Biomaterials Translational ›› 2021, Vol. 2 ›› Issue (4): 312322.doi: 10.12336/biomatertransl.2021.04.007
• RESEARCH ARTICLE • Previous Articles Next Articles
Yang Zhao, Qing Sun, Bo Huo^{*}()
Received:
20210930
Revised:
20211129
Accepted:
20211210
Online:
20211228
Published:
20211228
Contact:
Bo Huo
Email:huobo@bit.edu.cn
About author:
Bo Huo, huobo@bit.edu.cn.Abstract:
Focal adhesions are large macromolecular assemblies through which cells are connected with the extracellular matrix so that extracellular signals can be transmitted inside cells. Some studies have focused on the effect of cell shape on the differentiation of stem cells, but little attention has been paid to focal adhesion. In the present study, mesenchymal stem cells (MSCs) and osteoblastlike MC3T3E1 cells were seeded onto micropatterned substrates on which circular adhesive islands with different spacing and area were created for focal adhesion. Results showed that the patterns of focal adhesion changed cell morphology but did not affect cell survival. For MSCs cultured for 3 days, patterns with small circles and large spacing promoted osteogenesis. For MSCs cultured for 7 days, patterns with large circles and spacing enhanced osteogenesis. For MC3T3E1 cells, the patterns of focal adhesion had no effect on cell differentiation after 3 days of culture, but patterns with small circles and spacing improved osteogenic differentiation after 7 days. Moreover, the assembly of Factin, phosphorylation of myosin, and nuclear translocation of yesassociated proteins (YAP) were consistent with the expression of differentiation markers, indicating that the pattern of focal adhesion may affect the osteogenesis of MSCs and osteoblasts through changes in cytoskeletal tension and nuclear localisation of YAP.
Key words: Factin, focal adhesion, micropattern, osteogenesis, YAP
Zhao, Y.; Sun, Q.; Huo, B. Focal adhesion regulates osteogenic differentiation of mesenchymal stem cells and osteoblasts. Biomater Transl. 2021, 2(4), 312322.
Pattern  Diameter (μm)  Area (μm^{2})  Spacing (μm)  Pattern area/total area (%) 

LL  12  113  36  9 
SS  8  50  24  9 
SL  8  50  36  4 
Table 1 Geometric parameters of the three patterns.
Pattern  Diameter (μm)  Area (μm^{2})  Spacing (μm)  Pattern area/total area (%) 

LL  12  113  36  9 
SS  8  50  24  9 
SL  8  50  36  4 
Figure 1. Micropatterned substrate and cell culture. (A) The process of micropatterning. (B) Brightfield images of micropatterned substrates. (C) Brightfield images of cells cultured on the micropatterned or blank substrates for 3 days. The yellow lines show the outline of the cells. The freespreading cells showed polygons, while the spreading area of the patterned cells decreased. Scale bars: 50 μm. (D) Spread area at 3 days of cell culture. (E, F) Statistical analysis of the area and number of adhesive islands after 3 days of culture. Data are presented as mean ± SD (n = 3), and were analysed by oneway analysis of variance followed by Tukey’s post hoc analysis. CON: control (freely spreading group); LL: large circles with large spacing; MSC: mesenchymal stem cell; PDMS: polydimethylsiloxane; SL: small circles with large spacing; SS: small circles with small spacing; UV: ultraviolet.
Figure 2. Factin distribution in cells cultured on micropatterned substrates. (A, B) Fluorescent images of individual MSCs (A) and MC3T3E1 cells (B) stained with phalloidinlabelled Factin (green) with Hoechst 33342stained nuclei (blue) at 3 days after seeding. Cells could be spread out into triangles, dumbbells, etc. The yellow circles represent the location of micropatterned islands. The gray shape represents the spreading shape of cells. Scale bars: 50 μm. (C, D) Statistical results of Factin in MSCs (C) and MC3T3E1 cells (D). (E, F) Statistical results of Factin coherency in MSCs (E) and MC3T3E1 cells (F). The larger the value, the more ordered the actin, and the more consistent the direction of stress fibres. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). CON: control (freely spreading group); LL: large circles with large spacing; MSC: mesenchymal stem cell; PTN: pattern; SL: small circles with large spacing; SS: small circles with small spacing.
