NF-kB Inhibition in Mesenchymal Stem Cells Improves Diabetic Fracture Healing

Objectives: Diabetes leads to persistent inflammation and decreases the number of mesenchymal stem cells (MSCs) that participate in fracture healing. We therefore tested the hypothesis that activation of inflammatory NF-kappaB pathway in MSCs is responsible for delayed fracture healing in diabetes.
Methods: Cre recombinase was expressed under the promoter control of MSC-specific gene Prrx1 in transgenic mice that express IKK-beta^loxp/loxp, leading to a functional knockdown of NF-kB in MSCs. Littermate control mice carried IKK-beta^loxp/loxp. Type I diabetes was induced with low-dose streptozotocin injection, and normoglycemic controls received citrate buffer. Femoral fracture was induced, and the animals were euthanized at day 6 and 14 post-fracture, the time-points for active cartilage and bone formation. Paraffin-sections were prepared and stained, and new cartilage and bone formation was assessed. Specific antibody to Sca1 was used to immunostain and quantify MSC numbers. Comparison was made between control normoglycemic and diabetic animals, and transgenic and control littermates (n=5-9/each). Statistical significance was determined at p<0.05 by two-way ANOVA and post-hoc test.
Results: Diabetes significantly reduced callus and cartilage formation during fracture healing at day 6 by 39% and 58% compared to normoglycemic controls, respectively (p<0.05). In transgenic mice, the diabetes-mediated reduction in callus and cartilage size was significantly less such that the parameters measured were similar to normoglycemic controls (p>0.05). At 14 days, diabetes decreased the callus and bone formation by 36% and 47% respectively, compared to controls (p<0.05). Transgenic diabetic mice showed an increased callus and bone size by 33% and 50% respectively when compared to its diabetic control littermates (p<0.05). Diabetes also decreased MSC numbers by 57% compared to normoglycemic controls (p<0.05), which was rescued in transgenic diabetic mice (p<0.05).
Conclusions: Diabetes significantly reduced the amount of cartilage and bone formation 6-day and 14-day post fracture, respectively. IKK-beta knockdown in MSCs significantly improved the cartilage and bone formation, as well as MSC numbers in diabetic fracture healing. Thus, diabetes-mediated activation of NF-kB in MSCs has a negative impact on regenerative tissue formation.