Supporting Information

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1 Supporting Informtion Remote Control of Multi-Modl Nnoscle Lignd Oscilltions Regultes Stem Cell dhesion nd Differentition Heemin Kng,, Dexter Siu Hong Wong,, Xiohui Yn, Hee Joon Jung,,,# Sungkyu Kim,,,# Sien Lin, Kongchng Wei, Gng Li, Vinyk P. Drvid,,,#,* nd Liming in,δ,,,,* Deprtment of iomedicl Engineering, The Chinese University of Hong Kong, Hong Kong, Chin. Deprtment of Physics, The Chinese University of Hong Kong, Hong Kong, Chin. Deprtment of Mterils Science nd Engineering, Northwestern University, Evnston, IL, US. Interntionl Institute for Nnotechnology, Evnston, IL, US. # NUNCE Center, Northwestern University, Evnston, IL, US. Deprtment of Orthopedics nd Trumtology, Fculty of Medicine, The Chinese University of Hong Kong, Hong Kong, Chin. Δ Shun Hing Institute of dvnced Engineering, The Chinese University of Hong Kong, Hong Kong, Chin. Shenzhen Reserch Institute, The Chinese University of Hong Kong, Hong Kong, Chin. Chin Orthopedic Regenertive Medicine Group (CORMed), Hngzhou, Chin. Centre for Novel iomterils, The Chinese University of Hong Kong, Hong Kong, Chin. 1

2 Supplementry Figures nd Movies Supplementry Figure S1. Chrcteriztions of the bre SPIONs. () Trnsmission electron microscopic imge of the bre superprmgnetic iron oxide nnoprticle (SPION). Scle br represents 2 nm. () Dynmic light scttering nlysis of the size distribution of the bre SPION. (C) Vibrting smple mgnetometer hysteresis mesurement of the bre SPION. The mgnetiztion ws normlized to the dry weight of the SPION. Supplementry Figure S2. Chrcteriztions of RGD lignd tethering to the SPIONs. () Zet potentil of the bre SPION, mino-silic coted SPION (SPION@SiO2), PEGylted (Mn = 5 D) SPION-silic core-shell nnoprticles (PEG-SPION@SiO2), nd RGD lignd-bering SPION (RGD-PEG-SPION@SiO2). Dt re shown s men ± stndrd errors (n=3). () Fourier trnsform infrred spectr of the bre SPION nd RGD lignd-bering SPION (RGD-PEG- SPION@SiO2). Supplementry Figure S3. Substrte tethering of the oscilltory RGD lignd-spion vi flexible linker. Schemtic representtion of the surfce chemistry chnges on the glss substrte. The glss substrte ws thiolted with mercptopropylsiltrne (MPS), which ws further modified with PEGylted SPION (PEG-SPION@SiO2), to which RGD peptide ws grfted (RGD-PEG- SPION@SiO2). Supplementry Figure S4. Chrcteriztions of substrte chemistry chnges upon tethering of the oscilltory RGD lignd-spion. () Wter contct ngle imges nd () the corresponding quntifiction of the wter contct ngles (n=3). The glss substrte ws thiolted nd, modified with PEGylted SPION (PEG-SPION@SiO2), nd then the RGD peptide ws coupled (RGD-PEG-SPION@SiO2) to the PEGylted SPION. Dt re presented s men ± stndrd errors (n=4). Different letters indicte sttisticl significnces (p <.5), determined with one-wy NOV nd Tukey-Krmer post-hoc test. Supplementry Figure S5. Chrcterizing control of in situ tomic force microscopy (FM) imging of the motion of the tethered SPION on the substrte. () Trnsmission electron microscopic imge of gold nnorod, used s the non-mgnetic reference. Scle br represents 5 2

