Periodontitis is a prevalent chronic inflammatory disease characterized by the progressive destruction of tooth-supporting tissues, including the alveolar bone, periodontal ligament, and gingiva(Sanz et al., 2020). The pathogenesis of periodontitis is multifactorial, involving dysbiosis of the subgingival microbiota, host immune dysregulation, and environmental risk factors such as poor oral hygiene, tobacco use, diabetes, and genetic predisposition . A hallmark of the disease is the excessive host inflammatory response to microbial challenge, leading to the release of pro-inflammatory cytokines [e.g., interleukin (IL)-1β, tumor necrosis factor (TNF)-α] and matrix metalloproteinases (MMPs), which collectively promote extracellular matrix degradation and alveolar bone resorption(Abusleme et al., 2021; Xie et al., 2024). Current therapeutic strategies, including mechanical debridement, adjunctive antibiotics, and surgical interventions, aim to control infection and arrest disease progression(Kwon et al., 2021). However, these approaches often fail to fully regenerate lost periodontal tissues due to persistent microbial recolonization, unresolved chronic inflammation, and the irreversible nature of bone and ligament destruction.

A critical factor in periodontitis progression is the imbalance between osteogenic differentiation and inflammatory signaling(Cao et al., 2023; Cong et al., 2024). Under physiological conditions, mesenchymal stem cells (MSCs) differentiate into osteoblasts to maintain alveolar bone homeostasis(Larrouture et al., 2021). However, the inflammatory microenvironment in periodontitis disrupts this process by upregulating osteoclastogenic mediators (e.g., RANKL) while suppressing key osteogenic transcription factors such as Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN)(Jin et al., 2020; Pan et al., 2024). Pro-inflammatory cytokines further exacerbate bone loss by inducing oxidative stress, activating nuclear factor (NF)-κB signaling, and enhancing extracellular matrix degradation via MMPs(Hashim et al., 2025). This dysregulation creates a vicious cycle in which unresolved osteolytic activity perpetuates chronic inflammation, further impairing tissue regeneration. Thus, understanding the interplay between inflammation and osteogenic differentiation is essential for developing novel therapeutic strategies to restore periodontal tissue integrity. In this context, exosome-mediated intercellular communication has emerged as a potential key mechanism linking immune dysregulation and impaired osteogenesis in periodontitis.

Exosomes, nanosized extracellular vesicles (30–150 nm) secreted by virtually all cell types, play a pivotal role in intercellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids [e.g., microRNAs (miRNAs), mRNAs](Ahmad et al., 2025; Miron et al., 2024). Their role in inflammatory diseases is context-dependent, as exosomes can either propagate or mitigate inflammation depending on their cellular origin and cargo composition. In periodontitis, exosomes from various cell sources, including periodontal ligament stem cells and neutrophil-like cells, have been shown to regulate osteoclast differentiation and inflammation by delivering miRNAs(Kang et al., 2023; Zhang et al., 2023).

Macrophages, as key regulators of innate immunity, exhibit remarkable plasticity and play a central role in maintaining tissue homeostasis and modulating inflammatory responses(Varol et al., 2015). Their polarization into the alternatively activated M2 phenotype is crucial for inflammation resolution and tissue repair. For instance, gingival tissue-derived MSCs have been reported to suppress osteoclastogenesis and inflammation by promoting M2 macrophage polarization(Nakao et al., 2021). These findings highlight the therapeutic potential of modulating macrophage polarization in inflammatory diseases, including periodontitis. While the regenerative potential of MSCs-derived exosomes is increasingly recognized, the specific role of exosomes derived from M2-polarized macrophages (M2-exos) in modulating the osteogenic and inflammatory responses of human periodontal ligament stem cells (hPDLSCs) remains inadequately explored. Moreover, the key exosomal miRNAs responsible for these effects and their specific downstream targets in the context of periodontitis are largely unknown.

Calcium/calmodulin-dependent protein kinase II delta (CaMK2D) is a type of important signal transduction protein, which is widely involved in the intracellular calcium ion signal transmission process and regulates cell proliferation, apoptosis, and inflammatory responses(Rigter et al., 2024). The role of CAMK2D in inflammatory diseases has gradually been revealed. Studies have found that CAMK2D is expressed at higher levels in ulcerative colitis and can also serve as a biomarker for predicting treatment responses(Ye et al., 2023). However, the role of CAMK2D in periodontitis has not been reported yet.

Therefore, this study aimed to investigate the role of M2-exos in modulating the inflammatory response and osteogenic differentiation of hPDLSCs, with a specific focus on identifying the key functional exosomal miRNA and exploring its novel target, CAMK2D.