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

Abstract

Background: The complicated hyperglycaemic and chronic inflammation of diabetic wounds in orthopaedic surgery leads to dysregulated immune cell function and potential infection risk. Immune interventions in diabetic wounds face a possible contradiction between simultaneous establishment of the pro-inflammatory microenvironment in response to potential bacterial invasion and the anti-inflammatory microenvironment required for tissue repair. To study this contradiction and accelerate diabetic-wound healing, we developed a photocurable methacryloxylated silk fibroin hydrogel (Sil-MA) system, co-encapsulated with metformin-loaded mesoporous silica microspheres (MET@MSNs) and silver nanoparticles (Ag NPs).

Results: The hydrogel system (M@M-Ag-Sil-MA) enhanced diabetic-wound healing via spatiotemporal immunomodulation. Sil-MA imparts a hydrogel system with rapid in situ Ultra-Violet-photocurable capability and allows preliminary controlled release of Ag NPs, which can inhibit bacterial aggregation and create a stable, sterile microenvironment. The results confirmed the involvement of Met in the immunomodulatory effects following spatiotemporal dual-controlled release via the mesoporous silica and Sil-MA. Hysteresis-released from Met shifts the M1 phenotype of macrophages in regions of diabetic trauma to an anti-inflammatory M2 phenotype. Simultaneously, the M@M-Ag-Sil-MA system inhibited the formation of neutrophil extracellular traps (NETs) and decreased the release of neutrophil elastase, myeloperoxidase, and NETs-induced pro-inflammatory factors. As a result of modulating the immune microenvironmental, the M@M-Ag-Sil-MA system promoted fibroblast migration and endothelial cell angiogenesis in vivo, with verification of enhanced diabetic-wound healing accompanied with the spatiotemporal immunoregulation of macrophages and NETs in a diabetic mouse model.

Conclusions: Our findings demonstrated that the M@M-Ag-Sil-MA hydrogel system resolved the immune contradiction in diabetic wounds through spatiotemporal immunomodulation of macrophages and NETs, suggesting its potential as a promising engineered nano-dressing for the treatment of diabetic wounds in orthopaedic surgery.

Keywords: Macrophage polarisation; Metformin; Neutrophil extracellular traps; Orthopaedic wound; Spatiotemporal immunomodulation.

Fig. 1

Characterization of the synthesized nanoparticles. SEM micrographs showed the average particle size of a MSN and b MET@MSNs (Scale bar: 200 μm). BET and BJH indicated the surface area and pore diameter of c MSN and d MET@MSNs. e FT-IR spectra of MSN, Met, and MET@MSNs

Fig. 2

Characterization of the hydrogel system. a Photographs of the solution to hydrogel transition by photocuring and the stability of hydrogel was proved by placing in different orientations. b Adhesion and bending ability on different surfaces. c Representative SEM images showed the interconnected porous meshwork of Sil-MA and M@M–Ag–Sil-MA scaffolds (Scale bar: 50 μm). The black and red arrows represent MET@MSNs and Ag NPs encapsulated in hydrogels, respectively. Storage modulus (G′) surpassing the loss modulus (G″) and at different d time (lasting for 100 s) and e frequencies (from 65 to 255 Hz). f–h Swelling property of hydrogel system containing different components under the PBS with different pH values (pH = 6.0/7.4/8.0). i–k Degradation property of the different hydrogel system soaked into PBS until complete swelling under different pH values (pH = 6.0/7.4/8.0)

Fig. 3

Drug release assay of the hydrogel system in vitro. Cumulative release mass curves of Met and Ag NPs of hydrogel systems with different MET@MSNs and Ag NPs mass ratios (1: 1, 2:1, and 3:1) in 2 mL neural (pH = 7.4) (a–c), acidic (pH = 6.0) (d–f) and alkaline(pH = 8.0) (g–i) PBS at 37 ℃ using a shaker (200 rpm)

