Supporting Information

Cobalt-mediated multi-functional dressings promote bacteria-infected wound

healing

Qingying Shi a, Xin Luo a, Ziqi Huang a, Adam C. Midgley a, b, Bo Wang c, Ruihua Liu

a, Dengke Zhi a, Tingting Wei a, Xin Zhou d, Mingqiang Qiao c, Jun Zhang a, *, Deling

Kong a, b, *, Kai Wang a, *

a Key Laboratory of Bioactive Materials, Ministry of Education, College of Life

Science, Nankai University, Tianjin 300071, China

b Xu Rongxiang Regeneration Life Science Center, State Key Laboratory of

Medicinal Chemical Biology, College of Life Science, Nankai University, Tianjin

300071, China

c Key Laboratory of Molecular Microbiology and Technology, Ministry of Education,

College of Life Science, Nankai University, Tianjin 300071, China

d Department of Medical Imaging, Shanxi Medical University, Taiyuan 030001,

China

*Corresponding authors: Dr. Kai Wang, Dr. Deling Kong and Dr. Jun Zhang, Key

Laboratory of Bioactive Materials, Ministry of Education, College of Life Science,

Nankai University, 94 Weijin Road, Tianjin 300071, China. Tel/Fax: +86 22

23502111.

E-mail: wangkai@nankai.edu.cn (K. Wang), kongdeling@nankai.edu.cn (D. Kong)

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nankaizhangjun@nankai.edu.cn (J. Zhang)

1. Materials and Methods

1.1 Whole-blood clotting test

The dressings (n=5) were placed into polypropylene tubes and tubes without

dressings were used as control groups. The tubes were pre-warmed to 37℃. Blood

was drawn from rat vena cava and stored in anticoagulation BD Vacutainers (Nouhua

9NC, containing sodium citrate). Citrated whole blood (0.5 mL) was dispensed onto

the dressings and 20 μL of CaCl2 solution (0.2 mol/L) was added to initiate whole-

blood clotting. The tubes were shaken (30 rpm) at 37℃, and the blood-clotting time

was recorded.

1.2 Immunofluorescence and immunohistochemical staining

The sections were stained by immunohistochemical staining with anti- HIF-1α to

HIF-1α expression in wound skin tissues. The frozen sections were fixed in acetone at

-20 °C for 10 min, air-dried, and rinsed once with 0.01 mM PBS. Then the sections

were incubated with 3% H2O2 for 10 min and washed in PBS 3 times. Sections were

permeated with 0.5% Triton-PBS for 10 min, and then washed in PBS 3 times.

Sections were then blocked with 5% normal goat serum for 45 min at 4 °C and then

incubated with rabbit anti-HIF-1α antibody (1:200, Abcam, USA) for 12 h at 4 °C.

After washing in PBS for 5 times, slides were then incubated with HRP-labelled goat

anti- rabbit IgG (H+L) antibody (1:500, Bioworld, USA) for 1 h in darkness.

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Antibody-binding was visualized by incubation with a DAB chromogen kit (ZSGB-

Bio, China), then counterstained with hematoxylin for 5 minutes. Slides incubated

with secondary antibody only, served as negative controls. All slides were observed

and photographed using a light microscope (Leica DM 3000, Germany).

The sections were stained by immunofluorescent staining with anti-CD31, anti-

CD68 and anti-CD206 to assess vascularization, total macrophages amount and M2

macrophages amount in wound skin tissue amount, respectively. Frozen sections were

fixed in acetone at -20 °C for 10 min, air-dried, and rinsed once with 0.01 mM PBS.

Slides were blocked with 5% normal goat serum for 45 min at 4 °C and then

incubated with rat anti-CD31 antibody (1:200, BD biosciences, USA), or rabbit anti-

mannose receptor antibody (CD206, 1:200, Abcam, USA), or rabbit anti-CD68

(1:200, Abcam, USA) antibody for 12 h at 4°C. Slides were rinsed 5 times with PBS.

