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An injectable photo-cross-linking silk hydrogel system augments diabetic wound healing in orthopaedic surgery through spatiotemporal immunomodulation | Journal of Nanobiotechnology


Preparation and characterization of MET@MSNs

MET@MSNs were synthesized as previously described [22, 59]. Briefly, MSN (20 mg; XFNANO, Nanjing, China) and Met (60 mg; MACKLIN, Shanghai, China) were dispersed in ethanol and stirred at 37 °C for 24 h. The mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in ethanol and dried at 80 °C for ~ 8 h for further studies. The drug loading (DL%) and encapsulation efficiency (EE%) were determined by UV–Vis spectroscopy (Cary 300; Agilent Technologies, Santa Clara, CA, USA), and the total amount of unloaded MET was calculated according to the absorbance at 234 nm. The results were calculated, as follows:

$$\mathrm{DL}(\mathrm{\%})=\frac{Mass \, of \, total \, MET-Mass\, of \,unloaded \,MET\,(Encapsulated \,MET)}{Mass\, of\, total\, MSNs\, and\, encapsulated\, MET}\times 100$$


$$\mathrm{EE}(\mathrm{\%}) =\frac{Mass \,of\, total\, MET-Mass \,of\, unloaded \,MET\,(Encapsulated \,MET)}{Mass\, of\, total\, MET}\times 100$$


To determine the particle size and morphology of the MSN and MET@MSNs, SEM was performed (S4800; Hitachi, Tokyo, Japan). The BET method (ASAP2460; Micromeritics, Atlanta, GA, USA) was employed to assess the structural indicators (surface area, pore volume) related to the mesopores of MSN and MET@MSNs. The chemical bond of MSNs and MET@MSNs were analysed using FT-IR (Nicolet IS 10; Thermo Fisher Scientific, Waltham, MA, USA).

Preparation and characterization of the hydrogel system

The Sil-MA (EFL, Suzhou, China) solution was prepared according to manufacturer instructions. Briefly, Sil-MA (0.5 mg) was dissolved in 0.25% (w/v) initiator LAP solution (5 mL) with stirring at room temperature (23–24 °C) for 30 min. Ag NPs solution (100 ppm; XFNANO) and MET@MSNs with different mass ratios were mixed with Sil-MA solution and vortexed for 5 min, followed by ultasonication (200 W) for 10 min to ensure complete dispersion of the NPs in the Sil-MA solution. The mixture was then irradiated under a 405 nm light source for ~ 25 s to obtain the hydrogel system (Sil-MA, Ag-Sil-MA, M@M-Sil-MA, and M@M-Ag-Sil-MA).

The samples were fixed at the sample stage with conductive glue, and the surface was sprayed with gold. The surface morphology was then observed using SEM. A rotational rheometer was used to test the rheological properties of the hydrogel system (Discovery HR-2; TA instruments, New Castle, DE, USA).

To evaluate the swelling properties of the hydrogel, samples were weighed and the mass was recorded as W0. Phosphate-buffered saline (PBS) was added to the samples at different pH values (pH = 6.0, 7.4, and 8.0), after which the samples were removed at a predetermined time point and weighed after absorbing the residual PBS on the surface of the sample with Kimwipes (Kimberly-Clark Corp., Dallas, TX, USA), and the mass was recorded as Wt. The samples were returned to PBS after weighing, and the swelling ratio was calculated, as follows:

$$\mathrm{Swelling \,ratio }\,(\mathrm{\%}) =\frac{Wt-W0}{W0}\times 100$$

Degradation tests (%) were conducted by soaking the samples in PBS at different pH values at 37 °C until complete swelling, and then, the initial mass was recorded as Wi. Then, the samples were placed in PBS solution contained collagenase (Biosharp, Hefei, China) with shaking at 37 °C. At the pre-set time points, the samples were removed and dried with Kimwipes, and the remaining mass was recorded as Wp. The degradation (%) was calculated by the following formula:

$$\mathrm{Degradation }\,(\mathrm{\%}) =\frac{Wi-Wp}{Wi}$$

M@M-Ag-Sil-MA with different mass ratios of Ag NPs and MET@MSNs (1:1, 1:2, and 1:3) was immersed in 2 mL PBS at different pH values (pH = 6.0, 7.4, and 8.0) to evaluate the cumulative release of components. The release system was incubated at 37 °C using a shaker (200 rpm). At specific time points, 1 mL PBS was removed to analyse the concentrations of Ag NPs and Met, after which 1 mL of fresh PBS was added to the release system. Released levels of Ag NPs and Met were determined by measuring the absorbances at 234 nm and 400 nm by UV–Vis spectroscopy, respectively.

