Sox9 positive periosteal cells in fracture repair of the adult mammalian long bone

Abstract

Introduction: The phases of fracture healing have been well characterized. However, the exact source and genetic profile of the skeletal progenitors that participate in bone repair is somewhat unclear. Sox9 expression in skeletal elements precedes bone and cartilage formation and a Sox9⁺ cell type is retained in the adult periosteum. We hypothesized that Sox9⁺ periosteal cells are multipotent skeletal progenitors normally participating in fracture repair.

Methods: To test this hypothesis we used tamoxifen (TM)-mediated lineage tracing of Sox9⁺ cells in Sox9CreErt2:Td-Tomato mice. Intact femora were analyzed with immunostaining and RNA sequencing to evaluate the skeletal distribution and gene expression profile of Td-Tomato positive, Sox9-descendent cells in the adult femur. To assess the role of Td-tomato+cells in the fracture healing process, mice underwent a closed mid-diaphyseal femoral fracture. Fractured hind limbs were analyzed by X-ray, histology and immuno-staining at 3, 9 or 56days post-fracture.

Results: In the intact adult mouse femur, Td-Tomato-labeled cells were observed in the primary spongiosa, periosteum and endosteum. RNA sequencing showed that Td-Tomato positive periosteal cells were co-enriched for Sox9 transcripts, and mRNAs for osteoblast and chondrocyte specific genes. In a femoral fracture model, we showed that pre-labeled Td-Tomato positive descendent cells were mobilized during the early stages of bone repair (day 3 post-op) contributing to the fracture repair process by differentiating into chondrocytes, osteoblasts and osteocytes.

Conclusion: A Sox9⁺ skeletal progenitor population resides in the adult periosteum. Fate tracing studies show that descendants of the Sox9⁺ periosteal progenitors give rise to chondrocytes, osteoblasts and mature cortical osteocytes in repair of the fractured femur. To our knowledge this is the first report of a reparative Sox9⁺ progenitor population in the periosteum of the adult long bone. Taken together with developmental studies, our data suggest a broad role for Sox9⁺ osteochondroprogenitors in development and repair of the mammalian skeleton.

Keywords: Fracture healing; Periosteal cells; Skeletal progenitors; Sox9.