroughly in ethanol and dry them with argon. Store substrates at 4 °C.
3. Control Substrates for Chondrogenesis.
   1. Use pristine PLLA substrates as the negative control. Without any surface treatment, PLLA shows poor interfacing with living cells.³¹
   2. For the positive controls, prepare 5 mL of 0.1 mg/mL fibronectin in phosphate-buffered saline (PBS) and incubate each PLLA substrate with 1.6 mL of the fibronectin solution for 1 h at RT.
   3. Remove and discard the fibronectin solution, and wash the substrates with PBS. Store substrates at 4 °C.

4. Surface Characterization

1. AFM Imaging
   1. Perform AFM imaging of PLLA substrates for a roughness analysis. Since dendrimers have a diameter of 4-5 nm, roughness values of 1 nm should be obtained for the proper visualization of the nanopatterns. Also, perform AFM imaging of the nanopatterns. In both cases, perform AFM in tapping mode in air.
   2. Select a silicon cantilever with a spring constant k = 40 N/m and a resonant frequency ν = 300 kHz and mount it onto the AFM equipment.
   3. Mount the sample on the stage of the atomic force microscope. Depending on the set-up of the apparatus, tuning and imaging specifications may vary. Consult the manufacturer's handbook.
   4. Approach the sample with the tip of the microscope until contact.
   5. To image PLLA for roughness analysis, select at least four representative areas of 20x20 µm per substrate from three independent substrates. To image dendrimer nanopatterns, choose at least three representative images of 5×5 µm per substrate from three independent substrates per condition (initial dendrimer concentration in solution). To avoid damaging the dendrimer layer while imaging, adjust the set point to keep the force at a minimum.
      NOTE: The choice of the imaging area also depends on the working range of the piezoelectric scanner. Please consult the instrument manual in this regard.
   6. Process the AFM height images by fitting each scan line to polynomial leveling functions using the proprietary software of the manufacturer of the AFM apparatus, and analyze those from PLLA to calculate surface roughness. Root-mean square (RMS) analysis may provide a suitable option.
2. Local RGD Surface Density Measurement.
   1. Obtain the image thresholds of the processed AFM height images to select the dendrimers on the surface.
   2. Determine the particle positions using an image processing software and use them to obtain the minimum interparticle distances (d_min).
   3. Plot d_min values in z to the corresponding particle positions to obtain the probability contour maps for d_min. Adjust the color scale of the plot to visualize the regions of highest local RGD surface density (d_min < 70 nm).
   4. Quantify the area of the regions with the highest local RGD surface density using an image processing software. Include the areas of dendrimer aggregates in the calculation.
3. STM Imaging.
   Note: STM imaging can be performed only for nanopatterns on conductive Au(111) substrates. Measurements are done in air.
   1. Place the nanopatterned substrate in the sample holder of the STM equipment. Check the electrical connection between sample and holder with a tester.
   2. Etch a tip of the selected probe material (i.e. Pt 0.8: Ir 0.2) with a diameter that ensures proper fitting on the head of the STM equipment. Etching can be performed either by manual cutting or by electrochemical etching.³²
      NOTE: Electrochemical etching renders more symmetric tips.
   3. Mount the tip in the head of the STM equipment and connect the sample.
   4. Set "current" in the feedback channel (in this type of measurement, current will be constant by feedback tuning). Adjust the bias voltage and current set point and scan size to obtain a well resolved image once the tip is engaged.
      NOTE: The choice of the imaging area also depends on the working range of the piezoelectric scanner. Please consult the instrument manual in this regard.
   5. Process the STM topographical images by fitting each scan line to polynomial leveling functions using the proprietary software of the manufacturer of the STM apparatus.
4. CA Measurements.
   1. Measure CA on the nanopatterned substrates by the sessile-drop method at three different positions on three independent substrates per condition (initial dendrimer concentration in solution) with an optical CA system.
   2. Fill the microsyringe with deionized water and set the parameters in the CA software to produce drops of 1 µL.
   3. Place the sample on the stage so that the surface of the sample is clearly visible on the computer for imaging.
   4. Move the syringe with the micromanipulator until it gets close to the surface and dispense the drop onto the surface. Record the image immediately after droplet stabilization.
      NOTE: In CA measurements, it is very important to control humidity in order to achieve reproducible results.
   5. Analyze the CAs measured with the proprietary software of the manufacturer of the CA apparatus. The fitting method can be adjusted to user requirements. The elliptic fitting method can be an option.

5. Cell Culture

NOTE: All steps were performed in a tissue culture hood, and only sterile materials, solutions and techniques were used.

