Magnesium Nanocomposite Hydrogel Reverses the Pathologies to Enhance Mandible Regeneration

Abstract

The healing of bone defects after debridement in medication-related osteonecrosis of the jaw (MRONJ) is a challenging medical condition with impaired angiogenesis, susceptible infection, and pro-inflammatory responses. Magnesium (Mg) nanocomposite hydrogel is developed to specifically tackle multiple factors involved in MRONJ. Mg-oxide nanoparticles tune the gelation kinetics in the reaction between N-hydroxysuccinimide-functionalized hyperbranched poly (ethylene glycol) and proteins. This reaction allows an enhanced mechanical property after instant solidification and, more importantly, also stable gelation in challenging environments such as wet and hemorrhagic conditions. The synthesized hydrogel guides mandible regeneration in MRONJ rats by triggering the formation of type H vessels, activating Osterix+ osteoprogenitor cells, and generating anti-inflammatory microenvironments. Additionally, this approach demonstrates its ability to suppress infection by inhibiting specific pathogens while strengthening stress tolerance in the affected alveolar bone. Furthermore, the enhanced osteogenic properties and feasibility of implantation of the hydrogel are validated in mandible defect and iliac crest defect created in minipigs, respectively. Collectively, this study offers an injectable and innovative bone substitute to enhance mandible defect healing by tackling multiple detrimental pathologies.

Keywords: bone regeneration; hydrogel; magnesium; nanoparticle; osteonecrosis.

Figure 1

Design, fabrication, and controlled gelation of PBM hydrogels. A) Schematic illustration of crosslinking mechanism and design of PBM hydrogel. B) The gross‐view of the hydrogels and the internal microstructure observed by SEM. C) Photographs and D) quantification of the controllable gelation behavior tuned by MgO NPs (n = 5). PBR(pH = m) hydrogels were defined as hydrogels formed under a pH = m condition of hydrogel precursor B. PBM(n) hydrogels referred to hydrogels formed with the addition of n mg/mL MgO NPs in precursor B.

Figure 2

Optimized physiochemical properties and cytocompatibility of hydrogels by MgO NPs. A) The illustration depicted the design of PBM hydrogels functioning in hemorrhage. B) Photographs and C) quantification of the gelation behaviors of hydrogels within blood using the vial tilting method (n = 3). D) Weight loss and E) cumulative concentration of Mg²⁺ released from hydrogels during in vitro degradation tests (n = 3). F) Young's modulus (n = 4). G) Storage modulus (G′) and loss modulus (G′′) through strain and frequency sweep tests. H) Quantitative analysis of cell adhesion and viability (n = 5). I) Live (green) /dead (red) staining of MMSCs cultured with hydrogel systems. *p < 0.05, **p < 0.01, and ***p < 0.001, by one‐way ANOVA with Tukey's post hoc test.

Figure 3

Osteo‐ and angio‐promotive effects of PBM hydrogels in vitro. A) Quantitative osteogenic related‐gene expression of MMSCs determined by qPCR on Day 5 and 14 (n = 5). B, C) Representative images and quantification of fluorescent intensity (n = 5). D) ALP and alizarin red S (ARS) staining. E, F) Migration (scratch test; n = 5) and (G‐H) tube formation assay (n = 7‐8). *p < 0.05, **p < 0.01, and ***p < 0.001, by two‐way (A) or one‐way (C, F, H) ANOVA with Tukey's post hoc test.

Figure 4

PBM hydrogels augmented mandible bone regeneration in MRONJ. A, B) Schematic diagram and surgical photographs of MRONJ model establishment, and surgical procedures involving dental extraction, mandible defect creation, and implant implantation. C) Representative radiographs, D) Quantitative micro‐CT measurements (n = 6), E) 3D reconstructed images of mandible defects at 4, 8, and 12 weeks. F) Representative H&E staining of mandible defect, showing the histological morphology of newly‐formed bone (NB), old bone (OB), and the defect (Def). *p < 0.05 and **p < 0.01, by two‐way ANOVA with Tukey's post hoc test.

Figure 5

PBM hydrogel enhanced angiogenesis and stem cell recruitment in mandible regeneration. A–C) IHC staining and quantitative analysis on the expression of Osterix and Ocn (n = 6). D, E) IF staining and quantification of type H vessels (CD31+ Emcn+) at 4 weeks (n = 6). F, G) IF staining and quantification of Osterix+ osteoprogenitor cells and Ecmn+ vessels at 4 weeks (n = 6). H) Representative 3D reconstructed images of micro‐CT angiography, and quantification of vessel volume fraction (Vessel V/TV), vessel number (Vessel.N), and vessel thickness (Vessel.Th) through different treatments at 8 weeks (n = 3). I, J) IF staining and quantification of Postn+ area and active CD105+ stem cells in the newly‐formed bone at 4 weeks (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001, by two‐way (B) or one‐way (E, G, H, J) ANOVA with Tukey's post hoc test.

Figure 6

Underlying mechanisms uncovered by bulk‐RNA sequencing. A) Unsupervised hierarchical clustering of transcriptomes in different groups. B) Volcano plots of the upregulated and downregulated genes between Ctrl, PBR, and PBM group (False discovery rate < 0.05 and Fold change > 2). Gene ontology biological process enrichment of genes C) upregulated and D) downregulated in PBM group than Ctrl group; Gene ontology biological process enrichment of genes E) upregulated and F) downregulated in PBM than PBR group (Q value < 0.05).

Figure 7

The microbial changes in the defect area following PBM hydrogel treatment. A) Schematic diagram of sampling microbes from the alveolar‐mandible defect exposed to the oral cavity. B) Venn diagram of the unique and common amplicon sequence variants (ASVs) identified in different groups. C) PCoA and NMDS plots of the variation in microbial community composition. D) Histogram of species abundance and annotation in phylum level (n = 6). E) Heat map of species abundance and annotation in genus level (n = 6). F) Bacterial phenotype prediction (n = 4‐6). *p < 0.05, **p < 0.01, and ***p < 0.001, by one‐way ANOVA with Tukey's post hoc test.

Figure 8

Feasibility and efficacy of PBM hydrogel treatment in mandible and iliac crest defects of minipig. Surgical procedures of the treatment to A) mandible defect and B) iliac crest defects and hydrogel delivery. C) In vivo CT scan and D) quantification of defect gap at 2 and 4 weeks (n = 3). E) Representative H&E staining, IHC staining of Osterix, IF staining of Type H vessels in defect sites. F) Quantification of percentage of Osterix+ cells and area fraction of type H vessels in different treatment groups (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001, by two‐way ANOVA with Tukey's post hoc test (D) and unpaired two‐tailed Student's t‐test (F).