308 Targeting Cdk8 to improve Ischemic Fracture Healing

Abstract

OBJECTIVES/GOALS: There are178 million bone fractures globally each year, and 46% of fractures that have accompanying vascular damage (ischemia) will not heal without surgical intervention. Using single cell RNAseq we identified Cdk8 as a gene upregulated under ischemic fracture. Our work seeks to inhibit Cdk8 to assess its potential as a therapeutic target. METHODS/STUDY POPULATION: Most bone injuries heal through a cartilage intermediate that requires mesenchymal progenitor cells (hMSCs) to become cartilage forming chondrocytes. hMSCs underwent pelleted 3D chondrogenic differentiation in the presence of Cdk8 inhibitor, Senexin B. Chondrogenic gene expression was assessed via gene analysis of Aggrecan, Collagen II, and Collagen X. Content of sulfated glycosaminoglycans (sGAGs) was quantified through DMMB analysis. With IACUC approval, C57Bl/6 WT mice underwent femoral artery isolation and resection to create an ischemic environment prior to a transverse tibia fracture. Mice underwent intraperitoneal injections of Senexin B from day -1 to 8 and were harvested at 10 days post fracture. Control mice received vehicle injection. Calluses were analyzed through µCT and histomorphometry. RESULTS/ANTICIPATED RESULTS: At 14 days, Senexin B increased chondrogenic gene expression and improved sGAG content in hMSCs. This persisted to day 21, suggesting that Cdk8 inhibition via Senexin B promotes chondrogenesis and matrix deposition. Histomorphometric analysis reveals thatin vivotreatment with Senexin B increases cartilage content and reduces mineralization of the fracture callus compared to the Control. µCT analysis corroborates this, with distinctly less peri-cortical mineral present in Senexin B-treated calluses, and a decrease in total bone volume. These results suggest an altered progression of cartilage formation and endochondral ossification with Cdk8 inhibition. DISCUSSION/SIGNIFICANCE: Our findings reveal that increased Cdk8 is associated with poor healing in ischemic fractures. Inhibition of Cdk8 appears to increase chondrogenesis of hMSCsin vitroand in the murine fracture callusin vivo. Targeting Cdk8 offers potential to improve callus formation in impaired healing scenarios.