1. Fibroblast Adhesion Experiment.
   1. Culture NIH 3T3 mouse embryonic fibroblasts from early passages (< 10) at 37 °C and 4.6% CO₂ atmosphere in basal medium with high glucose supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin-streptomycin and 1% sodium pyruvate (growth medium). T75 flasks for a total seeding of 500,000 cells per flask in 10 mL of growth medium are recommended.
   2. Remove old medium with a 10 mL pipette every 2 days and replace with 10 mL of freshly prepared growth medium.
   3. Culture cells in the growth medium until they reach around 80% confluence, then remove the medium and add 5 mL of trypsin per flask. To ensure proper cell detachment from the bottom of the flask, maintain the trypsin solution in contact with the cells for 5 min at 37 °C.
   4. Add 5 mL of growth medium per flask and collect the cells in a centrifuge tube. Centrifuge the cells at 470 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 10 mL of growth medium. Determine the concentration of cells using a hemocytometer.
   5. Transfer the substrates to well plates not treated for tissue culture (non-adherent).
   6. Seed the cells on the substrates at a density of 4,000 cells/cm² in growth medium and incubate them for 4.5 h at 37 °C and 10% CO₂ atmosphere.
2. Chondrogenic Induction of MSCs.
   1. Culture human MSCs from early passages (<5) at 37 °C and 4.6% CO₂ atmosphere in MSC growth medium. T75 flasks for a total seeding of 500,000 cells per flask in 10 mL of growth medium are recommended.
   2. Change medium every 3 days (as described in 5.1.2).
   3. Trypsinize cells before 80% confluence is reached, and centrifuge and resuspend them in MSC growth medium (as described in 5.1.3). Determine the concentration of cells using a hemocytometer.
   4. Centrifuge the cells again and resuspend them in 10 mL of chondrogenesis-inducing medium.
   5. Transfer the substrates from the plates to new well plates not treated for tissue culture (non-adherent). Seed the cells on the substrates at a density of 3,000 cells/cm².
   6. Change the chondrogenic medium every 3 days (as described in 5.1.2).

6. Cell Fixation and Immunostaining

NOTE: The following steps can be performed in non-sterile conditions.

1. Remove the media and wash the cells gently with PBS. Fix them by adding a 10% formalin solution for 20 min at RT.
2. Remove the formalin and wash the cells with PBS.
3. Block the free aldehyde groups by adding a 50 mM solution of ammonium chloride (NH₄Cl) in PBS. Leave the cells for 20 min at RT. Remove NH4Cl solution and wash the cells with PBS.
   NOTE: The protocol can be paused here. Store samples in PBS at 4 °C.
4. Remove PBS and permeabilize the cells by adding a 0.1% solution of saponin in the blocking solution (1% albumin in PBS) for 10 min at RT. Wash the cells with PBS and transfer samples to a new well plate.
5. Incubate the cells with a solution of primary antibodies in the blocking solution (Table 3) for 1 h at RT. Then, remove the primary antibody solution and wash the cells with PBS.
6. Incubate the cells with secondary antibodies in the blocking solution (Table 3) for 1 h at RT.
   NOTE: Avoid light exposure.
7. Remove the secondary antibody solution and wash the cells with PBS and dry.
   NOTE: The protocol can be paused here. Store the samples in PBS at 4 °C in the dark.
8. Sample mounting for microscope observation: Using a diamond-tip cutter, cut coverslips into 1.25 cm x 1.25 cm. Apply 50 µL of microscopy mounting medium onto the samples and cover them gently with the cut coverslips.
9. Transfer the samples to a convenient recipient, cover it with aluminum foil and store in the dark at 4 °C until observation.

7. Cell Imaging and Data Analysis

NOTE: For opaque Au(111) and thick microslide-based substrates, an upright microscope must be used.

1. Fibroblast Adhesion Experiment.
   1. Use an epifluorescence microscope equipped with a digital camera and low (i.e. 10X) and high (i.e. 40X) magnification objectives. Perform measurements in air.
   2. Place the sample on the stage and image cell nuclei with the 10X objective using an ultraviolet excitation, longpass emission filter to visualize the Hoechst/DAPI stain.
   3. Image cell cytoskeleton and focal adhesions (FAs) with the 40X objective, selecting the microscope filter that matches the corresponding antibody fluorochrome specifications.
   4. For FA quantification, use an image processing software to convert images to 8-bit files. Remove the background and convert images to binary by setting a threshold. Compute at least 30 images per sample and consider FAs from 1 µm² for calculation.
2. Chondrogenic Induction of MSCs.
   1. Image cell condensates at the initial stages of chondrogenic induction (< 5 days) with an upright epifluorescence microscope equipped with a digital camera and a low (i.e. 10X) magnification objective. Use ultraviolet excitation and longpass emission filter to visualize the Hoechst/DAPI stain.
   2. For measuring the condensate area, use an image processing software, convert images to 8-bit files, remove the background and select a threshold that highlights the aggregate contour. Calculate the area of the particles.
   3. Image immunostained samples for FAs, cytoskeleton actin fibers, and cartilage-specific collagen type II alpha 1 (COL2A1) with an upright confocal microscope at high magnification (i.e. 40-60X). Collect sections at a representative interval (i.e. 0.5–1 µm).
   4. To process the stack, use an image processing software. Convert images to 8-bit files, remove the background, and make them binary by setting a threshold. Treat a minimum of three cell condensates per condition of three independent samples.
   5. Quantify the FA protein stained areas from the basal zone of cell condensates and express them as the corresponding percentage of area divided by the number of cell nuclei in the image.
   6. To measure COL2A1 staining, use confocal z-projections and quantify the sum of the projected area (maximum COL2A1 area per sample). Normalize the area value obtained against the area of the corresponding condensate.

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.

1. Isolate total RNA and pulverize it in a RNA disrupter. Treat RNA samples with DNase and convert them into cDNA using a cDNA synthesis kit.
2. Conduct qRT-PCR using primers for the transcription factor SOX9. Beta-2-microglobulin (B2M) and ribosomal protein L13a (RPL13a) can be used as housekeeping genes.
3. Calculate the expression levels and normalize them against the negative control (PLLA).

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 (

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 ...

The authors have nothing to disclose.

The authors acknowledge Oriol Font-Bach and Albert G. Castaño for their help in d_min 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).