Bioinspired soft-hard combined system with mild photothermal therapeutic activity promotes diabetic bone defect healing via synergetic effects of immune activation and angiogenesis

Background: The comprehensive management of diabetic bone defects remains a substantial clinical challenge due to the hostile regenerative microenvironment characterized by aggravated inflammation, excessive reactive oxygen species (ROS), bacterial infection, impaired angiogenesis, and unbalanced bone homeostasis. Thus, an advanced multifunctional therapeutic platform capable of simultaneously achieving immune regulation, bacterial elimination, and tissue regeneration is urgently designed for augmented bone regeneration under diabetic pathological milieu. Methods and Results: Herein, a photoactivated soft-hard combined scaffold system (PGCZ) was engineered by introducing polydopamine-modified zeolitic imidazolate framework-8-loaded double-network hydrogel (soft matrix component) into 3D-printed poly(ε-caprolactone) (PCL) scaffold (hard matrix component). The versatile PGCZ scaffold based on double-network hydrogel and 3D-printed PCL was thus prepared and features highly extracellular matrix-mimicking microstructure, suitable biodegradability and mechanical properties, and excellent photothermal performance, allowing long-term structural stability and mechanical support for bone regeneration. Under periodic near-infrared (NIR) irradiation, the localized photothermal effect of PGCZ triggers the on-demand release of Zn2+, which, together with repeated mild hyperthermia, collectively accelerates the proliferation and osteogenic differentiation of preosteoblasts and potently inhibits bacterial growth and biofilm formation. Additionally, the photoactivated PGCZ system also presents outstanding immunomodulatory and ROS scavenging capacities, which regulate M2 polarization of macrophages and drive functional cytokine secretion, thus leading to a pro-regenerative microenvironment in situ with enhanced vascularization. In vivo experiments further demonstrated that the PGCZ platform in conjunction with mild photothermal therapeutic activity remarkably attenuated the local inflammatory cascade, initiated endogenous stem cell recruitment and neovascularization, and orchestrated the osteoblast/osteoclast balance, ultimately accelerating diabetic bone regeneration. Conclusions: This work highlights the potential application of a photoactivated soft-hard combined system that provides long-term biophysical (mild photothermal stimulation) and biochemical (on-demand ion delivery) cues for accelerated healing of diabetic bone defects.

Table S1.Primer sequences used in qRT-PCR analysis.

Genes
Figure S1.TEM images of ZIF-8@PDA nanoparticles at different magnifications.The white arrows indicate the PDA layer.Scale bar: 50 and 10 nm for the left and right images, respectively.

Figure
Figure S5.(A) Infrared thermal images, (B) temperature curves, and (C) thermal cycle profiles of the prepared nanoparticle aqueous solutions (1 mg/mL) under NIR laser

Figure S7 .
Figure S7.Total porosity of different hydrogels.Data are presented as the mean ± SD (n = 3).

Figure S8 .
Figure S8.The distribution of ZIF-8@PDA nanoparticles in the hydrogel was observed by SEM.The red arrows represent the nanoscale particles on the hydrogel surface.Scale bar: 1 µm.

Figure
Figure S10.(A) Compressive stress-strain curves and (B) compressive strength of

Figure S12 .
Figure S12.Quantitative analysis of cell density based on live/dead staining assay.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the GMCS group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the GMCS/Z2 group.

Figure
Figure S13.(A) H&E staining, MST staining, and immunohistochemical staining images of decalcified bone tissue.FT: fibrous tissue.NB: newly formed bone tissue.The yellow asterisks represent the residual materials.Scale bar: 50 μm.Quantitative expression of (B) CD90, (C) Runx2, and (D) OPN.Data are presented as the mean ± SD (n = 3).Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the control group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the GMCS/Z2 group.

Figure S15 .
Figure S15.Micro-CT images of cross-sectional scaffolds.The yellow asterisks represent the separation gap between the hydrogel and the PCL.The yellow dotted lines indicate the interfacial contact of the hydrogel with the PCL.Scale bar: 200 µm.

Figure S16 .
Figure S16.The average (A) pore size and (B) porosity of the different scaffolds.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.

Figure
Figure S18.(A) SEM images of different scaffolds after mineralization.The yellow arrows indicate in situ mineralized hydroxyapatite nanocrystals.Scale bar: 2 µm.(B) SEM images of PGCZ scaffolds at different magnifications after mineralization.Scale bar: 2 µm and 400 nm for the left and right images, respectively.(C) FTIR spectra and (D) XRD patterns of different scaffolds after mineralization.

Figure S20 .
Figure S20.Quantitative analysis of cell spreading of MC3T3-E1 cells.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S21 .
Figure S21.Quantitative analysis of (A) ALP activity and (B) ECM mineralization in MC3T3-E1 cells.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S22 .
Figure S22.Relative mRNA expression of HSPa1a, HSPa1b, HSP47, and HSP25 in MC3T3-E1 cells cultured on different scaffolds with or without NIR treatment.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S24 .
Figure S24.Quantitative analysis of positive staining areas.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S26 .
Figure S26.Relative mRNA expression of (A) proinflammatory and (B) anti-inflammatory markers in macrophages after 3 days of co-culture.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the control

Figure S27 .
Figure S27.Secretion of angiogenic (VEGF and bFGF) and inflammatory (TNF-α and IL-10) cytokines by macrophages in the different groups.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S28 .
Figure S28.Statistical analysis of the survival ratio of S. aureus and E. coli based on the spread plate method.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S29 .
Figure S29.Statistical analysis of S. aureus and E. coli bacterial biofilms.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the control group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S31 .
Figure S31.Photothermal heating curves of the implantation site under NIR irradiation (1 W/cm 2 , 808 nm) with four on/off cycles.Data are presented as the mean ± SD (n = 3).

Figure S32 .
Figure S32.Quantitative analysis of iNOS-and CD206-positive staining areas after implantation.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences with the PCL group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S34 .
Figure S34.ELISA results of CD4 and CD8 in serum of rats.Data are presented as the mean ± SD (n = 3).

Figure S35 .
Figure S35.Photothermal heating curves of the implantation site under NIR irradiation (1 W/cm 2 , 808 nm) with four on/off cycles.Data are presented as the mean ± SD (n = 3).

Figure S36 .
Figure S36.H&E staining images of the major organs, including the heart, liver, spleen,

Figure
Figure S38.(A) Flow cytometry analysis and (B-C) corresponding quantification of macrophage phenotypes at 2 weeks after implantation.Relative mRNA expression of (D) proinflammatory and (E) anti-inflammatory markers at 2 weeks after implantation.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the control group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S40 .
Figure S40.Quantitative analysis of immunohistochemical staining at 8 weeks after implantation.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the control group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.

Figure S41 .
Figure S41.TRAP staining images of decalcified bone tissue and quantification of TRAPpositive cells.Scale bar: 100 μm.Data are presented as the mean ± SD (n = 3).*P < 0.05 and **P < 0.01 indicate significant differences compared with the control group.# P < 0.05 and # # P < 0.01 indicate significant differences compared with the PGCZ+NIR group.