Cells isolated and culture

The femur and tibia medullary cavity of C57/BL6 mice were rinsed with Roswell Park Memorial Institute (RPMI)-1640 medium (Biosharp, Hefei, China) to obtain primary cells. Neutrophils were isolated using the EasySep mouse neutrophil enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer instructions and cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 0.1 mg/mL primocin (InvivoGen, San Diego, CA, USA).

EA.hy926, L929, and RAW264.7 cells were incubated in high-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Verification of neutrophil cells

The obtained neutrophils were collected through centrifugation at 300g for 10 min at 4 ℃, and the pellets were resuspended with 100 μL PBS containing 0.25 μg of the allophycocyanin (APC)-conjugated anti-mouse/human CD11b antibody (Biolegend, San Diego, CA, USA) and the phycoerythrin (PE)-conjugated anti-mouse Ly-6G antibody (Biolegend), followed by incubation on ice for 30 min. Levels of CD11b and Ly-6G in the neutrophils were analysed by flow cytometry using the CytoFLEX system (Beckman Coulter, Pasadena, CA, USA).

Evaluation of in vitro cytotoxicity

RAW264.7, EA.hy926, and L929 cells were used to evaluate in vitro cytotoxicity. M@M-Ag-Sil-MA and the three cells were co-cultured in 6-well plates at 37 ℃, after which M@M-Ag-Sil-MA and the medium were removed and replaced with DMEM containing 10% Cell Counting Kit-8 reagent (Biosharp). After incubating for 2 h, the absorbance was measured at 450 nm using a microplate reader (Epoch; BioTEK, Winooski, VT, USA).

Additionally, RAW264.7 cells were stained using the LIVE/DEAD cell imaging kit (Invitrogen, Carlsbad, CA, USA) for 15 min, and EA.hy926 cells were subsequently stained with 100 nM TRITC-phalloidin (Yeasen, Shanghai, China) and 4′,6-diamidino-2-phenylindole (DAPI, Biosharp) to observe cell morphology. Stained cells were observed under an inverted fluorescence microscope (ECLIPSE Ts2; Nikon, Tokyo, Japan).

CM and MCM preparation

Briefly, four samples including Sil-MA, M@M-Sil-MA, Ag-Sil-MA, and M@M-Ag-Sil-MA were soaked with PBS with shaking at 37 °C. Based on the observed release of Met and Ag NPs, the PBS solution was collected on days 1 and 7, centrifuged, filtered using a 0.22-μm filter, and mixed with DMEM at a ratio of 1:2 (v/v). This CM was then prepared for further testing. For MCM preparation, macrophages were cultured with CM for 24 h, followed by replacement of CM with complete medium (DMEM). After another 24-h incubation, the medium was collected to prepare MCM using the same methods as those for CM.

NETs formation and extraction

Round coverslips were placed on 12-well plates, onto which isolated neutrophils were seeded (1 × 105cells/well) in RPMI-1640 medium containing phorbol (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 2 h at 37 °C. The cells were then fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. The NETs were stained with 0.5 μM SYTOX Green nucleic acid stain (Invitrogen) and observed using confocal laser scanning microscopy (LSM710; Carl Zeiss, Oberkochen, Germany). NETs extracts were collected using the same methods as those for CM collection.

In vitro antibacterial test

S. aureus (ATCC43300) and E. coil (ATCC 35218) cultured in tryptic soy broth were used for antibacterial tests. Bacterial suspensions (1 × 107 CFU/mL) were spread onto Mueller–Hinton agar plates. Sil-MA containing different materials (Sil-MA, M@M-Sil-MA, Ag-Sil-MA, or M@M-Ag-Sil-MA) were photocured on curing rings, which were then placed on the agar plates containing the bacteria and incubated 24 h at 37 °C. The diameter of the inhibition zone around the samples was subsequently measured.

