Pyroptosis-responsive microspheres modulate the inflammatory microenvironment to retard osteoporosis in female mice

Abstract

The treatment of osteoporosis and related bone defects remains challenging. This study identifies pyroptosis-driven inflammation as a key disruptor of bone homeostasis. To address this, we develop a magnesium-gelatin composite microsphere scaffold (GelMa/Mg/DMF MS) that exploit pyroptosis blockade and hydrogen-mediated inflammation regulation for osteoporosis treatment. This porous microsphere scaffold is implanted into bone defects to achieve the sustained release of hydrogen gas, magnesium ions (Mg²⁺), and dimethyl fumarate (DMF). DMF act by activating the nuclear factor erythroid-related factor 2 to prevent osteoblast pyroptosis, and combine with the antioxidant effects of hydrogen, effectively remodel the inflammatory microenvironment and create favorable conditions for the restoration of bone homeostasis. Mg²⁺ further expedite bone tissue repair. These results demonstrate that the GelMa/Mg/DMF MS effectively reverse inflammatory microenvironments both in vivo and in vitro, resulting in significant tissue repair. These results suggest the combination of hydrogen therapy and pyroptosis blockade as a potential therapeutic strategy.

Fig. 1. Pyroptosis disrupts bone homeostasis in osteoporosis.

A Pyroptosis disrupts bone homeostasis in patients with osteoporosis. (created with BioRender.com) B Inflammatory cytokine levels in the serum of patients. C Correlations between the serum levels of the cytokines interleukin-1β (IL-1β) and IL-18 and the T-score of bone mineral density (BMD) in patients. D Immunohistochemical staining of GSDMD, CASP1 and NLRP3 in patient bone tissue samples. Scale bars: 100 μm. E X-ray image of c57 mice (the white dashed box illustrated the microcomputed tomography (micro-CT) image of the lumbar spine). F The BMD of the C57 mouse lumbar spine and tibia was determined via dual-energy X-ray absorptiometry (DXA) method (n = 5 samples). G Changes in the level of inflammatory factors in c57 serum were detected via enzyme-linked immunosorbent assay (ELISA) (n = 3 samples). H Images of tibial sections after immunohistochemistry for GSDMD. Scale bars: 100 μm. I Immunofluorescence staining of OPN (green) and propidium iodide (PI, red) in the tibial. Scale bars: 100 μm. J Co-localization of OPN with PI was calculated via ImageJ. K Pyroptosis-related protein expression in bone marrow mesenchymal stromal cells (BMSCs) was detected by Western blotting. L Expression of GSDMD in BMSCs was detected by confocal laser scanning microscopy (CLSM). Scale bars: 50 μm. M Expression of NLRP3 in BMSCs was detected by CLSM. Scale bars: 50 μm. N Transmission electron microscopy (TEM) image of the BMSCs. Scale bars: 5 μm and 1 μm. O Quantitative analysis of the expression of NLRP3 and GSDMD in BMSCs was detected by CLSM (n = 5 independent experiments). P Alkaline phosphatase (ALP) staining of BMSCs after 14 days. Scale bars: 200 μm. Q Alizarin red S (ARS) staining of BMSCs after 21 days. Scale bars: 200 μm. R Tartrate-resistant acid phosphatase (TRAP) staining of bone marrow-derived macrophages (BMDMs) for 7 days. Scale bars: 50 μm. S Osteoclast ring of BMDMs was detected via CLSM. Scale bars: 100 μm. T Number of osteoclasts per field of view (n = 5 independent experiments). The data are presented as the means ± SD. Statistical significance was calculated by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 2. Material characterization of magnesium-gelatin composite microsphere scaffold (GelMa/Mg/DMF MS).

A Scanning electron microscopy (SEM) images of the different samples. Scale bars: 20 μm. B Element mapping images of the GelMa/Mg/DMF MS. Scale bars: 20 μm. C Energy dispersive spectroscopy (EDS) spectrum of the GelMa/Mg/DMF MS. D X-ray diffraction (XRD) patterns of the different samples. E Mg ion release of GelMa/Mg/DMF MS detected by inductively coupled plasma-optical emission spectrometry (ICP) (n = 3 independent experiments). F Microsphere morphology of GelMa/Mg/DMF MS under light microscopy at different times of degradation (schematic diagram is shown in the upper right corner, created with BioRender.com). Scale bars: 20 μm. G Detection of dimethyl fumarate (DMF) release via UV-vis spectroscopy (n = 5 independent experiments). H Detection of hydrogen release from materials by reduction of methylene blue. I Detection of the total antioxidant capacity of GelMa/Mg/DMF MS via the 3, 3’, 5, 5’-tetramethylbenzidine (TMB) method. The data are presented as the means ± SD. Source data are provided as a Source Data file.

Fig. 3. Modulation of the inflammatory microenvironment (IME) by the GelMa/Mg/DMF MSs.

