The Functional Response of Mesenchymal Stem Cells to Electron-Beam Patterned Elastomeric Surfaces Presenting Micrometer to Nanoscale Heterogeneous Rigidity

Abstract

Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential cellular function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with a range of Young's modulus values for bulk rigidity, at the subcellular level, tissues are comprised of heterogeneous distributions of rigidity. Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, it is shown for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric poly(dimethylsiloxane) substrates and a new class of 2D elastomeric substrates with controlled patterned rigidity ranging from the micrometer to the nanoscale is described. The mechanoresponse of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces.

Keywords: electron beam; focal adhesions; mechanotransduction; polydimethylsiloxane; rigidity; stem cells.

Figure 1. Electron-beam interaction with PDMS thin-films

(A) A 120 μm layer of PDMS was deposited onto 22 mm square microscopy cover-glasses by a spin-coating process. Substrates were treated with an oxygen plasma process and coated with a final polymeric discharge layer (AquaSAVE) prior to e-beam patterning. A focused e-beam was rastered over the substrate surface to create arrays of defined surface features (spots) possessing a sub-surface rigidity gradient. (B) Monte Carlo simulations identified the electron trajectory and scatter profile in PDMS substrates. (C,D) Peak-force quantitative AFM nanomechanical mapping (PF-QNM) of 2 μm spots indicated the e-beam exposure of the PDMS film causes an increase in the elastic modulus of the polymer as a function of e-beam dose. (E) shows the function relating Young's modulus changes due to the e-beam exposure dose.

Figure 2. Chemical modulation of PDMS substrates by focused electron-beam patterning

(A) Surface wettability analysis following PDMS treatment with an oxygen plasma. (B)High resolution X-ray photoelectron spectroscopy of (C) O 1s (D) C 1s and (E) Si 2p3. (F) Raman spectra of (F) Raman spectra of e-beam exposed and non-exposed PDMS regions. The spectra below 2600 cm^-1 was enhanced five-fold, while spectra above 2600 cm^-1 was reduced two-fold, for better visibility of the peaks. (G) Component Discriminant least squares analysis, using peaks from e-beam exposed (green) and unexposed (red), as the spectra of the components, shows spectral differences on PDMS patterned with 1μm spots. The figure shows that the peaks that define the control region are also found in the inter-spot region (red), while the e-beam exposed 1 μm spots are associated with significant peaks (green) that are not present in non-exposed regions.

Figure 3. Focal adhesion formation on 2 μm spots of modulated rigidity

(A) e-beam spots of ∼350 MPa induced differential focal adhesion co-localization in hMSCs. (B) This effect was lost on f 50 MPa spots. High magnification insert of paxillin staining within the boarded area indicated in (a,b). (C) E-beam exposure induced a linear increase in focal adhesion co-localization to spots of altered rigidity. (D) Cellular spreading was not affected in MSCs cultured on 2 μm diameter spots of modulated rigidity. (E) Significant changes in mean FA area were induced by increasing the elastic modulus of 2 μm diameter spots. For statistical analysis of significance see Supplementary Table 1 and Supplementary Table 2. Results are SEM, green = actin, blue = paxillin, red = nucleus, bar = 10 μm.

Figure 4. Focal adhesion formation on ∼350 MPa spots of varying diameter

(A) 1 μm spots of ∼350 MPa (formed with doses of 3,000 μC/cm²) induced differential focal adhesion co-localization in hMSCs as a function of spot diameter. This effect was lost on 100 nm spots, high magnification insert of paxillin staining within the boarded area indicated in (a,b). (B) Mander's coefficient of co-localization indicated a linear increase in FA co-localization to the e-beam exposed regions with increasing spot diameter. (D) Cellular spreading was not significantly different in MSCs cultured on spots of modulated rigidity as a function of spot diameter relative to unexposed PDMS, yet significant reductions in cell spreading were noted relative to hMSCs cultured on glass control substrates. (E) Significant reductions in mean FA area were also induced by reducing the spot diameter. For statistical analysis of significance see Supplementary Table 2 and Supplementary Table 3. Results are SEM, green = actin, blue = paxillin, red = nucleus, bar = 10 μm.

Figure 5. Functional analysis of hMSCs cultured on PDMS substrates patterned with 2 μm spots of increasing rigidity for 12 hours

(A) Functional pathway analysis of hMSCs cultured on e-beam patterned 2 μm spot substrates with different rigidities revealed significant activation of signaling pathways as a function of spot dose relative to cells cultured on non-exposed homogenous rigidity substrates in (A) chondrogenic and (B) Osteogenic media. Red indicates an increase in pathway activation and green indicates a decrease in pathway activation relative to controls as shown in the Activation Score bar. Statistical significance of pathway modulation was calculated via a right-tailed Fisher's exact test.

Figure 6. Functional analysis of hMSCs cultured on PDMS substrates patterned with ∼350 MPa spots of increasing spot diameter for 12 hours

Functional pathway analysis of hMSCs cultured on e-beam patterned spots with diameters ranging from 500-2000 nm revealed significant activation of signaling pathways as a function of spot size relative to cells cultured on control homogeneous rigidity substrates in (A) chondrogenic and (B) Osteogenic media. Red indicates a increase in pathway activation and green indicates a decrease in pathway activation relative to controls as shown in the Activation Score bar. Statistical significance of pathway modulation was calculated via a right-tailed Fisher's exact test.