To explore the effects of artesunate on wear particle-induced inflammatory osteolysis explored by in vitro and in vivo experiments.

In vitro experiments: healthy mouse embryonic osteoblast precursor cells MC3T3-E1 cells were selected and divided into four groups for culture: blank control group; Ti particle group, where titanium particles were added for osteogenic differentiation culture; artesunate group, where 10 μmol/L artesunate ester was added for osteogenic differentiation culture; combination group, where titanium particles and 10 μmol/L artesunate ester were added simultaneously for osteogenic differentiation culture. Cell activity was determined using the cell counting kit-8 (CCK-8), while apoptosis was analyzed by flow cytometry. In vivo experiments: forty C57BL/6J mice were randomly divided into four groups using a random number table. In the Ti particle group and the Ti + artesunate group, the mice’s scalps were incised and titanium was implanted to establish a mouse skull bone resorption model. In the sham group and the artesunate group, the same surgical procedures were performed without implanting titanium. After establishing the artesunate group and the titanium plus artesunate group, artesunate was administered by gavage at a dose of 50 mg/(kg·d). The mice were executed after two weeks, and hematoxylin-eosin method (HE) staining was used to observe the pathological changes of the skull; enzyme-linked immunosorbent assay (ELISA) was performed to measure the expression levels of inflammatory factors, tumor necrosis factor (TNF) α, interleukin (IL)-1β, and IL -6. Real-time quantitative polymerase chain reaction (RT-PCR) was used to assess the expression of osteogenic differentiation-related genes including alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (Runx-2), and collagen type-Ⅰ (COL I). Two weeks after dosing, Micro-CT of the skull was conducted. For comparisons between groups of measurement data, independent samples t test or one-way analysis of variance was used; for multiple comparisons, LSD test was used; and for comparisons between groups of count data, chi square test was employed.

In vitro experiments: CCK8 results showed that at artesunate concentrations ≤10 μmol/L, there was no significant effect on osteoblast proliferation. Titanium particle concentrations≤0.5 mg/ml did not significantly affect the proliferation of MC3T3-E1 cells. Flow cytometry results showed that titanium particles promoted apoptosis in MC3T3-E1 cells (t=57.46, P<0.001); artesunate inhibited the effect of titanium particles (t=19.64, P<0.01). In vivo experiments: HE staining showed that the number of inflammatory cells and the degree of bone destruction in the peri-cranial tissues of the Ti granules group were significantly higher than those of the Ti+artesunate, artesunate, and Sham groups; the inflammatory response was reduced and bone destruction was significantly improved after artesunate intervention. ELISA and PCR results showed that compared to sham group, the Ti particles stimulated a significant increase in the concentration of the three inflammatory factors (t=8.872, 15.6, 18.71, all P<0.05) and a decrease in the mRNA expression of osteogenesis-related genes (t=18.31, 20.47, 23.95, 27.22, all P<0.05). In contrast, compared with the Ti particles group, the Ti + artesunate group showed different degrees of down-regulation of the expression of the three inflammatory factors (t=4.672, 4.805, 3.405, all P<0.01) and increased expression of osteogenesis-related genes (t=12.2, 15.15, 22.02, 16.99, all P<0.05). Micro-CT scans showed that titanium particles led to an increase in bone destruction and induced osteolysisaround the skull, and that the titanium particle-induced osteolysis could be mitigated by the intervention of artesunate.

Artesunate can improve titanium particles induced osteolysis and bone destruction by inhibiting inflammatory factors and up-regulating the function of osteoblasts.