Size-Dependent Layered Double Hydroxide Nanoparticles Promote Osteogenesis and Bone Regeneration via METTL3-Dependent N6-Methyladenosine Modification of Runx2 mRNA

  • Int J Nanomedicine. 2026 Jun 15:21:611081. doi: 10.2147/IJN.S611081.
Yang Zhu  #  1 ,  Weiwei Sun  #  1 ,  Yaoyu Huang  1 ,  Jianxin Li  1 ,  Haozhe Jiang  1 ,  Chao Luo  1 ,  Fuyin Wan  1 ,  Zhenyu Zhou  1
Affiliations
  • 1. Department of Orthopaedics, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, Jiangsu, People's Republic of China.
  • # Contributed equally.
PMID: 42325402 DOI: 10.2147/IJN.S611081
Abstract

Background: The osteogenic efficacy of mesenchymal stem cell (MSC)-based bone regeneration is often limited by insufficient osteogenic differentiation. Layered double hydroxide (LDH) nanoparticles are promising biomaterials, but whether their intrinsic osteoinductive activity is regulated by particle size and epitranscriptomic mechanisms remains unclear. This study investigated whether LDH nanoparticles promote osteogenesis in bone marrow-derived MSCs (BMSCs) through METTL3-dependent N6-methyladenosine (m6A) modification.

Methods: BMSCs were treated with 50 nm or 100 nm LDH nanoparticles under osteogenic induction. Osteogenic differentiation was evaluated by Alkaline Phosphatase activity, mineralization staining, osteogenic gene/protein expression, and cytoskeletal morphology. Global m6A levels and m6A regulator expression were assessed, and the role of METTL3 was examined using METTL3 knockdown. RUNX2 mRNA m6A enrichment and stability were analyzed by MeRIP-qPCR and actinomycin D chase assays. Bone regeneration was further evaluated using a GelMA-LDH hydrogel in a murine calvarial defect model.

Results: LDH nanoparticles promoted BMSC osteogenesis in a size-dependent manner, with 100 nm LDH producing stronger ALP activity, mineralization, and osteogenic marker expression than 50 nm LDH. Mechanistically, 100 nm LDH increased global m6A methylation and selectively upregulated METTL3. METTL3 knockdown markedly impaired osteogenesis and abolished the pro-osteogenic effects of LDH. LDH enhanced m6A modification of RUNX2 mRNA and prolonged RUNX2 transcript stability, thereby supporting RUNX2-mediated osteogenic programming. In vivo, GelMA-LDH implantation significantly enhanced calvarial bone repair and increased RUNX2 and METTL3 expression within defect regions.

Conclusion: Among the two tested particle sizes, 100 nm LDH nanoparticles exhibited superior pro-osteogenic activity and promoted bone regeneration through a METTL3-dependent m6A mechanism that stabilizes RUNX2 mRNA.

Keywords
METTL3; layered double hydroxide; m6A RNA methylation; osteogenic differentiation; size-dependent.
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