Supplementary Information

Modulation of the Mechanosensing of Mesenchymal Stem Cells by Laser-Induced Patterning for the Acceleration of Tissue Reconstruction through the Wnt/β-catenin Signaling Pathway Activation

Jieni Fu1, Xiangmei Liu1*, Lei Tan1, Zhenduo Cui2, Yanqin Liang2, Zhaoyang Li2, Shengli Zhu2, Yufeng Zheng3, Kelvin Wai Kwok Yeung4, Paul K. Chu5, Shuilin Wu1,2*

Figures and tables

Fig. S1. Surface morphology of the selective laser-melted Ti plate (Ti-LA). (a) The actual picture

of Ti-LA. (b) The SEM picture of Ti-LA.

Table S1. The composition of the 1.5-SBF solution.

Table S2. The primer sequences of the MC3T3-E1 cells in the qRT-PCR gene expression

analysis.

Table S3. The primer sequences of the MSCs cells in the qRT-PCR gene expression analysis.

Fig. S2. Surface morphology of the selective laser-melted Ti plate (Ti-LA). a) The groove of Ti-

LA. b) The ridge of Ti-LA.

Fig. S3. XPS Ti 2p spectra.

Fig. S4. XPS O 1s spectra.

Fig. S5. (a) Nyquist plots of different samples. (b) XRD for Untreated Ti, Ti (0.9 % NaCl 15

days) and Ti-LA-AT2 (0.9 % NaCl 15 days).

Fig. S6. Surface morphology of the samples after immersion in 1.5-SBF for 3 days at low

magnification: (a) Ti-AT2; (b) Ti-LA-AT1; (c) Ti-LA-AT2; (d) Ti-LA-AT4; (e), (f) and (g)

SEM/EDS of Ti-LA-AT2 after immersion for 3 days at high magnification.

1

Fig. S7. The roughness of different samples: (a) Ti-LA (70 A); (b) Ti-LA (80 A); (c) Ti-LA (90

A).

Fig. S8. The MSCs number (%) quantitative analysis of (a) Ti-LA (70 A); (b) Ti-LA (80 A); (c)

Ti-LA (90 A); (d) Quantitative analysis of cell area of Ti-LA (70 A) Ti-LA (80 A) and Ti-LA

(90 A). (e) Quantitative analysis of cell aspect ratio of Ti-LA (70 A) Ti-LA (80 A) and Ti-LA

(90 A). (f) Quantitative analysis of protrusion Frequencies/μm of 70A-2 M, 80A-2 M and 90A-2

M. (g, h) The definition of aspect ratio for cells and orientation angle. (a-f) Data are presented as

mean ± SD.; *p<0.05, **p <0.01, ***p <0.001; n = 3, One-way analysis of variance (ANOVA)

with Dunnett's multiple comparisons test..

Fig. S9. Different samples (Ti, Ti-AT2, Ti-LA-AT1, Ti-LA-AT2, and Ti-LA-AT4) can regulate

cell nuclei orientation for MC3T3-E1. (a) Fluorescence microscopy images of MC3T3-E1 on

after 48 h in culture on various samples. The green color represents F-actin and the blue color

represents cell nuclei. The scale bar is 100 μm. (b) SEM images of MC3T3-E1 after culturing for

24 h on Ti, Ti-AT2, Ti-LA-AT1, Ti-LA-AT2, and Ti-LA-AT4. (c) Cell nucleus spreading area in

different samples. (d) Cell nucleus in different angles. Mean values and standard deviations from

200 randomly chosen cell. (e) Cell nucleus areas of various samples. Mean values and standard

deviations from 50 randomly chosen cell. (f) Cell nucleus aspect ratio of various samples. (g)

MTT for MC3T3-E1. (d - f) Data are presented as mean ± SD.; *p<0.05, **p <0.01, ***p

<0.001; One-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test. (g)

Two-way ANOVA with Tukey's multiple comparisons test.

Fig. S10. The cell adhesive density on day 2 on different samples. n=4, data are presented as

mean ± SD.; *p<0.05, **p <0.01, ***p <0.001; One way ANOVA with Dunnett's multiple

comparisons test.

2

Fig. S11. The ALP for MC3T3-E1 on different samples, n=3. Data are presented as mean ± SD.;

*p<0.05, **p <0.01, ***p <0.001; Two way ANOVA with Dunnett's multiple comparisons test.