Figure 3. PMLC2 in cells cultured on micropatterned substrates. (A, B) Fluorescent images of individual MSCs (A) and MC3T3E1 cells (B) stained with TRITClabeled PMLC2 (red) with Hoechst 33342stained nuclei (blue) at 3 days after seeding. The yellow circles represent the location of micropatterned islands. The gray shape represents the spreading shape of cells. Scale bars: 50 μm. (C, D) Statistical results of PMLC2 in MSCs (C) and MC3T3E1 cells (D). Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). CON: control (freely spreading group); LL: large circles with large spacing; MSC: mesenchymal stem cell; PMLC2: phosphorylated myosin light chain 2; PTN: pattern; SL: small circles with large spacing; SS: small circles with small spacing; TRITC: tetraethyl rhodamine isothiocyanate.
Figure 4. FITClabeled fluorescent images of the osteogenic differentiation markers ALP, COL I, and OCN in MSCs and MC3T3E1 cells cultured on a micropatterned substrate after 3 (ALP) or 7 (COL I, OCN) days of culture. Cells might spread out into triangles, dumbbells, etc. The yellow circles indicate the location of micropatterned islands. The gray shape represents the spreading shape of cells. Scale bars: 50 μm. ALP: alkaline phosphatase; COL I: type I collagen; FITC: fluorescein isothiocyanate; MSC: mesenchymal stem cell; OCN: osteocalcin.
Figure 5. Intensity of osteogenic differentiation markers in cells cultured on a micropatterned substrate. (AF) Relative fluorescent intensity of ALP, COL I, and OCN in MSCs (A, C, E) and MC3T3E1 cells (B, D, F). Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). ALP: alkaline phosphatase; COL I: type I collagen; CON: control (freely spreading group); LL: large circles with large spacing; MSC: mesenchymal stem cell; OCN: osteocalcin; PTN: pattern; SL: small circles with large spacing; SS: small circles with small spacing.
Figure 6. YAP staining in cells cultured on a micropatterned substrate. (A) Fluorescent images of individual cells stained with fluorescein isothiocyanatelabeled YAP (green) with Hoechst 33342stained nuclei (blue) after 3 or 7 days of culture on the micropatterned substrate. Yellow circles indicate the location of micropatterned islands, and yellow dotted circles show the outlines of nuclei. The gray shape represents the spreading shape of cells. Scale bars: 50 μm. (B, C) Statistical analyses of the fluorescence intensity of YAP in MSCs and MC3T3E1 cells, respectively. (D, E) Statistical analyses of the nuclear/cytoplasmic ratio of YAP in MSCs and MC3T3E1 cells, respectively. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). CON: control (free spreading group); LL: large circles with large spacing; MSC: mesenchymal stem cell; PTN: pattern; SL: small circles with large spacing; SS: small circles with small spacing; YAP: yesassociated proteins.
Figure 7. Summary of the mechanism via which focal adhesion distribution regulates osteogenic differentiation. LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing; YAP: yesassociated proteins.
Additional Figure 1. Statistical analysis of the fluorescence intensity of TUNELstained cells cultured on micropatterned substrates. (AD) MSCs and MC3T3E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.
