The role of medium-rough implant surface topography on macrophages polarization in age-independent manner.

backgroundAging is well recognized to impact the immunological and metabolic processes, including aging-related reduction in macrophage function. We previously shown the titanium implant surface topography influences macrophage polarization and osteogenic differentiation of mesenchymal stromal cells. However, the impact of implant surface roughness of the polarization of aged-macrophages is currently unknown. The study aims to investigate aged-macrophage behavior on medium-roughness titanium and its subsequent effect on the osteogenesis of periodontal ligament mesenchymal stromal cells (PDL MSCs).

methodsMurine-bone barrow-derived macrophages (BMDMs) were obtained from the bone marrow cells of the femur and tibia of male BALB/c mice aged 8 weeks (young) and 18 months (aged) and were distributed into 4 groups (n = 6) of untreated BMDMs obtained from young mice (YNT), aged mice (ANT), titanium treated BMDMs obtained from young mice (YT), and aged mice (AT). Ten disk-shaped specimens were distributed into YT and AT groups (n = 4). After 48 h of culture, macrophage adhesion and polarization were evaluated by immunofluorescence microscopy, gene expression profiling, and flow cytometry. Cytokine secretion (IL-1β and IL-10) was quantified in culture supernatants using ELISA. The conditioned medium (CM) collected from each macrophage group was subsequently applied to periodontal ligament mesenchymal stromal cells (PDL-MSCs) to evaluate proliferation (MTT assay) and osteogenic differentiation (Alizarin Red staining and osteogenic gene expression). Data were analyzed using the Kruskal-Wallis test with Dunn’s post hoc test, or one-way ANOVA with Bonferroni post hoc test, as appropriate.

resultsGene expression analysis of M1 and M2 demonstrated no significant differences across different age groups. However, treatment with titanium disk in both young and aged BMDMs significantly increased the expression of TBF-β, VEGF, and IL-10. Consistently, ELISA measurements showed no significant differences in IL-1β levels among groups, whereas IL-10 concentrations were significantly higher in YNT compared with AT. Flow cytometry results indicate differences in the proportions of CD86⁺ and CD163⁺ cells across different age groups and treatment conditions. The PDL MSCs proliferation was affected by conditioned medium of both young and aged BMDMs treated with medium roughness of titanium disk at 48 and 72 h of culture. Furthermore, PDL MSCs osteogenic differentiation was affected by BMDMs conditioned medium (CM) compared to control. Cell morphology observation showed slight difference in cell adhesion behavior. However, cell aspect ratio analysis indicates no significant difference among all groups.

conclusionsExposure to medium-roughness titanium promoted polarization toward the M2 phenotype in both young and aged macrophages and improved PDL MSCs osteogenesis. While this effect was observed across age groups, subtle differences in gene expression and surface marker distribution suggest age-related variability in the magnitude of the response.