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. 2020 Jun 10;12(23):25591-25603.
doi: 10.1021/acsami.0c05012. Epub 2020 May 27.

Synergistic Effect of Cell-Derived Extracellular Matrices and Topography on Osteogenesis of Mesenchymal Stem Cells

Liangliang Yang  1   2 , Lu Ge  1   2 , Patrick van Rijn  1   2
Affiliations

Synergistic Effect of Cell-Derived Extracellular Matrices and Topography on Osteogenesis of Mesenchymal Stem Cells

Liangliang Yang et al. ACS Appl Mater Interfaces. .

Abstract

Cell-derived matrices (CDMs) are an interesting alternative to conventional sources of extracellular matrices (ECMs) as CDMs mimic the natural ECM composition better and are therefore attractive as a scaffolding material for regulating the functions of stem cells. Previous research on stem cell differentiation has demonstrated that both surface topography and CDMs have a significant influence. However, not much focus has been devoted to elucidating possible synergistic effects of CDMs and topography on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this study, polydimethylsiloxane (PDMS)-based anisotropic topographies (wrinkles) with various topography dimensions were prepared and subsequently combined with native ECMs produced by human fibroblasts that remained on the surface topography after decellularization. The synergistic effect of CDMs combined with topography on osteogenic differentiation of hBM-MSCs was investigated. The results showed that substrates with specific topography dimensions, coated with aligned CDMs, dramatically enhanced the capacity of osteogenesis as investigated using immunofluorescence staining for identifying osteopontin (OPN) and mineralization. Furthermore, the hBM-MSCs on the substrates decorated with CDMs exhibited a higher percentage of (Yes-associated protein) YAP inside the nucleus, stronger cell contractility, and greater formation of focal adhesions, illustrating that enhanced osteogenesis is partly mediated by cellular tension and mechanotransduction following the YAP pathway. Taken together, our findings highlight the importance of ECMs mediating the osteogenic differentiation of stem cells, and the combination of CDMs and topography will be a powerful approach for material-driven osteogenesis.

Keywords: extracellular matrix; mechanotransduction; mesenchymal stem cells; osteogenic differentiation; topography.

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Conflict of interest statement

The authors declare the following competing financial interest(s): P.v.R. also is co-founder, scientific advisor, and share-holder of BiomACS BV, a biomedical oriented screening company. The authors declare no other competing interests.

Figures

Figure 1
Figure 1
Schematic representation of the preparation process of the CDM. Fibroblast-derived extracellular matrices were obtained through a decellularization process of cultured fibroblasts. Then, onto the matrix, hBM-MSCs were seeded to investigate the co-effect of topography and CDMs on osteogenesis.
Figure 2
Figure 2
Representative AFM images of the substrate and topography profiles (height) of the structured PDMS substrates obtained (A) after imprinting and (B) after ECM deposition by fibroblasts with subsequent decellularization. W0.5, W3, and W10 stand for W0.5/A0.05, W3/A0.7, and W10/A3.5, respectively, and W is the abbreviation of wavelength.
Figure 3
Figure 3
Representative immunofluorescence image of macromolecular ECM components (A: Fn; B: Col I) after decellularization. The white color arrows refer to the direction of the wrinkle. The scale bar is 40 μm. (C) Corresponding angular graph of the Col I orientation on different substrates, (D) statistical analysis of the Col I orientation, and (E) quantified fluorescence intensity of Col I compared to the mean values of the Flat substrate. Five images for each substrate were analyzed. Data are shown as mean ± standard deviation (SD), and N.S represents not significant, and **P < 0.01, ***P < 0.001.
Figure 4
Figure 4
Representative fluorescence microscopy images of hBM-MSCs grown on (A) pristine topography and (B) substrates deposited with the CDM for 1 day, respectively. The row below the fluorescence image shows the corresponding angular graph of the cell cytoskeleton orientation on different substrates. The cytoskeleton (red) and the nucleus (blue) were visualized using TRITC-labeled phalloidin and DAPI as stains, respectively. The arrow represents the direction of the wrinkle. The scale bar is 100 μm for all the images. Statistical analysis of (C) cell orientation, (D) area per cell, and (E) cell aspect ratio (n ≥ 60 cells, three independent experiments). Data are shown as mean ± standard deviation (SD), and *P < 0.05.
Figure 5
Figure 5
(A) Immunofluorescence labeling of the osteogenic marker OPN of hBM-MSCs cultured on the original substrate and CDM substrates cultured for 14 days in OM. hBM-MSCs were labeled for nuclei (DAPI, blue) and OPN (red). The scale bar is 100 μm for all images. (B) Quantification of OPN expression in the cells cultured in OM at day 14, normalized by the cell number (n ≥ 100 cells, three independent experiments). Data are shown as mean ± standard deviation (SD), and **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
(A) Representative photographs of Alizarin Red-stained calcium nodules indicating extracellular calcium deposits by osteoblasts derived from hBM-MSCs cultured for 21 days in OM. (B) Mineralization quantification by elution of Alizarin Red S from the stained mineral bone matrix. Data are shown as mean ± standard deviation (SD), and **P < 0.01. The scale bar is 5 mm.
Figure 7
Figure 7
(A) Representative images of hBM-MSCs residing on different surfaces and the location of YAP after 24 h of seeding. Blue: nucleus, Green: F-actin, Red: YAP. The arrows refer to the YAP location. The scale bar is 20 μm. (B) The percentage of cells with YAP localized in the nucleus. Data are indicated as mean ± standard deviation (SD) (n ≥ 30 cells, three independent experiments), and *P < 0.01.
Figure 8
Figure 8
Fluorescence images of cell tension on the various substrates. (A) Representative images of single stem cells on the various substrates with and without CDMs in the growth medium cultured for 24 h. Nucleus (Blue), F-actin (Green), and pMLC (Red). The scale bar is 20 μm. (B) Integrated fluorescence intensity of pMLCs and that compared via normalization for the Flat substrate. Data are given as mean ± standard deviation (SD) (n ≥ 30 cells, three independent experiments), and *P < 0.05.
Figure 9
Figure 9
(A) Immunofluorescence staining of hBM-MSCs for vinculin after culturing for 1 day on various substrates. Blue: nucleus, Green: F-actin, and Red: vinculin. Grayscale image for vinculin is shown in Figure S4. The scale bar is 20 μm. Quantification of (B) FA area per cell and (C) FA elongation. The white arrows refer to the vinculin spots that are well-defined dashlike in structure. Data are displayed as mean ± standard deviation (SD) (n ≥ 30 cells, three independent experiments), and *P < 0.05.
Figure 10
Figure 10
Schematic representation the effects of topography and Flat substrates coated with CDMs to direct osteogenic differentiation. Compared with the pristine W3 substrate, the wrinkle substrate coated with the CDM will enhance the formation and elongation of focal adhesions and strengthen cell contractility, resulting in the activation of YAP and translocation into the nucleus, therefore improving osteogenesis of hBM-MSCs.
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