Directional Topography Influences Adipose Mesenchymal Stromal Cell Plasticity: Prospects for Tissue Engineering and Fibrosis

Abstract

Introduction: Progenitor cells cultured on biomaterials with optimal physical-topographical properties respond with alignment and differentiation. Stromal cells from connective tissue can adversely differentiate to profibrotic myofibroblasts or favorably to smooth muscle cells (SMC). We hypothesized that myogenic differentiation of adipose tissue-derived stromal cells (ASC) depends on gradient directional topographic features.

Methods: Polydimethylsiloxane (PDMS) samples with nanometer and micrometer directional topography gradients (wavelength (w) = 464-10, 990 nm; amplitude (a) = 49-3, 425 nm) were fabricated. ASC were cultured on patterned PDMS and stimulated with TGF-β1 to induce myogenic differentiation. Cellular alignment and adhesion were assessed by immunofluorescence microscopy after 24 h. After seven days, myogenic differentiation was examined by immunofluorescence microscopy, gene expression, and immunoblotting.

Results: Cell alignment occurred on topographies larger than w = 1758 nm/a = 630 nm. The number and total area of focal adhesions per cell were reduced on topographies from w = 562 nm/a = 96 nm to w = 3919 nm/a = 1430 nm. Focal adhesion alignment was increased on topographies larger than w = 731 nm/a = 146 nm. Less myogenic differentiation of ASC occurred on topographies smaller than w = 784 nm/a = 209 nm.

Conclusion: ASC adherence, alignment, and differentiation are directed by topographical cues. Our evidence highlights a minimal topographic environment required to facilitate the development of aligned and differentiated cell layers from ASC. These data suggest that nanotopography may be a novel tool for inhibiting fibrosis.

Figure 1

Schematic illustration of the operational process for the fabrication of gradient linear topographies using PDMS via prolonged plasma oxidation. Adapted from “Screening Platform for Cell Contact Guidance Based on Inorganic Biomaterial Micro/nanotopographical Gradients” by Zhou et al. [57]. Adapted with permission.

Figure 2

Surface topography characterization. (a) AFM images of the ten different ranges (small to large: R1-R11) of the two topographical directional gradients (nanometer scale and micrometer scale) along the PDMS substrate. Also shown is the flat PDMS control. Scale bars are 4 μm and apply to all images. (b) Dependence of the wavelength and amplitude of created wrinkle gradients. The microgradient surface (blue) starts where the nanogradient surface (green) ends with respect to wavelength and amplitude. Data are reported as the mean ± standard deviation (n = 30). (c) XPS spectra of PDMS wrinkle gradients.

Figure 3

Cell alignment (24 h of culture). (a) Overview of the Phalloidin staining (red) for F-actin on the two different wrinkle gradients (R1-R5 and R6-R11), as well as on the flat PDMS control. (b) Cell alignment quantification as the mean cell angle relative to the directional topography. The dotted line represents the 10° cutoff defining the limit for cellular alignment. Black represents the flat PDMS control, red represents the nanotopography gradient, blue represents the microtopography gradient, and green represents R11; ^∗ p < 0.05 vs. PDMS flat control. Values represent mean ± SEM of 3 independent experiments.

Figure 4

Focal adhesions (24 h of culture). (a) Fluorescence staining for vinculin (white) on the different wrinkle sizes of nano- (R1-R5) and microtopography (R6-R11) gradients, as well as on the flat PDMS control. Yellow dots represent the areas recognized as focal adhesions by the Focal Adhesion Analyze Server. (b) Number of focal adhesions per cell. (c) Area of each single focal adhesion. (d) Total focal adhesion area per cell. (e) Focal adhesion alignment to the directional topography. For all the graphs, black represents the flat PDMS control, red represents the nanotopography gradient, blue represents the microtopography gradient, and green represents R11; ^∗ p < 0.05 vs. PDMS flat control. Values represent mean ± SEM of 3 independent experiments.

Figure 5

Cell differentiation (7 days of culture). (a) Fluorescence staining for SM22α (red) and DAPI (blue) in the different wrinkle sizes of nano- (R1-R5) and microtopography (R6-R11) gradients, as well as in the flat PDMS control. (b) SM22α expression in ASC induced by TGF-β1 as the fold change of the unstimulated flat PDMS control. Black represents the flat PDMS control, red represents the nanotopography gradient, blue represents the microtopography gradient, and green represents R11; ^∗ p < 0.05 vs. TGF-β1-induced flat PDMS control. Values represent mean ± SEM of 3 independent experiments.

Figure 6

Influence of micrometer-sized topography and TGF-β1 stimulation on mesenchymal gene expression in adhered ASC over seven days. Expression of ACTA2 (a) and TAGLN (b) increased over 3 days, independent of TGF-β1 stimulation and topography (flat vs. 1 or 11 μm wavelength). For the net influence of TGF-β1 and topography, the area under the curve was determined for total expression of ACTA1 (c) and TAGLN (d), respectively. This showed no differences between flat material and topographies, irrespective of TGF-β1. Values represent mean ± SEM of 3 independent experiments. Statistical analysis was performed comparing the flat topography to both of the linear topography patterns for both TGF-β1-stimulated and not stimulated groups.

Figure 7

Protein expression of myogenic markers (7 days of culture). (a) Relative protein expression of αSMA with and without TGF-β1 induction in flat and uniform wrinkle (1 μm and 11 μm) topographies. Representative WB results are shown below the graph. (b) Relative protein expression of SM22α with and without TGF-β1 induction in flat and uniform wrinkle (1 μm and 11 μm) topographies. Representative WB results are shown below the graph. For all the graphs, black represents the flat PDMS control, red represents the 1 μm uniform features, and green represents the 11 μm uniform features. Values represent mean ± SEM of 3 independent experiments. Statistical analysis was performed comparing the flat topography to both of the linear topography patterns for both TGF-β1-stimulated and not stimulated groups.

Figure 8

Scatter plot showing the correlation between cell and focal adhesion alignment. Pearson correlation, r ² = 0.8237, p < 0.0001. Values represent the mean of 3 independent experiments. Data from the experiment using all 11 topographical ranges in which cells were cultured for 24 hours without TGF-β1.

Figure 9

Heat map interpolating focal adhesion area per cell, cell alignment, and expression of SM22α data. Data for focal adhesions and cell alignment were from the experiment using all 11 topographical ranges in which cells were cultured for 24 hours without TGF-β1. Data for differentiation were from the experiment in which cells were cultured for 7 days under stimulation with TGF-β1, also with all 11 topographical ranges.