3 nm. () FM imges scnned on the sme re under sttionry pplied on the sme side of the scn re. Sphericl shpe of the prticle indictes SPION, wheres the nnorod ws used s the reference. lck dotted lines re drwn long the long xes of the nnorods nd the centers of the SPION to confirm the negligible movement of the SPION. Scle brs indicte 5 nm. Supplementry Figure S6. Experimentl set-up for in situ mgnetic high-ngle nnulr drkfield scnning trnsmission electron microscopy (HDF-STEM) imging of the nnoscle motion of the oscilltory RGD lignd-spion coupled onto the substrte. () Photogrph of the mounted TEM grid smples nd the mgnetic tip on the modified electricl bising holder (Nnofctory ST-1) where the mgnetic tip position cn be chnged inside the STEM chmber. () STEM imges of the smple loction (RGD-SPION coupled substrte; red dotted circle) corresponding to the in situ mgnetic STEM imges shown in Figure 2D displying the reltive positionl chnges (left, bottom, right) of the mgnetic tip (blue dotted line). Scle brs indicte 1 µm. Supplementry Figure S7. In situ mgnetic high-ngle nnulr drk-field scnning trnsmission electron microscopy (HDF-STEM) imges of four individul SPIONs (RGD lignd-spion coupled onto the substrte) under three different conditions corresponding to Supplementry Figure S6, demonstrting the cler chnges in the inter-prticle distnces nd movement directions of the SPIONs under different locl s. Gry dotted lines re drwn long the centers of the lowest SPIONs to show the reltive movement of the upper SPIONs. Red dotted lines re drwn cross the centers of the upper SPIONs to show the reltive positionl chnges of the SPIONs. Red rrows represent the direction of movement of the upper SPIONs from the left to the middle to the right imge. Plese note tht lthough the loction of the mgnetic tip ginst the imged SPIONs ws ccurtely controlled with piezo-bsed movement, the from the curved tip does not bsolutely define how exctly the locl is formed ner smples nd it is obvious tht the strengths nd directions of the locl mgnetic fields re not the sme. Scle brs indicte 5 nm. 3

4 Supplementry Figure S8. () Fluorescent stining imges of ctin (Red) nd nuclei (blue) fter 12 h of hmsc culture under the oscilltion frequencies (.2 nd.1 Hz) of the of the RGD-bering SPION nd () corresponding quntifiction of the dherent cell density. Scle brs indicte 2 µm. Dt re displyed s men ± stndrd errors (n=3). No sttisticlly significnt differences (p <.5) were found between the groups s determined by two-tiled Student s t-test. (C) Fluorescent stining microgrphs of ctin (Red) nd nuclei (blue) following 12 h of hmsc culture under the oscilltion frequencies (2 nd 5 Hz) of the of the RGD-bering SPION nd (D) corresponding quntifiction of the dhered cell density. Scle brs represent 2 µm. Dt re displyed s men ± stndrd errors (n=3). No sttisticlly significnt differences (p <.5) were found between the groups s determined by two-tiled Student s t-test. (E) Fluorescent stining imges of ctin (Red) nd nuclei (blue) fter 12 h of hmsc culture under Sttionry,, nd Fst oscilltions of the substrte-tethered SPION, but with the blocking of poly(ethylene glycol) nd (F) corresponding quntifiction of the dherent cell density. Scle brs indicte 2 µm. Dt re shown s men ± stndrd errors (n=3). Different letters indicte sttisticl significnces (p <.5), determined with one-wy NOV nd Tukey-Krmer post-hoc test. Supplementry Figure S9. () Schemtic representtion of the control experimentl set-up for ltering non-lignd conjugted SPION oscilltions s tethered onto the substrte. () Fluorescent stining microgrphs of ctin (Red) nd nuclei (blue) fter 12 h of hmsc culture under Sttionry,, Moderte, nd Fst non-lignd tethered SPION oscilltions by pplying vrious oscilltion frequencies (,.1,.5, nd 2 Hz) of, respectively, with No mgnet control. Scle brs indicte 2 µm. () Corresponding quntifiction of dhered cell density. Dt re presented s men ± stndrd errors (n=3). No sttisticlly significnt differences (p <.5) were found mongst ll groups s determined by one-wy NOV with Tukey-Krmer post-hoc test. Supplementry Figure S1. Fluorescent stining microgrphs of ctin (Red) nd nuclei (blue) fter 12 h of hmsc culture under nd Fst oscilltions of the substrte-tethered SPION, but with the grfting of the scrmbled RGD, () RD or (C) RGE, by pplying vrious oscilltion frequencies (.1 nd 2 Hz) of, respectively. (, D) Corresponding quntifiction of 4

