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.

Figure 1

Short term Sox9CreErt2:Td-Tomato lineage tracing in representative frozen sections of intact adult mouse femora. The contribution of Td-Tomato positive cells was examined in the femurs of uninjected control (A) and TM-injected mice (B) 14 days after a final TM injection. In the primary spongiosa/growth plate area, expression and cellular localization of CD31+ endothelial cells (C), Col1a1+ osteoblasts (D), Sp7+ osteoblast progenitors and osteoblasts (E) and endogenous Sox9+ cell types (F). Periosteal and endosteal localization of Sox9 descendant Td-Tomato+ cells (red) was compared with endogenous Sox9+ cells (green in G, H) or Ocn+ cells (green in I, J). Nuclei were labeled by DAPI (blue). The left panel (a) shows a high magnification overlay of Td-Tomato, Sox9 or Ocn and DAPI signals. The middle panel (b) separates the Td-Tomato and DAPI signal, and the right panel (c) the Sox9/Ocn and DAPI signal from panel (a). The white arrow indicates subpopulations of Td-Tomato+/Sox9+ (G, H) and Td-Tomato+/Ocn+ (I) cells. GP: growth plate, PS: primary spongiosa, EO: endosteum, pPO: proximal diaphyseal periosteum, mPO: medial diaphyseal periosteum

Figure 2

RNA sequencing of Td-Tomato positive periosteal cells. The top 20 gene functions predicted from DAVID GO analysis of RNA-seq profiling of Td-Tomato + periosteal cells. Td-Tomato positive periosteal cells are associated with various osteogenic and chondrogenic functions. The X axis depicts the -log10 transformation of p-values from Supplementary Table S2.

Figure 3

Representative images of femoral fracture site 3 days post-operatively, demonstrating the expansion and activation of Td-Tomato+ periosteal cells on injury, in lower (A) and higher magnification (B) views. Td-Tomato+ cells co-express Sox9 (C) or Sp7 (D) within the fracture site. Panels C and D present higher magnification views of the overlap in Td-Tomato, Sox9 (Ea–Ec) and Sp7 (Fa–Fc) activity at the fracture site. The left panel (a) presents an overlay of Td-Tomato, Sox9 or Sp7, and DAPI (nuclear) signals. The middle panel (b) separates the Sox9 or Sp7 signal and the right panel (c) the nuclear signal (DAPI) from the overlay in (a). The bone cortices are demarcated with dashed lines.

Figure 4

Representative images of two mouse femora 9-days post fracture, showing the contribution of Td-Tomato+ cells to bone repair in the fracture-induced callus. Td-Tomato signal within cells was compared with Sox9 (A and A′), Col1a1 (B and B′) and Sp7 (C and C′) by immunostaining. (Da–Dc) presents a magnified view of the soft callus visualizing Td-Tomato (red), Sox9 (green) and nuclear (DAPI, blue) signals. (Ea-Ec) presents a magnified view of the hard callus visualizing Td-tomato (red), Sp7 (green) and nuclear (DAPI, blue) signals. As expected, Td-Tomato + cells in the hard callus were negative for Sox9 (F). The majority of Td-Tomato+ cells in the soft callus were negative for Sp7, except for a population of pre-hypertrophic chondrocytes located at the proximal and distal ends of the soft callus (G). These findings in (F) and (G) demonstrate the specificity for the overlapping signals observed in (D) and (E), respectively.

Figure 5

Representative frozen sections of mouse femora 2 months post-fracture. Distribution of Td-Tomato+ cells in the healed femur (B, D) and contralateral intact femur (A, C) in lower and higher magnification views, respectively. Phalloidin labelling of F-actin shows a distinct osteocyte organization comparing normal cortical bone (E) with cortices generated on fracture repair of the femur (F). Double headed arrows indicate the long axis of osteocyte orientation.