Thermodynamic 2D Silicene for Sequential and Multistage Bone Regeneration

Abstract

Bone healing is a multistage process involving the recruitment of cells, revascularization, and osteogenic differentiation, all of which are modulated in the temporal sequence to maximize cascade bone regeneration. However, insufficient osteoblast cells, poor blood supply, and limited bone induction at the site of critical-sized bone defect broadly impede bone repair. 2D SiO₂ -silicene@2,2'-,azobis(2-[2-imidazolin-2-yl] propane) (SNSs@AIPH) with inherent thermodynamic property and osteoinductive activity is therefore designed and engineered for sequentially efficient bone repair. By means of controllable NIR-II irradiation, the integrated SNSs@AIPH stimulates the generation of appropriate intracellular reactive oxygen species, which accelerates early bone marrow mesenchymal stem cells (BMSCs) proliferation and angiogenesis remarkably. Importantly, as silicon-based 2D nanoparticles, the engineered SNSs@AIPH with high biocompatibility features distinct bioactivity to significantly promote BMSCs osteogenesis differentiation by activating TGFβ and BMP pathways. In a rat cranial defect model, SNSs@AIPH-NIR-II leads to a comparable increase of BMSCs proliferation and local vascularization at an early stage, followed by significant osteogenic differentiation, synergically resulting in a highly effective bone repair. Collectively, the fascinating characteristics and exceptional bone repair efficiency of NIR-II-mediated SNSs@AIPH allow it to be a promising bionic-oriented strategy for bone regeneration, broadening a new perspective in the application of cell-instructive biomaterials in bone tissue engineering.

Keywords: angiogenesis; proliferation; sequential bone repair; silicene; thermodynamics.

Scheme 1

Schematic illustration. After implantation of the SNSs@AIPH/CPC‐BMSCs at the site of bone defect, controllable 1064 nm NIR (NIR‐II) stimulated moderate increase of intracellular ROS which induced BMSCs proliferation, vascular cells proliferation and migration by activating of MAPK signaling pathway in the cell proliferation phase. Subsequently, the sprouting of new blood vessel formation occurred in the early reparative phase and SNSs@AIPH facilitated BMSCs osteogenic differentiation by targeting TGFβ and BMP signaling pathways afterward, which synergically in an effort to promote bone regeneration.

Figure 1

Characterization of silicene nanosheets, SiO₂‐silicene and SiO₂‐silicene@AIPH (SNSs@AIPH) nanoparticles. A) TEM image of multilayer silicene MXene, B) single‐layer silicene nanosheets, and C) its corresponding SAED pattern. D) SEM image and E,F) TEM images of single SiO₂‐silicene NPs. G) High‐resolution TEM image obtained from SNSs@AIPH and its corresponding electron energy loss spectroscopy (EELS) mapping, with the superimposed images clearly showing the distribution of N in AIPH. H) UV–vis spectra, I) DLS, and J) zeta potential of silicene nanosheets, SiO₂‐silicene, and SNSs@AIPH nanoparticles. Scale bars are as follows: (A,F) 50 nm, (B) 200 nm, (C) 5 1/nm, (D,E,G) 100 nm.

Figure 2

Photothermal performance and the free radicals generation of SNSs@AIPH nanoparticles under NIR‐II laser irradiation. A) XPS spectrum of SNSs@AIPH. B) UV–vis spectra of SNSs@AIPH under various concentrations and its corresponding mass extinction coefficient at NIR‐II. C,D) Photothermal heating curves of dispersed SNSs@AIPH NPs under irradiation of NIR‐II at various concentrations (0, 12.5, 25, 50, 100 µg mL⁻¹) and different power densities (0.25, 0.5, 0.75, 1.0 W cm⁻²). E) Calculation of the photothermal conversion efficiency at NIR‐II. Red line: photothermal effect of an aqueous dispersion of SNSs@AIPH NPs under irradiation with a NIR‐II laser for certain periods, and then the laser was switched off. Blue line: time constant (τs) for the heat transfer from the system determined by applying the linear time data from the cooling period. F) Heating curve of SNSs@AIPH NPs suspension under NIR‐II laser irradiation in six lasers on/off cycles (1.0 W cm⁻²). G) EPR spectra of POBN in AIPH, SNSs and SNSs@AIPH NPs solution after NIR‐II irradiation for 5 min. H) The formation of ABTS^+• induced by free radicals generated in SNSs@AIPH NPs solution at various temperatures and time points. I) UV–visible spectra of ABTS^+• in dispersed SNSs@AIPH NPs under the various irradiation‐processing durations and the statistical plot of the relationship between the absorbance of ABTS^+• and the different irradiation durations in SNSs, AIPH, and SNSs@AIPH NPs solution.