5 the dherent cell density. Scle brs represent 2 µm. Dt re presented s men ± stndrd errors (n=3). No sttisticlly significnt differences (p <.5) were found mong ll groups s determined by two-tiled Student s t-test. Supplementry Figure S11. () Trnsmission electron microgrph of RGD lignd-tethered pure silic nnoprticles. Scle br indictes 5 nm. () Scnning electron microgrph for monolyer of RGD lignd-silic nnoprticle tht ws tethered to the substrte. The scle brs represent 1 µm. (C) Fluorescent stining imges of ctin (Red) nd nuclei (blue) fter 12 h of hmsc culture under Sttionry,, nd Fst oscilltions of the substrte-tethered RGD-silic nnoprticles, under the ppliction of vrious oscilltion frequencies (,.1, nd 2 Hz) of, respectively, with No mgnet s control. Scle brs indicte 2 µm. (D) Corresponding quntifiction of the dhered cell density. Dt re displyed s men ± stndrd errors (n=3). No sttisticlly significnt differences (p <.5) were found mongst ll groups s determined by one-wy NOV with Tukey-Krmer post-hoc test. Supplementry Figure S12. ltering lignd oscilltions regulte focl dhesion nd morphology of stem cells. Time-dependent quntifictions of () focl dhesion number nd () cell spect rtio (mjor/minor xes) fter 12 h nd 48 h of hmsc culture under sttionry, slow, moderte, fst lignd-spion oscilltions under Sttionry,, Moderte, nd Fst lignd-spion oscilltions by pplying vrious oscilltion frequencies (,.1,.5, nd 2 Hz) of, respectively, with No RGD nd No mgnet controls. Dt re shown s men ± stndrd errors (n=3). Different letters indicte sttisticl significnces (p <.5) t the sme culture time s determined by one-wy NOV with Tukey-Krmer post-hoc test. Supplementry Figure S13. Modulting lignd oscilltions regultes the mechnosensing of stem cells. () Immunofluorescent stining imges ginst TZ (green), ctin (red), nd nuclei (blue) fter 2 d of hmsc culture, under Sttionry,, nd Moderte lignd oscilltions by pplying vrious oscilltion frequencies (,.1, nd.5 Hz) of, respectively, with No mgnet control. Scle brs indicte 1 µm. () Corresponding rtiometric nlyses of nucler to cytoplsmic fluorescence intensities for TZ. Dt re shown s men ± stndrd errors 5

6 (n=3). Different letters indicte sttisticl significnces (p <.5) s determined by one-wy NOV with Tukey-Krmer post-hoc test. Supplementry Figure S14. Modulting lignd oscilltions regultes mechnosensing of in vivo dhered stem cells. () Immnuofluorescent stining imges ginst YP (green), ctin (red), nd nuclei (blue) following 4 h of in vivo hmsc injection onto the implnted substrte under Sttionry,, nd Fst lignd oscilltions by pplying vrious oscilltion frequencies (,.1, nd 2 Hz) of, respectively, with No RGD nd No mgnet control groups. Scle brs represent 1 µm. () Corresponding quntifiction of nucler to cytoplsmic fluorescence rtio. Dt re presented s men ± stndrd errors (n=2). Different letters represent sttisticl significnces (p <.5) s determined by one-wy NOV with Tukey-Krmer posthoc test. Supplementry Movie S1. Negligible motion of red-fluorescent RGD-bering SPION under sttic. right-field imge ws overlin s reference. Supplementry Movie S2. Oscilltory motion of red-fluorescent RGD-grfted SPION under n oscillting t frequency of 2.5 mhz. right-field imge ws overlin s reference. Supplementry Movie S3. Oscilltory motion of red-fluorescent RGD-tethered SPION under n oscillting t frequency of 5 mhz. right-field imge ws merged s reference. Supplementry Movie S4. Oscilltory motion of red-fluorescent RGD-coted SPION under n oscillting t frequency of 12.5 mhz. right-field imge ws merged s reference. 6

