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

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.