DataSheet_1_Single-Cell RNA Sequencing Reveals B Cells Are Important Regulators in Fracture Healing.docx

The bone marrow microenvironment is composed primarily of immune and stromal cells that play important roles in fracture healing. Although immune cells have been identified in mouse bone marrow, variations in their numbers and type during the fracture healing process remain poorly defined. In this study, single-cell RNA sequencing was used to identify immune cells in fracture tissues, including neutrophils, monocytes, T cells, B cells, and plasma cells. The number of B cells decreased significantly in the early stage of fracture healing. Furthermore, B cells in mice fracture models decreased significantly during the epiphyseal phase and then gradually returned to normal during the epiphyseal transformation phase of fracture healing. The B-cell pattern was opposite to that of bone formation and resorption activities. Notably, B-cell–derived exosomes inhibited bone homeostasis in fracture healing. In humans, a decrease in the number of B cells during the epiphyseal phase stimulated fracture healing. Then, as the numbers of osteoblasts increased during the callus reconstruction stage, the number of B cells gradually recovered, which reduced additional bone regeneration. Thus, B cells are key regulators of fracture healing and inhibit excessive bone regeneration by producing multiple osteoblast inhibitors.

骨髓微环境（bone marrow microenvironment）主要由免疫细胞与基质细胞（stromal cells）构成，在骨折愈合过程中发挥关键作用。尽管已有研究在小鼠骨髓中鉴定出免疫细胞，但骨折愈合进程中免疫细胞的数量与类型变化仍未得到明确阐释。本研究采用单细胞RNA测序（single-cell RNA sequencing）技术对骨折组织中的免疫细胞进行鉴定，所涉细胞类型包括中性粒细胞（neutrophils）、单核细胞（monocytes）、T细胞、B细胞及浆细胞（plasma cells）。研究发现，骨折愈合早期阶段，B细胞数量显著降低。进一步分析显示，小鼠骨折模型中的B细胞在骨折愈合的骨骺阶段大幅减少，随后在骨折愈合的骨骺转化阶段逐渐恢复至正常水平。B细胞的动态变化模式与骨形成及骨吸收活动恰好相反。值得注意的是，B细胞衍生的外泌体（exosomes）可抑制骨折愈合过程中的骨稳态。在人体中，骨骺阶段B细胞数量减少可促进骨折愈合；而在骨痂重建阶段，随着成骨细胞（osteoblasts）数量增多，B细胞数量亦逐渐恢复，进而抑制过度骨再生。综上，B细胞是骨折愈合的核心调控因子，可通过分泌多种成骨细胞抑制剂来阻遏过度骨再生。