Supporting Information

A novel photothermally controlled multifunctional scaffold for

potential clinical treatment of osteosarcoma and tissue

regeneration

Liang Maa ,1 , Xiaobo Feng a ,1 , Hang Liang a , Kun Wang a , Yu Song a , Lei Tan b ,

Bingjin Wanga , Rongjin Luo a , Zhiwei Liao a , Gaocai Li a , Xiangmei Liu b, *, Shuilin

Wu c,* ,Cao Yanga , *

a Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430022, China

b Hubei Key Laboratory of Polymer Materials, Ministry-of-Education Key Laboratory

for the Green Preparation and Application of Functional Materials, School of

Materials Science & Engineering, Hubei University, Wuhan 430062, China

c The Key Laboratory of Advanced Ceramics and Machining Technology by the

Ministry of Education of China, School of Materials Science & Engineering, Tianjin

University, Tianjin 300072, China

1

Figure captions

Fig. S1. Morphological and structural characterization. (A) SEM and (B) TEM

images of 30% nHA/GO particles. (C) XRD and (D) FTIR data of GO and 30%

nHA/GO particles.

Fig. S2. Flow cytometry analysis and differentiation ability of hBMSC. (A) Flow

cytometry analysis of cell surface markers (CD73, CD105, CD90, CD34, and HLA-

DR) of hBMSC. (B) The ability of hBMSC to differentiate into the osteogenic,

chondrogenic, and adipogenic lineages as confirmed through Alizarin red staining

(left panel; scale bar, 100 μm), oil Red O staining (middle panel; scale bar, 50 μm),

and Alcian blue staining (right panel; scale bar, 100 μm).

Fig. S3. Relative proliferation rates of MC3T3-E1 cells and HOS. (A-C) Relative

proliferation rates of MC3T3-E1 cells incubated with DMEM/F12, 10%nHA/GO,

30%nHA/GO, or 50%nHA/GO particles at concentrations of 50, 100, and 200 μg/ml

for 1, 3, and 5 days (**p < 0.01, comparison between control and 50 μg/mlin same

composite particles group; #p < 0.05, comparison between 50 μg/ml in 30%nHA/GO

group and 50 μg/ml in 50%nHA/GO; ns denotes no significance, comparison between

2

30%nHA/GO group; and 50% nHA/GO group at concentrations of 50 μg/ml). (D)

Photothermal performance of the different compound particles at a concentration of

50 μg/ml. (E) Relative proliferation rates of HOS cells co-cultured with GO or 30%

nHA/GO particles at concentrations of 50 μg/ml with or without irradiation, examined

using CCK8. (F) Temperature measurements of HOS cells co-cultured with GO or

30% nHA/CS particles under NIR irradiation; the control group was set without

adding any particles.

Fig. S4. Relative proliferation rates of MC3T3-E1cells. (A) Relative proliferation

rates of MC3T3-E1 cells co-cultured with GO or 30% nHA/GO particles at

concentrations of 50 μg/ml with or without irradiation, as examined using CCK8. (B)

Live/dead assay of MC3T3-E1 cells co-cultured with GO or 30% nHA/GO particles

at concentrations of 50 μg/ml with or without irradiation. (C) Temperature

measurements of hBMSC co-cultured with GO or 30% nHA/CS particles under NIR

irradiation and with the control group set without the addition of any particles.

Fig. S5. Morphological and structural characterization. (A) FTIR and (B) XPS data of

the CS, GO/CS, and nHA/GO/CS scaffolds. (C) Element map analysis of the CS and

GO/CS scaffolds. (D) Transient thermal measurements of the nHA/GO/CS scaffolds

under repeated on-off cycles of NIR light irradiation. Five cycles of irradiation at 808

nm were carried out, and each cycle consists of 60 s irradiation followed by a 60 s

3

cooling phase. (E) Photographs of compression process of nHA/GO/CS scaffolds and

compression strength of various scaffolds (**p < 0.01, comparison between CS

scaffolds and nHA/GO/CS scaffolds; #p < 0.05, comparison between GO/CS

scaffolds and nHA/GO/CS scaffolds ， ns stands for no significant statistical

significance).

