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

Abstract

Aims: Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated 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 analysis. 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 incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, 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 fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients.

Fig. 1

A representative radiograph showing fracture healing of the type 2 diabetes mellitus group compared to non-diabetic controls three weeks post-femoral osteotomy, with external fixators in situ.

Fig. 2

Toluidine blue histology of whole femora of control (A) and Zucker diabetic fatty (B) rats three weeks post-fracture fixation. A´ and B´ show respective magnified regions indicated by black rectangles. Osteotomy sites are indicated by black triangles. The black arrow indicates the location of the fracture site. The black bars in A and B represent a length of 5 mm, while in A´ and B´ they represent 250 µm.

Fig. 3

Volcano plot with significant genes shown in red (FDR < 0.05). Genes to a FDR of 10.0% are additionally labelled in grey. FDR, false discovery rate; Mmp, matrix metallopeptidase; Pnpla2, Patatin-like phospholipase domain-containing protein 2.