Fig. 4

Antibacterial properties of the hydrogel systems. a Representative culture images of bacterial colonies formed by S. aureus (the top row) and E. coli (the bottom row) after exposure to PBS, CMs of Sil-MA, M@M-Sil-MA, Ag-Sil-MA, M@M-Ag-Sil-MA and the relative amounts of the corresponding colonies of S. aureus (b) and E. coli (c) determined by spread plate method. d Representative SEM images of S. aureus and E. coli treated with PBS or different CMs. Yellow spheres indicate S. aureus, and violet rods indicate E. coli. *P < 0.05, **P < 0.01 and ***P < 0.001) (Scale bar: 1 μm)

Fig. 5

Immunomodulatory function of macrophages in vitro. a Representative flow cytometry results after 1 and 7 d of co-culture with CM of hydrogel systems of RAW264.7. CD 86 represents M1 macrophages surface markers and CD 206 represents M2 macrophages surface markers. b, c Expression of M1-related gene, iNOS, and M2-related genes,Arg-1after the RAW264.7 cells were treated by the day 1 and day 7 CM containing different components for 24 h. d, e ELISA results for TNF-α and IL-10 after the RAW264.7 cells were treated by different CM for 24 h. f In vitro angiogenesis of Ea.hy 926 cells in different MCM and comparison of circle and junction generated in 6 h of culture at 37 ℃(Scale bar: 200 μm). And the quantitative results of h circle and i junction generated were counted manually. g In virto migration of L929 cells were treated by the day 1 and day 7 different MCM containing different components(Scale bar: 100 μm) and j comparison of quantitative results of cell migration ratio in 24 h of culture at 37 ℃. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 6

Immunomodulation of NETs in vitro. a Fluorescence micrographs of high glucose (50 mM)-treated and LPS-treated neutrophils and stained with SYTOX green on Day 1 and Day 7.The dispersed green area was indicated as NETs (scale bar: 25 μm). b Fluorescence intensity of NETs with high glucose-treated neutrophils. c, d ELISA results for MPO and NE of NETs of high glucose-treated neutrophils. e Representative flow cytometry results after co-culture with LPS, extracts of NETs, extracts of M@M-Ag-Sil-MA₇ (the extracts of M@M-Ag-Sil-MA on day 7), or DNase I of RAW264.7.CD 86 and CD 206 represents M1 macrophages surface markers and M2 macrophages surface markers, respectively. f–i ELISA results for TNF-α and IL-10 of high glucose-treated and LPS-treated neutrophils

Fig. 7

Hydrogel system promoted diabetic wound repair and regeneration in vivo. a Schematic diagram of diabetic wound and in situ hydrogel system photocuring. b Photographs of the wounds with different treatments on days 0 (before and after photocuring), 3, 7, 10 and 14. c Skin wound healing rate of the diabetic wound model at different time points. d Weight of the all-diabetic mice at different time points. e The overlaid images of the wound healed by different treatments on days 0 (red), 3 (yellow), 7 (green), 10 (blue) and 14 (purple). f H&E staining of the wound area on days 1, 3, 7, and 14 reflected the tissue-repair. (scale bar: 50 μm). g Masson’s trichrome staining on days 1, 3, 7, and 14 reflected collagen deposition (Scale bar: 50 μm)

Fig. 8

Immunomodulation of macrophages and NETs and tissue regeneration in vivo. a Immunofluorescence staining of CD86 (red, M1 macrophage surface marker) and CD206 (green, M2 macrophage surface marker) on days 1, 3, 7, and 14. (Scale bar: 50 μm). b Immunofluorescence staining of the markers of NETs formation, CitH3(red) and MPO (green), on days 1, 3, 7, and 14 (Scale bar: 50 μm). c CD31 and d α-SMA immunohistochemical staining images of the number of CD31-positive and α-SMA -positive cells in the skin tissues of diabetic mice wounds in different treatment groups (Scale bar: 50 μm)

Scheme 1

a Synthesis of MET@MSNs and Ag NPs-loaded Sil-MA hydrogel (M@M–Ag–Sil-MA). b Preliminary controlled release of Ag NPs inhibits bacterial aggregation and creates a sterile microenvironment. c Dual-controlled release of Met promotes wound healing through spatiotemporal immunomodulation of macrophages and NETs