The different CMs (Sil-MA, M@M-Sil-MA, Ag-Sil-MA, and M@M-Ag-Sil-MA) were co-cultured with bacterial suspension (1 × 107 CFU/mL) and incubated at 37 ℃, with the control group treated with PBS. At a predetermined time point, 1 mL of the suspension was used to generate a tenfold gradient dilution, with 100 μL of the dilution spread onto sheep blood agar plates. After overnight incubation at 37 °C, bacterial colonies on the plates were counted.

To observe bacterial morphology, sterile titanium sheets, different CM, and bacterial suspension were co-cultured in 6-well plates 24 h at 37 °C, after which the titanium sheets were subjected to SEM analysis. Briefly, the sheets were fixed with 2.5% glutaraldehyde overnight at 4 °C, and samples were dehydrated using an ethanol gradient (50%, 70%, 80%, 90%, 95%, and 100%) for 10 min at room temperature (23–24 °C). After freeze-drying, the surface of the sheets was sprayed with gold, followed by SEM analysis.

Flow cytometry

RAW264.7 cells were seeded onto 6-well plates at a concentration of 2 × 105cells/well. After incubation for 24 h at 37 ℃, the medium was removed and washed three times with PBS, followed by the addition of different CM to each well. To mimic the in vivo inflammatory conditions of diabetic wounds and explore the effect of NETs and DNase I on macrophage repolarization, LPS (Sigma-Aldrich), NETs extracts, and DNase I (Sigma-Aldrich) were added into each well, respectively. After a 24-h culture, RAW264.7 cells were collected via centrifugation at 1000 rpm for 5 min, and the pellets were resuspended with 100 μL PBS containing 0.25 μg APC-conjugated anti-mouse CD86 antibody (Biolegend) and 0.5 μg PE-conjugated anti-mouse CD206 antibody (Biolegend), followed by incubation on ice for 30 min. CD86 and CD206 levels on RAW264.7 cells were analysed by flow cytometry using the CytoFLEX system (Beckman Coulter).


RAW264.7 cells treated with different CM for 24 h were evaluated for iNOS and Arg-1 expression by RT-PCR. Total RNA was extracted and purified using the EZ-press RNA purification kit (EZBioscience, Roseville, MN, USA) and then reverse transcribed into cDNA using a Colour reverse transcription kit (EZBioscience). Quantitative RT-PCR was performed using 2 × Colour SYBR Green qPCR master mix (EZBioscience) and relative expression was calculated using the 2−ΔΔCt method. Primer sequences for glyceraldehyde 3-phiosphate
dehydrogenase (Gapdh), iNOS, and Arg-1 are shown in Additional file 1: Table S1.


RAW264.7 cells treated with different CM for 24 h, and the medium was collected and centrifuged at 3000 rpm for 20 min. The supernatant was then used to determine inflammatory cytokine expression using ELISA Kits (Dakewe Biotech, Guangzhou, China) according to manufacturer instructions.

Tube formation assay

Briefly, EA.hy926 cells (1 × 104 cells/well) pre-treated with different MCM for 24 h were seeded onto μ-slide plates (IBIDI GmbH, Munich, Germany) pre-coated with Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA). After incubation for 6 h at 37 °C, the formed tubes were fixed and stained with 100 nM fluorescein isothiocyanate-phalloidin (Yeasen) and observed using an inverted fluorescence microscope. The numbers of junctions and circles were counted manually.

Scratch assay

L929 cells (2 × 105cells/dish) were seeded in 35-mm cell culture dishes and incubated at 37 ℃ to 90% confluence. Subsequently, 200-μL pipette tips were used to draw a line at the bottom of the dishes, which were then washed three times with PBS. The cells were then co-cultured with MCM in five groups for 24 h at 37°. At 0 h and 24 h, the cells in each group were fixed, washed, stained with Crystal Violet for 3 min, and observed using an optical microscope. Cell-migration rates in each group were assessed using ImageJ software (v1.52; NIH, Bethesda, MD, USA).