Additional Figure 2. Statistical analysis of the area and circularity of focal adhesions. (A) Area of focal adhesion of MSCs. (B) Area of focal adhesion of MC3T3E1 cells. (C) Circularity of MSCs. (D) Circularity of MC3T3E1 cells. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). CON: control; LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 3. Statistical results of fluorescence intensity of Factin. (AD) MSCs and MC3T3E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 4. Statistical results of Factin coherency. (AD) MSCs and MC3T3E1 cells cultured for 3 or 7 days. The larger the value, the more ordered the actin, and the more consistent the direction of stress fibres. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 5. Statistical results of fluorescence intensity of PMLC2. (AD) MSCs and MC3T3E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; PMLC2: phosphorylated myosin light chain 2; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 6. Fluorescence intensity of osteogenic differentiation markers in MSCs cultured on micropatterned substrates. (AF) Statistical results of fluorescent intensity of ALP, COL I, and OCN, respectively, after culture for 3 days (A, C, E) or 7 days (B, D, F). Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). ALP: alkaline phosphatase; COL I: type I collagen; LL: large circles with large spacing; MSC: mesenchymal stem cell; OCN: osteocalcin; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 7. Fluorescence intensity of osteogenic differentiation markers in MC3T3E1 cells cultured on micropatterned substrates. (AF) Statistical results of fluorescent intensity of ALP, COL I, and OCN in cells cultured for 3 days (A, C, E) or 7 days (B, D, F), respectively. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). ALP: alkaline phosphatase; COL I: type I collagen; LL: large circles with large spacing; OCN: osteocalcin; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 8. Statistical results of fluorescence intensity of YAP. (AD) MSCs and MC3T3E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing; YAP: yesassociated proteins.
Additional Figure 9. Statistical analyses of the nuclear/cytoplasmic ratio of YAP. (AD) MSCs and MC3T3E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing; YAP: yesassociated proteins.
Additional Figure 10. Cell phenotypes and area after CD treatment for 24 hours. (A) Brightfield images of the cell on the micropatterned substrates. Cells were shrunk obviously. Scale bars: 50 μm. (B) Statistical analysis of the spreading area of the cells. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). CD: cytochalasin D; LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 11. SStatistical results of Factin staining after CD treatment for 24 hours. (A) Fluorescent images of Factin in shrunk cells. Scale bars: 50 μm. (B) Statistical results of fluorescence intensity of Factin. (C) Statistical results of Factin coherency. The larger the value, the better the order of actin, and the more consistent the direction of stress fibre. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). CD: cytochalasin D; LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 12. Fluorescent images of osteogenic differentiation markers (green, FITClabeled) in individual cells treated with CD and cultured on the micropatterned substrates for 3 (ALP) or 7 days (COL I, OCN). The yellow circles represent the location of micropatterned islands. Scale bars: 50 μm. ALP: alkaline phosphatase; CD: cytochalasin D; COL I: type I collagen; FITC: fluorescein isothiocyanate; MSC: mesenchymal stem cell; OCN: osteocalcin.
Additional Figure 13. Fluorescence intensity of osteogenic markers in cells cultured on micropatterned substrates after CD treatment. Data are presented as mean ± SD (n = 3). *P < 0.05 (oneway analysis of variance followed by Tukey’s post hoc analysis). ALP: alkaline phosphatase; CD: cytochalasin D; COL I: type I collagen; LL: large circles with large spacing; MSC: mesenchymal stem cell; OCN: osteocalcin; PTN: pattern; SL: small circles with large spacing; SS: small circles with small spacing.
Additional Figure 14. Statistical results of YAP localisation after CD treatment. (A) Fluorescent images of YAP. Nuclear transfer of YAP could not be observed. The yellow circles represent the location of micropatterned islands. Scale bars: 50 μm. (B) Statistical analyses of the nuclear/cytoplasmic ratio of YAP in MSCs and MC3T3E1 cells. Data are presented as mean ± SD (n = 3), and were analysed by oneway analysis of variance followed by Tukey’s post hoc analysis. CD: cytochalasin D; LL: large circles with large spacing; MSC: mesenchymal stem cell; SL: small circles with large spacing; SS: small circles with small spacing; YAP: yesassociated proteins.
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