A Live/dead assay of BMSCs and Raw 264.7 cells for 72 h. Scale bars: 200 μm. B Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay of BMSCs and Raw 264.7 cells (n = 5 independent experiments). C Detection of macrophage polarization levels via flow cytometry. D Quantitative analysis of the expression of dichlorodihydrofluorescein diacetate (DCF) in Raw 264.7 cells was detected by CLSM (n = 5 independent experiments). E DCF expression in Raw 264.7 cells was detected via CLSM. Scale bars: 10 μm. F Detection of DCF in Raw 264.7 cells by flow cytometry. Scale bars: 10 μm. G The expression of inducible nitric oxide synthase (iNOS) in Raw 264.7 cells was detected via CLSM. Scale bars: 10 μm. H Arginase-1 (Arg1) expression in Raw 264.7 cells was detected via CLSM. I Quantitative analysis of the expression of iNOS in Raw 264.7 cells was detected by CLSM (n = 5 independent experiments). J Quantitative analysis of the expression of Arg1 in Raw 264.7 cells was detected by CLSM (n = 5 independent experiments). K The GelMa/Mg/DMF MSs promote macrophage polarization levels and ROS scavenging and thereby orchestrate the IME. (created with BioRender.com) The data are presented as the means ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 4. Modulation of the pyroptosis-induced IME enhances osteoblast differentiation.

A TEM image of BMSCs after different treatments. Scale bars: 5 μm. B The expression of pyroptosis-related proteins in BMSCs was detected by Western blotting. C The expression of GSDMD in BMSCs was detected via CLSM. Scale bars: 50 μm. D Quantitative analysis of the expression of GSDMD in BMSCs was detected by CLSM (n = 5 independent experiments). E Changes in the levels of inflammatory factors within cell supernatants were detected via ELISA (n = 5 independent experiments). F ALP staining of BMSCs after 7 days and 14 days. Scale bars: 200 μm. G ARS staining of BMSCs after 14 days and 21 days. Scale bars: 200 μm. H The expression of osteogenic differentiation-related proteins in BMSCs was detected by Western blotting. I The GelMa/Mg/DMF MSs promoted osteogenic differentiation by targeting the N-GSDMD to inhibit pyroptosis in BMSCs and thereby orchestrated the IME. (created with BioRender.com) The data are presented as the means ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 5. Regulation of the pyroptosis-induced IME facilitates the phosphorylation of ERK in osteoclasts.

A The GelMa/Mg/DMF MSs coordinate bone homeostasis by inhibiting the pyroptosis of BMSCs, thereby suppressing the activity of BMDMs. (created with BioRender.com) B TRAP staining of BMDMs after 7 days. Scale bars: 50 μm. C Quantification of the number of nuclei per osteoclast (n = 5 independent experiments). D SEM images of BMDMs on the surface of bovine bone slices after 7 days of incubation. Scale bars: 20 μm. E Quantitative analysis of the surface resorbed area of SEM images of bovine bone slices (n = 5 independent experiments). F pH levels of the co-culture medium (n = 5 independent experiments). G The gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in BMDMs. H Gene set enrichment analysis of DEGs. I Protein expression in BMDMs was detected via Western blotting analysis. J Osteoclast ring formation and p-ERK expression in BMDMs were detected via CLSM. Scale bars: 100 μm. K GelMa/Mg/DMF MSs inhibit pyroptosis-induced IME promoting phosphorylation of ERK in osteoclasts. (created with BioRender.com) The data are presented as the means ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 6. In vivo GelMa/Mg/DMF MSs enhance osteoporosis treatment.

A Coronal and three-dimensional reconstructed images eight weeks after surgery for cranial defects viewed by micro-CT. Quantitative results of BMD B and bone volume-to-total volume (BV/TV) C in the cranial defect of C57 mice determined by micro-CT (n = 5 samples). D Cross-sectional image of the tibial observed via micro-CT. E Cross-sectional image of a representative spine viewed via micro-CT. Quantitative results of BMD in the tibial F and spine G of C57 mice determined by micro-CT (n = 5 samples). Quantitative results of the number of trabeculae (Tb.N) H and the degree of separation of trabeculae (Tb.Sp) I in the cranial defect of C57 mice determined by micro-CT (n = 5 samples). J Fluorescence microscope observation of a typical image of calcein and alizarin red staining. Scale bars: 200 μm. K Quantification of the mineralization rate (MAR) (n = 5 samples). L Compression experimental diagram. (created with BioRender.com) M Stress-strain curve of the femur. N Quantitative analysis of the modulus of elasticity of the femur of C57 mice (n = 5 samples). The data are presented as the means ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 7. GelMa/Mg/DMF MSs promotes tissue repair and reconstruction in osteoporosis by inhibiting pyroptosis and remodeling the IME.

Coronal images of the cranial defect after H&E staining A and Masson staining B 8 weeks after surgery. Scale bars: 100 μm. C Coronal images of the cranial defect after TRAP staining 8 weeks after surgery for the cranial defect. Scale bars: 100 μm and 20 μm. Coronal images of the cranial defect after immunohistochemistry for OCN D and GSDMD F 8 weeks after surgery for the cranial defect. Scale bars: 100 μm and 20 μm. Quantitative analysis of the percentage area of immunohistochemical staining for OCN E and GSDMD G 8 weeks after surgery for the cranial defect. (n = 5 samples). H Detection of inflammatory cytokines in the C57 serum at one week post-operation (n = 5 samples). I Images of cranial defects after immunofluorescence staining for Arg1⁺ (red) and iNOS⁺ (green). Scale bars: 100 μm and 20 μm. J Images of scalp tissue surrounding cranial defects after immunofluorescence staining for Arg1⁺ (red) and iNOS⁺ (green). Scale bars: 500 μm. K GelMa/Mg/DMF MSs inhibits pyroptosis and remodels the IME in osteoporosis. (created with BioRender.com) The data are presented as the means ± SD (n = 5 samples). Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 8. The GelMa/Mg/DMF MS exploited pyroptosis blockade and hydrogen-mediated inflammation regulation for osteoporosis treatment.

(created with BioRender.com).