Inhibition of NET formation

To explore Met inhibit NETs formation in the infective microenvironment of diabetic wounds, neutrophils were cultured with CM in three groups containing glucose or LPS for 2 h, after which the cells were stained with 1 μM SYTOX Green (Invitrogen) and observed by confocal laser scanning microscopy.

Streptozotocin (STZ)-induced diabetic mice

All animal experiments were approved by the Animal Welfare Ethics Committee of The First Hospital Affiliated University of Science and Technology of China. C57BL/6 mice (6–8 weeks, 23–26 g) were used to induce diabetes. Briefly, 100 mg/kg STZ (Sigma-Aldrich) was injected intraperitoneally into mice fasted from food and water for 1 day prior to injection. Blood glucose levels were determined using glucose meters (Roche, Penzberg, Germany), and all mice with glycaemia (≥ 16.7 mM) were considered diabetic.

Evaluation of in vivo wound healing

We randomly divided 45 diabetic mice into five groups: control, Sil-MA, M@M-Sil-MA, Ag-Sil-MA, and M@M-Ag-Sil-MA. For consistency in animal experiments, each group contained 9 mice. These diabetic mice were anesthetized by inhalation of CO2. After shaving and disinfecting the dorsal skin of the mice, a pouch with a diameter of 8 mm was used to create a skin wound, after which 0.2 mL of hydrogel (Sil-MA, M@M-Sil-MA, Ag-Sil-MA, or M@M-Ag-Sil-MA) was dropped onto the wound and photocured under 405 nm UV light for 25 s. On days 0, 3, 7, 10, and 14 after surgery, the weight of the mice was obtained, and the condition of the wounds were recorded to assess the wound-healing rate.

Histological analysis

On days 1, 3, 7, and 14 after surgery, random mice from each group were euthanized. The wounded skin tissues were collected and fixed in 4% paraformaldehyde, dehydrated using an ethanol gradient, embedded in paraffin wax, and cut into sections using an RM2016 microtome (Leica, Wetzlar, Germany). Sections were stained with H&E (Solarbio, Beijing, China) and Masson’s trichrome (Solarbio) to assess the degree of inflammatory cell infiltration, collagen deposition, and bacterial infection. For immunofluorescence staining, primary CD86 (1:3000; Bioss, Beijing, China), CD206 (1:400; Servicebio, Wuhan, China), MPO (1:400, Servicebio), CitH3 (1:3000; Abcam, Cambridge, UK), secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (1:500; Servicebio), and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (1:400; Servicebio) antibodies were used to observe the degree of inflammatory response, macrophage phenotype, and NETs in wounded skin tissues. To observe angiogenesis in wound skin tissues, sections were incubated with primary antibodies for IHC analysis [mouse anti-α-SMA (1:300; Bioss) and rabbit anti-VEGF antibody (1:200, Bioss, China)] at 4 ℃ overnight, followed by incubation with the secondary antibodies HRP-conjugated goat anti-mouse IgG (H+L) (1:200; Servicebio) and HRP-conjugated goat anti-rabbit IgG (H+L) (1:200; Servicebio) for 1 h. The binding sites were visualized with a 3,3′-diaminobenzidine detection kit (DAKO, Glostrup, Denmark), counterstained with haematoxylin (Servicebio), and mounted with neutral resinto (Servicebio). All stained sections were observed using a microscope (Ci-S; Nikon).

Evaluation of in vivo biocompatibility

The major organs (heart, liver, spleen, lung, and kidney) from each group were collected, fixed, dehydrated, embedded, cut, and stained with H&E. The sections were then observed using an optical microscope to assess the in vivo biocompatibility of the samples.

Statistical analysis

All data were exhibited as the mean ± standard deviation and analysed using GraphPad Prism software (version 8.0) and Origin (Version 2019b). Statistically significant values were assessed using two-sided student’s t test and one-way analysis of variance (ANOVA) test. P value < 0.05 was considered statistically significant.


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