Abstract: BACKGROUND: The pathogenesis of osteoporosis is closely related to the immune system. A comprehensive and in-depth study of the relationship between immunity and osteoporosis is crucial for understanding and treating the disease. 
OBJECTIVE: To investigate the role of immune cells in osteoporosis using single-cell sequencing technology.
METHODS: Femoral head tissue samples from osteoporosis and non-osteoporosis patients were downloaded from GEO database and analyzed using single-cell sequencing. Data analysis, including cell clustering, functional enrichment, pseudotime trajectory, and cell interaction analyses, was performed using R4.3.0 and software packages such as Seurat v.4.3, monocle (2.28.0), and CellChat. The femoral head tissues of patients with femoral neck fracture who underwent artificial hip replacement surgery were obtained, including two cases of osteoporosis patients and two cases of non-osteoporosis patients. Immunohistochemical staining was used to detect the protein expression of CCL13 and CCL18. qPCR was used to detect the immunoglobulin heavy constant γ-4, immunoglobulin λ constant 3, human class II major histocompatibility complex DRβ1, and CD83 mRNA expression. Western blot was used to detect the protein expression of receptor-type tyrosine protein phosphatase C, CD22, and CD99.
RESULTS AND CONCLUSION: Transcriptomic analysis identified 10 cell clusters, including osteoclasts, myeloid cells, T cells, osteoblasts, macrophages, monocytes, erythrocytes, B cells, bone marrow mesenchymal stem cells, and mast cells. There was an increase in the ratio of osteoclasts to T cells and a decrease in the ratio of osteoblasts to B cells in the femoral head tissue of the osteoporosis group. Among the B-cell subpopulations, the proportion of B-cells of taxa 1,3 (BC1, BC3) in the femoral head tissue of the osteoporosis group was higher than that of the non-osteoporosis group, and the proportion of B-cells of taxa 2 (BC2) was less than that of the non-osteoporosis group. BC1 was enriched significantly for labels such as regulation of adaptive immune response, somatic recombination of immune receptors, and modulation of lymphocyte-mediated immunity, while BC3 was enriched significantly for labels such as regulation of immunoglobulin production, response to type II interferon, apoptotic processes involving cysteine endopeptidases, and cytotoxicity. The communication intensity between B-cell subtype BC1 and osteoblasts in the femoral head tissue of the osteoporosis group was higher than that of the non-osteoporosis group, while the communication intensity between BC3 and BC1 was also increased. The communication between BC3 and BC1 was significantly enriched in the CD22-receptor-type tyrosine protein phosphatase C pathway; the communication between BC1 and osteoblasts was mainly enriched in the CD99-CD99 pathway; and the communication between BC3 and osteoblasts was also highly enriched in the CD99-CD99 pathway. Protein expression of CCL13, CCL18, receptor-type tyrosine protein phosphatase C, CD22, CD99, immunoglobulin heavy constant γ-4, immunoglobulin λ constant 3, human class II major histocompatibility complex DRβ1, and CD83 mRNA were higher in femoral tissues of the osteoporosis group than those of the non-osteoporosis group (P < 0.05). To conclude, specific B cell subpopulations can influence the differentiation and apoptosis of osteoblasts in the femoral tissue of osteoporosis patients.

Key words: single-cell sequencing, osteoporosis, B cells, osteoblasts, cellular interactions, engineered tissue construction