Fig. S12. Alizarin Red S staining of MC3T3-E1 after culturing 10 days. And quantitative

analysis of ECM mineralization. (n = 3); scare bar= 4 mm, Data are presented as mean ± SD.;

*p<0.05, **p <0.01, ***p <0.001; One-way analysis of variance (ANOVA) with Dunnett's

multiple comparisons test.

Fig. S13. Alizarin Red S staining of MSCs after culturing 3 days, 7 days. And quantitative

analysis of ECM mineralization. (n = 3), scale bar= 4 mm. n=3, data are presented as mean ±

SD.; *p<0.05, **p <0.01, ***p <0.001; One-way analysis of variance (ANOVA) with Dunnett's

multiple comparisons test.

Fig. S14. qRT-PCR analysis of the expression of osteoblast-specific genes for MC3T3-E1 after

culturing for 3 days normalized to Gapdh expression of cells on Ti on that day, n=3, t tests.

Fig. S15. Immunofluorescence staining of cahherin 2 for MC3T3-E1culturing on the surface of

Ti and Ti-LA-AT2 at 3 days.

Fig. S16. Immunofluorescence staining of osteogenic markers: (a, c) OCN and OPN of MSCs on

Ti and Ti-LA-AT2 after culturing for 3 days and 7 days (scale bars =100 μm). (b, d) The MSCs

quantitative analysis of OCN and OPN, and normalized to OPN or OCN expression of cells on

Ti on that day, n=3, t tests.

Fig. S17. The pictures of implantation of samples and X-ray picture of titanium rods after 4

weeks.

Fig. S18. Immunohistochemical staining of iNOS and TGFβ in various samples. The yellow

color is the positive expression of related protein.

3

Table S1 Composition of the 1.5-SBF solution.

Ion(mM) Na+ K+ Mg2+ Ca2+ Cl- HCO3- HPO4

- SO42-

1.5-SBF 213 7.5 2.25 3.75 154.5 15 1.5 0.75

Table S2. Primer sequences of the MC3T3-E1 cells in the qRT-PCR gene expression analysis.

gene 5’-3’ primers

Alpforward GCCCTCCAGATCCTGACCAA

reverse GCAGAGCCTGCTGGTCCTTA

Runx2forward TGCCCAGGCGTATTTCAG

reverse TGCCTGGCTCTTACTGOG

Ocnforward AGCAGCTTGGCCCAGACCTA

reverse TAGCGCCGGAGTCTGTTCACTAC

β-actinforward TGCTATCCAGAAAACCCCTCAA

reverse GCGGGTGGAACTGTGTTACG

Table S3. The primer sequences of the MSCs in the qRT-PCR gene expression analysis.

gene 5’-3’ primers

Opnforward TCCTGTCTCCCGGTGAAAGT

reverse GGCTACAGCATCTGAGTGTTTGC

Runx2forward AATGCCTCCGCTGTTATG

reverse CCGGAGTCTATTCACCACCACCTTACT

integrinα5forward GTGACGGGACTCAACAACTGC

reverse GAAGGCTGAACGGCTGGTATT

integrinβ1forward GAAGGACATTGATGACTGCTGGTT

reverse GCGTGTCCCACTTGGCATT

cadherin 2forward GAGGAGCCGATGAAGGAACC

reverse TGATGAAGATGCCCGTTGGA

Ocnforward AAGCCCAGCGACTCTGAGTCT

reverse CCGGAGTCTATTCACCACCTTACT

Gapdh forward GCCTCGTCTCATAGACAAGATGGT

4

reverse GAAGGCAGCCCTGGTAACC

Fig. S1. Surface morphology of the selective laser-melted Ti plate (Ti-LA). (a) The actual picture

of Ti-LA. (b) The SEM picture of Ti-LA.

5

Fig. S2. Surface morphology of the selective laser-melted Ti plate (Ti-LA). (a) The groove of Ti-

LA. (b) The ridge of Ti-LA.

6

Fig. S3. Ti 2p XPS spectra.

7

Fig. S4. XPS O 1s spectra.

8

Fig. S5. (a) Nyquist plots of different samples. (b) XRD for Untreated Ti, Ti (0.9 % NaCl 15 days) and

Ti-LA-AT2 (0.9 % NaCl 15 days).

9

Fig. S6. Surface morphology of the samples after immersion in 1.5-SBF for 3 days at low

magnification: (a) Ti-AT2; (b) Ti-LA-AT1; (c) Ti-LA-AT2; (d) Ti-LA-AT4; (e), (f) and (g)

SEM/EDS of Ti-LA-AT2 after immersion for 3 days at high magnification.