7 Supplementry Figure S1 C Intensity (%) Size (d, nm) Mgnetic moment (emu/g) Mgnetic field strength (Oe) 1

8 Supplementry Figure S2 Zet potentil (mv) SPION SPION@SiO 2 PEG-SPION@SiO 2 RGD-PEG-SPION@SiO 2 Trnsmittnce (.u.) O=C-NH Fe-O Si-O RGD-PEG-SPION@SiO 2 SPION Wvenumber (cm -1 ) 2

9 Supplementry Figure S3 GCGYGRGDSPG SPION (Superprmgnetic iron oxide nnoprticle) mino-silic GCGYGRGDSPG Substrte Substrte Substrte Substrte Non-thiolted Thiolted 2 2 3

10 Supplementry Figure S4 Non-thiolted Thiolted 2 2 Contct ngle (degree) b Non-thiolted Thiolted PEG-SPION@SiO 2 RGD-PEG-SPION@SiO 2 4

11 Supplementry Figure S5 5

12 Supplementry Figure S6 RGD-SPION coupled substrte (TEM grid) Mgnetic tip RGD-SPION substrte RGD-SPION substrte RGD-SPION substrte Mgnetic tip (flexibly curved) Mgnetic tip (flexibly curved) Mgnetic tip (flexibly curved) 6

13 Supplementry Figure S7 SPIONs SPIONs SPIONs 7

14 Oscillting Oscillting Oscillting Supplementry Figure S8 ctin/nuclei f =.2 Hz f =.1 Hz dhered cell density (Cells/cm 2 ) Oscillting.2 Hz.1 Hz C D ctin/nuclei f = 2 Hz f = 5 Hz dhered cell density (Cells/cm 2 ) Oscillting 2 Hz 5 Hz E F ctin/nuclei Sttionry Fst dhered cell density (Cells/cm 2 ) b c 8

15 Oscillting Supplementry Figure S9 ctin/nuclei No mgnet Sttionry Moderte Fst C dhered cell density (Cells/cm 2 ) Oscillting No mgnet Sttionry Moderte Fst 9

16 Oscillting Oscillting Supplementry Figure S1 RD ctin/nuclei Fst dhered cell density (Cells/cm 2 ) Oscillting Fst C RGE ctin/nuclei D Fst dhered cell density (Cells/cm 2 ) Oscillting Fst 1

17 Oscillting Supplementry Figure S11 C ctin/nuclei No mgnet Sttionry Fst D dhered cell density (Cells/cm 2 ) Oscillting No mgnet Sttionry Fst 11

18 Supplementry Figure S12 Focl dhesion number f b c d e e 12 h 48 h e g Oscillting No RGD No mgnet Sttionry Moderte Fst spect rtio (Mjor/minor xis) b c c c d c e Oscillting No RGD No mgnet Sttionry Moderte Fst 12 h 48 h 12

19 Moderte Oscillting Sttionry No mgnet Supplementry Figure S13 TZ ctin/nuclei Nucler/cytoplsmic TZ rtio 5 b

20 Fst Oscillting Sttionry Supplementry Figure S14 YP ctin/nuclei No mgnet No RGD Nucler/cytoplsmic YP rtio 2.5 b In vivo dhesion 14