Figure 3

Cytocompatibility of the SNSs@AIPH‐NIR‐II. A) Cell counting experiments for assessing the proliferative ability of BMSC after SNSs@AIPH treatment under different NIR‐II irradiation time, n = 3. B) Live/Dead staining for the detection of BMSC apoptosis. C) Phalloidin staining graphically representing the cytoskeleton of BMSCs. D) qPCR demonstrating expression of Caspase‐3 and IL‐6 in BMSCs, n = 3. E) Quantification of intracellular ROS accumulation using carboxyH₂ DCFDA (ex/em: 488/52 nm), n = 12. F) Immunofluorescence positive staining of Ki‐67 and EdU representing cells with active proliferation and positive ratio of EdU and Ki‐67 obviously upregulated in the SNSs@AIPH‐NIR‐II group in comparison to the other groups. G) Total RNA was isolated from BMSC treated with SNSs@AIPH‐NIR‐II and SNSs@AIPH, followed by RNA sequencing analysis. Volcano plot displays global gene expression in the SNSs@AIPH‐NIR‐II and SNSs@AIPH sets. Two‐sided test. Orange represents upregulated genes, and purple represents downregulated genes. H) Gene ontology (GO) enrichment analysis of upregulated genes (>1.5‐fold) in the SNSs@AIPH‐NIR‐II and SNSs@AIPH sets. I) Heatmap analysis of proliferation‐associated genes in the SNSs@AIPH‐NIR‐II, SNSs@AIPH and negative control sets. Red indicates upregulated and blue indicates downregulated genes. J) Relative protein expression levels for the fos were analyzed by Western blot. K) NIR‐II triggered SNSs@AIPH to increase BMSCs in the S and G2 phases by flow cytometry analysis. L) Mechanism pattern diagram of BMSCs proliferation‐related pathways triggered by SNSs@AIPH‐NIR‐II. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis was performed by one‐way ANOVA.

Figure 4

Effects of SNSs@AIPH‐NIR‐II on angiogenesis in vitro and in vivo. A) EdU staining for detecting the increased positive ratio of EdU in the group of SNSs@AIPH‐NIR‐II, n = 3. B) Cell counting experiment displaying the significantly higher cell numbers in the SNSs@AIPH‐NIR‐II group as compared to the other groups in 48 and 72 h, n = 3. C) HUVEC scratch wound migration in response to SNSs@AIPH‐NIR‐II. D.E) HUVECs treated with SNSs@AIPH‐NIR‐II as indicated. Tube formation in matrigel was evaluated 10 h later. The histogram shows the quantification of the branch length per field using ImageJ, n = 3. F,G) Relative mRNA expression and protein levels for VEGF as analyzed by qPCR and western‐blot, n = 3. H) Blood vessel formation in the defect region of rats as evaluated by Microfil perfusion between groups, n = 5. I) Immunohistochemistry (IHC) analysis of VEGF and CD31 in the defect region of rats in each group (red lines indicate representative areas), n = 3, **P < 0.01, ***P < 0.001. Statistical analysis was performed by one‐way ANOVA.

Figure 5

Effects of SNSs@AIPH on BMSCs osteogenesis differentiation in vitro. A) ALP staining for evaluating the effect of SNSs@AIPH on ALP activity at osteogenesis day 7. B) ARS staining for evaluating the effect of SNSs@AIPH on the ECM mineralization at osteogenesis day 21. C,D) Immunocytochemistry detection to assess intracellular protein localization and expression of osteogenic markers OPN, BSP, RUNX2, and OSX in different groups, n = 3. E,F) Relative mRNA and quantitative protein expression levels for OPN, BSP, RUNX2, and OXS as analyzed by qPCR and Western blot, n = 3. G) Total RNA was isolated from BMSC treated with SNSs@AIPH, followed by RNA sequencing analysis. Volcano plot displays global gene expression in the SNSs@AIPH and negative control sets. Two‐sided test. Orange represents upregulated genes, and purple represents downregulated genes. H) Gene ontology (GO) enrichment analysis of upregulated genes (>1.5‐fold) in the SNSs@AIPH and negative control sets. I) Heatmap of gene set enrichment analysis of top gene signatures that are upregulated (orange) or downregulated (purple) in BMSCs treated with SNSs@AIPH and NC for 7 d culture. J,K) The mRNA expression levels and protein levels for the TGF‐β3 and BMP2 as analyzed by Western blot, n = 3. L) Western‐blot analysis of SNSs@AIPH‐induced SMAD2/3 and SMAD1/5 phosphorylation. M) Mechanism pattern diagram of TGFβ and BMP pathways triggered by SNSs@AIPH. *P < 0.05, **P < 0.01, ***P < 0.001, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. Statistical analysis was performed by one‐way ANOVA.

Figure 6

Effects of SNSs@AIPH‐NIR‐II on new bone formation in vivo. A) SEM images showing presence of BMSCs on the SNSs@AIPH‐NIR‐II/CPC and CPC. B) 3D rendering showing micro‐CT analysis of new bone formation in the defects area of each group. C) Locally enlarged area as used for new bone volume calculation. D) Quantitative analysis of BMD, BV/TV, and Tb.N in new bone area, n = 5. E) Sequential fluorescence labeling of Calcein, Tetracycline, and Alizarin Red showing the new bone formation of each group. F) Immunohistochemical analysis of OSX, OPN, and BSP in the defect area at eight weeks post‐operation, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. Statistical analysis was performed by one‐way ANOVA.