Biomaterials Translational ›› 2021, Vol. 2 ›› Issue (4): 312-322.doi: 10.12336/biomatertransl.2021.04.007
• RESEARCH ARTICLE • Previous Articles Next Articles
Yang Zhao, Qing Sun, Bo Huo*(
)
Received:2021-09-30
Revised:2021-11-29
Accepted:2021-12-10
Online:2021-12-28
Published:2021-12-28
Contact:
Bo Huo
E-mail:huobo@bit.edu.cn
About author:Bo Huo, huobo@bit.edu.cn.Zhao, Y.; Sun, Q.; Huo, B. Focal adhesion regulates osteogenic differentiation of mesenchymal stem cells and osteoblasts. Biomater Transl. 2021, 2(4), 312-322.
| Pattern | Diameter (μm) | Area (μm2) | 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 (μm2) | 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) Bright-field images of micropatterned substrates. (C) Bright-field images of cells cultured on the micropatterned or blank substrates for 3 days. The yellow lines show the outline of the cells. The free-spreading 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 one-way 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. F-actin distribution in cells cultured on micropatterned substrates. (A, B) Fluorescent images of individual MSCs (A) and MC3T3-E1 cells (B) stained with phalloidin-labelled F-actin (green) with Hoechst 33342-stained 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 F-actin in MSCs (C) and MC3T3-E1 cells (D). (E, F) Statistical results of F-actin coherency in MSCs (E) and MC3T3-E1 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 (one-way 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. P-MLC2 in cells cultured on micropatterned substrates. (A, B) Fluorescent images of individual MSCs (A) and MC3T3-E1 cells (B) stained with TRITC-labeled P-MLC2 (red) with Hoechst 33342-stained 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 P-MLC2 in MSCs (C) and MC3T3-E1 cells (D). Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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; P-MLC2: 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. FITC-labeled fluorescent images of the osteogenic differentiation markers ALP, COL I, and OCN in MSCs and MC3T3-E1 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. (A-F) Relative fluorescent intensity of ALP, COL I, and OCN in MSCs (A, C, E) and MC3T3-E1 cells (B, D, F). Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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 isothiocyanate-labeled YAP (green) with Hoechst 33342-stained 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 MC3T3-E1 cells, respectively. (D, E) Statistical analyses of the nuclear/cytoplasmic ratio of YAP in MSCs and MC3T3-E1 cells, respectively. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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: yes-associated 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: yes-associated proteins.
Additional Figure 1. Statistical analysis of the fluorescence intensity of TUNEL-stained cells cultured on micropatterned substrates. (A-D) MSCs and MC3T3-E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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 MC3T3-E1 cells. (C) Circularity of MSCs. (D) Circularity of MC3T3-E1 cells. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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 F-actin. (A-D) MSCs and MC3T3-E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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 F-actin coherency. (A-D) MSCs and MC3T3-E1 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 (one-way 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 P-MLC2. (A-D) MSCs and MC3T3-E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc analysis). LL: large circles with large spacing; MSC: mesenchymal stem cell; P-MLC2: 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. (A-F) 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 (one-way 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 MC3T3-E1 cells cultured on micropatterned substrates. (A-F) 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 (one-way 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. (A-D) MSCs and MC3T3-E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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: yes-associated proteins.
Additional Figure 9. Statistical analyses of the nuclear/cytoplasmic ratio of YAP. (A-D) MSCs and MC3T3-E1 cells cultured for 3 or 7 days. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way 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: yes-associated proteins.
Additional Figure 10. Cell phenotypes and area after CD treatment for 24 hours. (A) Bright-field 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 (one-way 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 F-actin staining after CD treatment for 24 hours. (A) Fluorescent images of F-actin in shrunk cells. Scale bars: 50 μm. (B) Statistical results of fluorescence intensity of F-actin. (C) Statistical results of F-actin 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 (one-way 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, FITC-labeled) 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 (one-way 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 MC3T3-E1 cells. Data are presented as mean ± SD (n = 3), and were analysed by one-way 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: yes-associated proteins.
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