Fig. S6. Live/dead staining, SEM of hBMSC and cell density. (A) Viability of the

hBMSC on different scaffolds with or without NIR under irradiation evaluated with

Live/Dead staining and SEM after 1 days, with CS group set as control. (B) Live cell

density of hBMSC seeded on different scaffolds at 1 and 3 days of culture (ns stands

for no significant statistical significance; **p < 0.01, comparison between control and

GO/CS+ scaffolds or nHA/GO/CS+ scaffolds).

Fig. S7. Western blotting and quantitative analysis. (A, C) Western blotting of (A)

HSP47 and HSP74 proteins, (C) BMP2 and BMP7 proteins, and (B, D) their

quantitative analysis, with or without NIR irradiation and after osteogenic culture for

14 days (**p < 0.01, comparison between control and GO/CS+ group or

nHA/GO/CS+ group. ns stands for no significant difference).

Fig. S8. Western blotting and quantitative analysis. (A) Expression levels of Smad1,

Smad5, P-Smad1/5, and Noggin proteins and their quantitative analysis, (B) with or

without NIR irradiation and after osteogenic culture for 14 days (ns stands for no

4

significant statistical significance; **p < 0.01, comparison between control and

GO/CS+ scaffolds or nHA/GO/CS+ scaffolds).

Fig. S9. Illustration of the osteogenic mechanism under NIR irradiation.

Fig. S10. Infrared thermal image and Hematoxylin and eosin (H&E) staining images.

(A) Representative infrared thermal images of the tumor and surrounding tissue. (B)

Hematoxylin and eosin(H&E) staining images of HOS tumor tissue implanted with

different scaffolds with or without NIR laser irradiation after 10 days (scale bar, 100

μm). (C) H&E staining of slices of major organs (heart, liver, spleen, lung, and

kidney) implanted with different scaffolds with or without NIR laser irradiation after

10 days (scale bar, 100 μm).

Fig. S11. In vivo infrared thermal images and osteogenesis. (A) In vivo infrared

thermal images and (B) temperature increase curves of cranial bone after implanting

different scaffolds and during 808 nm laser irradiation (1.5 W/cm2). (C)

Representative CT coronal and sagittal views of cranial defect area 4 weeks post

implantation (scale bar, 1 mm). (D) BV/TV ratio based on the micro-CT analysis (**p

< 0.01, comparison between control group and GO/CS+ or nHA/GO/CS+ group; *p <

0.05, comparison between control group and GO/CS+ or nHA/GO/CS+ group; #p <

0.05, comparison between GO/CS+ and nHA/GO/CS+; ns, no significance difference

between GO/CS+ group and nHA/GO/CS at 8 W).

Fig. S12. Low-magnification images in the eight groups (scale bar, 1 mm) after H&E

5

staining and Masson’s trichrome staining.

Fig. S13. Histomorphometry for bone formation. (A) New bone area rate according to

the H&E staining. (B) Collagen area rate according to Masson’s trichrome staining

(**p < 0.01, comparison between control group and GO/CS+ or nHA/GO/CS+ group;

#p < 0.05, comparison between GO/CS+ and nHA/GO/CS+ group).

Fig. S14. Immunohistochemical staining of COL1 on the newly formed bone tissue

after 8 weeks, and CS group was set as control (scale bars, 100 μm).

Fig. S15. Curves of temperature increase of different dressings after implanting

different scaffolds and under 808 nm laser irradiation (1.5 W/cm2).

Figure

6

FigureS1

7

Figure S2

8

Figure S3

9

Figure S4

10

Figure S5

11

Figure S6

12

Figure S7

13

Figure S8

14

Figure S9

15

Figure S10

16

Figure S11

17

Figure S12

18

Figure S13

19

Figure S14

20

Figure S15

21