β-Tricalcium phosphate (β-TCP) is commonly employed as a clinical artificial bone substitute. It has good biocompatibility and osteoconductivity (Putri et al., 2020). However, bone regeneration begins with inflammation (Li et al., 2020). Upon implantation, immune cells such as monocytes and macrophages are the first to encounter osteogenic materials. This interaction triggers an almost immediate inflammatory response, initiated by the recruitment and activation of numerous immune cells at the site of the bone defect (Rao et al., 2012, Cheung et al., 1997, Laquerriere et al., 2003). Macrophages carry out fundamental functions in this process, as biomaterials can influence their polarization (Chen et al., 2021). M1 and M2 macrophages strongly influence the microenvironment at the site of bone regeneration by secreting cytokines (Chen et al., 2021). M1 macrophages secrete inflammatory cytokines, such as interleukin (IL)-6, IL-1β, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) (Abdelmagid et al., 2015, Pathak et al., 2021, Li et al., 2017, Hu et al., 2023). However, persistent activation of M1 macrophages may cause chronic inflammation and tissue damage (Sica and Mantovani, 2012). M2 macrophages release anti-inflammatory cytokines, such as transforming growth factor-β (TGF-β), IL-4, and IL-10 (Pathak et al., 2021, Lee et al., 2020). They can decrease the inflammatory response and increase tissue healing (Sica and Mantovani, 2012). The M1 phenotype is more strongly associated with aseptic inflammation than the M2 phenotype (Rao et al., 2012), suggesting that osteogenic materials implanted into the organism may be more compatible with the host. β-TCP has been demonstrated to enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the regulation of macrophage polarization. Compared with β-TCP, β-TCP nanoparticles (NPs) have several advantages, including small size and surface area, rapid degradation, etc (Bohner et al., 2013).. It remains unclear whether β-TCP NPs alter macrophage polarization phenotype.

A substantial volume of research has demonstrated that mitochondria have a close involvement in regulating immune and inflammatory mechanisms (Xu et al., 2015, Thapa and Lee, 2019). Mitochondrial function is intimately linked to the macrophage immune response and its overall function, with macrophage polarization being mediated by various signaling pathways associated with mitochondrial metabolism (Xu et al., 2015, Thapa and Lee, 2019). Several studies found that mitochondrial dysfunction prevents repolarization of the M1 phenotype (Van den Bossche et al., 2016) and stimulates the release of the inflammatory cytokine IL-1β (Xu et al., 2022). It has also been demonstrated that mitochondrial metabolism acts as a key "controller" in determining macrophage phenotypic function during wound healing (Willenborg et al., 2021). Changes in mitochondrial metabolism affect the M1/M2 polarization phenotype of macrophages. TCP particles influence intracellular homeostasis by altering mitochondrial function (Yang et al., 2023). Nevertheless, the precise involvement of mitochondria in β-TCP NPs-induced macrophage inflammation and polarization remains elusive.

Here, we aimed to ascertain whether β-TCP NPs induce macrophage polarization and trigger inflammatory responses. Furthermore, the potential involvement of mitochondria in regulating polarization was also explored. Our results highlight the inflammatory risks associated with the use of β-TCP NPs in osteogenic applications, offering new insights and strategies for the improvement of these materials.