10

Fig. S7. The roughness of different samples: (a) Ti-LA (70 A); (b) Ti-LA (80 A); (c) Ti-LA (90

A).

11

Fig. S8. The MSCs number (%) quantitative analysis of (a) Ti-LA (70 A); (b) Ti-LA (80 A); (c)

Ti-LA (90 A); (d) Quantitative analysis of cell area of Ti-LA (70 A) Ti-LA (80 A) and Ti-LA

(90 A). (e) Quantitative analysis of cell aspect ratio of Ti-LA (70 A) Ti-LA (80 A) and Ti-LA

(90 A). (f) Quantitative analysis of protrusion Frequencies/μm of 70A-2 M, 80A-2 M and 90A-2

M. (g, h) The definition of aspect ratio for cells and orientation angle. (a-f) Data are presented as

mean ± SD.; *p<0.05, **p <0.01, ***p <0.001; n = 3, One-way analysis of variance (ANOVA)

with Dunnett's multiple comparisons test..

12

Fig. S9. Different samples (Ti, Ti-AT2, Ti-LA-AT1, Ti-LA-AT2, and Ti-LA-AT4) can regulate

cell nuclei orientation for MC3T3-E1. (a) Fluorescence microscopy images of MC3T3-E1 on

after 48 h in culture on various samples. The green color represents F-actin and the blue color

represents cell nuclei. The scale bar is 100 μm. (b) SEM images of MC3T3-E1 after culturing for

24 h on Ti, Ti-AT2, Ti-LA-AT1, Ti-LA-AT2, and Ti-LA-AT4. (c) Cell nucleus spreading area in

different samples. Mean values and standard deviations from 50 randomly chosen cell. (d) Cell

nucleus in different angles. Mean values and standard deviations from 50 randomly chosen cell.

(e) Cell nucleus areas of various samples. Mean values and standard deviations from 50

13

randomly chosen cell. (f) Cell nucleus aspect ratio of various samples. (g) The MTT for MC3T3-

E1, n=3. (d - f) Data are presented as mean ± SD.; *p<0.05, **p <0.01, ***p <0.001; One-way

analysis of variance (ANOVA) with Dunnett's multiple comparisons test. (g) Two way ANOVA

with Dunnett's multiple comparisons test.

14

Fig. S10. The cell adhesive density on day 2 on different samples. n=4, data are presented as

mean ± SD.; One way ANOVA with Dunnett's multiple comparisons test.

15

Fig. S11. The ALP for MC3T3-E1 on different samples. n=4, data are presented as mean ± SD.;

**p <0.01, ***p <0.001，****p <0.0001; Two way ANOVA with Dunnett's multiple

comparisons test.

16

Fig. S12. Alizarin Red Staining of MC3T3-E1 after culturing 10 days. And quantitative analysis

of ECM mineralization. (n = 3), scale bar= 4 mm. Data are presented as mean ± SD.; *p<0.05,

**p <0.01, ***p <0.001; One-way analysis of variance (ANOVA) with Dunnett's multiple

comparisons test.

17

Fig. S13. (a) Alizarin Red S staining of MSCs after culturing 3 days, 7 days. And quantitative

analysis of ECM mineralization. (n = 3), scale bar= 4 mm. n=3, data are presented as mean ±

SD.; *p<0.05, **p <0.01, ***p <0.001; One-way analysis of variance (ANOVA) with Dunnett's

multiple comparisons test.

18

Fig. S14. qRT-PCR analysis of the expression of osteoblast-specific genes for MC3T3-E1 after

culturing for 3 days normalized to Gapdh expression of cells on Ti on that day, n=3, t tests.

19

Fig. S15. Immunofluorescence staining of cahherin 2 for MC3T3-E1 at 3 days.

20

Fig. S16. Immunofluorescence staining of osteogenic markers: (a, c) OCN and OPN of MSCs on

Ti and Ti-LA-AT2 after culturing for 3 days and 7 days (scale bars =100 μm). (b, d) The MSCs

quantitative analysis of OCN and OPN, and normalized to OPN or OCN expression of cells on

Ti on that day, n=3, t tests.

21

Fig. S17. (a) The pictures of shaving the hair near the femur. (b) The picture of implanting

implant. (c) The picture of stitching the wound. (d) X-ray picture of titanium nail after 4 weeks.

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Fig. S18. Immunohistochemical staining of iNOS and TGFβ in various samples. The yellow

color is the positive expression of related protein.

23