Transcriptional profiling of intramembranous and endochondral ossification after fracture in mice

Bone fracture repair represents an important clinical challenge with nearly 1 million non-union fractures occurring annually in the U.S. Gene expression differs between non-union and healthy repair, suggesting there is a pattern of gene expression that is indicative of optimal repair. Despite this, the gene expression profile of fracture repair remains incompletely understood. In this work, we used RNA-seq of two well-established murine fracture models to describe gene expression of intramembranous and endochondral bone formation. We used top differentially expressed genes, enriched gene ontology terms and pathways, callus cellular phenotyping, and histology to describe and contrast these bone formation processes across time. Intramembranous repair, as modeled by ulnar stress fracture, and endochondral repair, as modeled by femur full fracture, exhibited vastly different transcriptional profiles throughout repair. Stress fracture healing had enriched differentially expressed genes associated with bone repair and osteoblasts, highlighting the strong osteogenic repair process of this model. Interestingly, the PI3K-Akt signaling pathway was one of only a few pathways uniquely enriched in stress fracture repair. Full fracture repair involved a higher level of inflammatory and immune cell related genes than did stress fracture repair. Full fracture repair also differed from stress fracture in a robust downregulation of ion channel genes following injury, the role of which in fracture repair is unclear. This study offers a broad description of gene expression in intramembranous and endochondral ossification across several time points throughout repair and suggests several potentially intriguing genes, pathways, and cells whose role in fracture repair requires further study Overall design: Following either a femur full fracture or ulnar stress fracture injury 70 female C57BL/6J mice were sacrificed at 4?h, 1, 3, 5, 7, or 14?days post-injury, and processed for RNA-seq. RNA-seq analysis began with RNA extraction from pulverized bone tissue. RNA was sequenced and reads were mapped to mm10 genome. Differential expression analysis was performed to create lists of differentially expressed genes (DEGs) for each time point. Comparison to previously generated qPCR data of stress and full fracture callus was used to validate RNA-seq data. Callus component analysis using transcriptional profiling of callus cells was performed at each time point. Pathway analysis and GO annotation were performed on the DEG lists of each time point.