Changjiang Liu,1,2,* Yifeng Yu,3,* Liang Tian,1,2 Yuting Liu,1,2 Dong Zhang,1,2 Aixi Yu1,2 

1Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071, People’s Republic of China; 2Hubei Clinical Medical Research Center of Trauma and Microsurgery, Wuhan, Hubei, 430071, People’s Republic of China; 3Department of Orthopedics, Renmin Hospital, Wuhan University, Wuhan, Hubei, 430071, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Dong Zhang, Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071, People’s Republic of China, Email zhangdongemail@whu.edu.cn Aixi Yu, Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071, People’s Republic of China, Email yuaixi@whu.edu.cn

Background: Fracture healing can be delayed or impaired in individuals with abnormal conditions, including Type 2 Diabetes Mellitus (T2DM). Mesenchymal stem cells (MSCs) are critical to the process of fracture healing and are found to be impaired in T2DM. Although some research has been conducted to address this, the specific mechanisms remain poorly understood and warrant further exploration.
Methods: We downloaded transcriptomic and single-cell RNA sequencing (scRNA-seq) data, performed multiple analyses (differential expression, ssGSEA, co-expression, GO, KEGG, GSEA, and cell clustering identification), and utilized tools (GeneMINIA and Metascape) to investigate alterations in MSCs under diabetic condition. Further validation and exploration were carried out through in vitro experiments (cell transduction, flow cytometry, ALP staining, ARS, qPCR, and Western blotting) and in vivo experiments (micro-CT, histological staining, and immunohistochemistry).
Results: Our study identified differentially expressed genes from fracture healing and non-union cases in human samples, suggesting abnormal immune infiltration and disrupted biological processes. ScRNA-seq analysis further revealed significant alterations in MSCs under diabetic conditions with enriched pathways, including MAPK, TGF-β, and P53 signaling pathways. Integrating with transcriptomic analysis, we identified Rbms3, which was significantly upregulated in diabetic MSCs and further validated in bone samples from patients at our institution. The upregulation of Rbms3 impaired fracture healing by modulating the MAPK signaling pathway, leading to reduced MSC osteogenic differentiation in vitro and impaired bone regeneration in vivo.
Conclusion: The upregulation of Rbms3 in MSCs under diabetic conditions contributes to impaired fracture healing by modulating the MAPK signaling pathway.

Keywords: bioinformatics, single-cell RNA-sequencing analysis, T2DM, fracture non-union, bone healing