Abstract: BACKGROUND: Cystic degeneration is a critical pathological feature of steroid-induced osteonecrosis of the femoral head and exhibits unusual reparative responses. Studies have shown that immune cell infiltration and upregulation of inflammatory cytokines in cystic areas may be closely related to bone regeneration during repair. Therefore, investigating the role of the immune microenvironment in cystic lesion repair has important clinical significance.
OBJECTIVE: To explore the immune microenvironment in cystic degeneration of steroid-induced osteonecrosis of the femoral head based on single-cell sequencing and to elucidate the role of inflammation in the repair process as well as to validate the effects of glucocorticoids on macrophage polarization and inflammatory responses via in vitro cellular experiments.
METHODS: (1) Single-cell RNA sequencing was used to analyze the immune microenvironment of cystic regions in steroid-induced osteonecrosis of the femoral head. Pathologic observation of cystic degeneration specimens was also performed. (2) In addition, an in vitro cell model was established. In vitro cultured RAW 264.7 macrophages were divided into control group (normal culture of RAW cells), lipopolysaccharide group (lipopolysaccharide intervention for 24 hours), and dexamethasone+lipopolysaccharide group (dexamethasone pretreatment for 72 hours and lipopolysaccharide intervention for 24 hours). RT-qPCR was used to detect the expression of cellular M1 polarization markers (inducible nitric oxide synthase, tumor necrosis factor α, and interleukin 6) and M2 polarization markers (arginase 1 and interleukin 10). ELISA was used to detect the levels of inflammatory factors in the cell culture supernatants. Flow cytometric assay was performed to detect the levels of cellular reactive oxygen species.
RESULTS AND CONCLUSION: (1) Single-cell sequencing results revealed a diverse infiltration of immune cells in the cystic region, with significant upregulation of inflammatory cytokines such as tumor necrosis factor α and interleukin 6. (2) Observation of the pathologic specimen showed the presence of multiple inflammatory cell infiltrates in the cystic degeneration. (3) In vitro experiments demonstrated that combined treatment with dexamethasone and lipopolysaccharide significantly induced M1 polarization of RAW 264.7 macrophages, inhibited the expression of anti-inflammatory M2 phenotype and promoted the generation of reactive oxygen species, thereby enhancing the local inflammatory response (significant upregulation of tumor necrosis factor α, interleukin 6 and interleukin 1β). Overall, the immune microenvironment in the cystic regions of steroid-induced osteonecrosis of the femoral head plays a critical role in bone repair. Immune cell infiltration and inflammatory cytokine expression may exert dual effects during the reparative process. Excessive immune activation may lead to repair failure. Thus, modulating immune responses—especially balancing M1/M2 macrophage polarization—may serve as a potential therapeutic strategy for enhancing cystic repair in steroid-induced osteonecrosis of the femoral head.

Key words: steroid-induced osteonecrosis of the femoral head, cystic degeneration, repair, inflammation, single-cell sequencing