Alexa Fluor 488-conjugated goat anti-rat IgG (1:200, Invitrogen) or goat anti-rabbit

IgG (1:200, Invitrogen) antibodies were applied for 2 h at room temperature, in

darkness. The cell nuclei were stained with a DAPI containing mounting solution.

Slides incubated with secondary antibody only were served as negative controls.

Slides were observed and photographed using a fluorescence microscope (Zeiss Axio

Imager Z1, Germany).

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Supplemental Figures

Fig. S1. Ion release of Ion-SA dressings and Ion-GSA composited dressings. The cumulative release amount of Co2+ and Ca2+ from 4 separate parts of the (A) Ion-SA dressings (Co2+0.25-Ca2+4 SA) and (B) Ion-GSA composite dressings at day 7. *P < 0.05.

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Fig. S2. SEM images of the surface of Ion-SA dressings, Ion-G and Ion-GSA composite dressings with different Co2+/Ca2+ ratios.

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Fig. S3. Photographs of the special-shape Ion-GSA composite dressings (Co2+0.25-Ca2+4 GSA).

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Fig. S4. Mechanical characterization of Ion-GSA composited dressings. (A) Tensile strength, (B) Tensile modulus and (C) Ultimate strain of gauzes (labelled as ‘G’ on graph axis) and Ion-GSA composite dressings.

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Fig. S5. Swelling ratio of plain gauze and Ion-GSA composite dressings with different Co2+/Ca2+ ratios. #P < 0.001.

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Fig. S6. Moisture retention capacity of plain gauze and Ion-GSA composite dressings with different Co2+/Ca2+ ratios. #P < 0.001, G versus other groups.

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Fig. S7. HUVECs migration of culture in Ion-GSA composited dressings without 3T3 cells. (A) Representative images of the trans-well migration assay of HUVECs after 12 h of co-culture with Ion-GSA composite dressings, without 3T3 cells. (B) Quantitative analysis of the migrated HUVECs per microscopic field in different groups. *P < 0.05.

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Fig. S8. Proliferation of macrophage cell (RAW 264.7 cell) on different Ion-GSA composite dressings. *P < 0.05, #P < 0.001.

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Fig. S9. In vitro antibacterial effects at 24h. Photos of surviving bacterial colonies of (A) S. aureus and (B) P. aeruginosa on agar plates after contact with different Ion-GSA composite dressings. The calculated number of colonies of (C) S. aureus and (D) P. aeruginosa. *P < 0.05, #P < 0.001.

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Fig. S10. Fluorescent images of (A) S. aureus and (B) P. aeruginosa after treatment on various materials. SYTO 9: staining for live and dead bacteria, PI: staining for dead bacteria, and the merged image of SYTO 9 and PI.

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Fig. S11. The hemostatic ability of Ion-GSA composite dressings. (A) Photographs of whole-blood incubated with non-dressings (control), G and Ion-GSA composite dressings with different Co2+/Ca2+ ratios of at 37 ℃ within 2 min. (B) Images of the hemostatic properties of G and different Ion-GSA composite dressings in the hemorrhaging liver rat model. (C) Whole-blood clotting time of G and different Ion-GSA composite dressings with different ratios of Co2+/Ca2+. (D) Total blood loss from the damaged livers after 3 min. *P < 0.05, #P < 0.001.

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Fig. S12. The protein expression analysis of IL-10 in wound sites on day 6 post-

wounding by ELISA. #P < 0.001.

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Fig. S13. Collagen deposition in wounds. (A) Representative images of collagen fibers stained by Masson’s trichrome method, within the wounded skin tissues, following the treatments on day 6 and 12. (B) The quantitative analysis of collagen-positive pixels per field. (C) Highly magnified view of the red rectangular part in the image of Co2+0.25-Ca2+4 GSA group on day 12. (D) Representative images of Masson’s trichrome staining of natural skin tissue. *P < 0.05, #P < 0.001.

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