Unbiased gene expression analysis of the delayed fracture healing observed in Zucker diabetic fatty rats

Aims Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully under-stood. In this study, we aimed to characterize the local changes in gene expression (GE) as-sociated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild -type (WT) littermates at three weeks following femoral osteotomy. Methods Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high -fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analy-sis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq).Results Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incom-plete bony bridge formation and an influx of intramedullary inflammatory tissue. In compar-ison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA- Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation.Conclusion These findings suggest that elevated or prolonged inflammation contributes to delayed frac -ture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients.