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

Highlights

• A Sox9⁺ osteochondroprogenitor population resides in the periosteum of adult long bones.

• RNA-seq analysis of the periosteal Sox9⁺ cells is consistent with an osteochondroprogenitor phenotype.

• Bone fracture stimulates the mobilization of Sox9 + cells to affect fracture repair.

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 56 days 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.

Introduction

Fracture healing is a complex process that involves the well-orchestrated participation of growth factors, cytokines and several cell types [1]. Most fractures are treated with a form of fixation that provides stability, but allows for some degree of motion (sling or cast immobilization, external fixation, intramedullary fixation). Thus the majority of fractures heal by secondary or indirect bone healing, a process that involves both intramembranous and endochondral ossification [2]. Secondary bone healing involves three major phases: the reactive phase (hematoma and inflammatory response), the reparative phase (soft and hard callus formation) and the remodeling phase [3]. Briefly, a fracture leads to surrounding soft tissue trauma, damage to local blood vessels and disruption of the bone marrow structure. Wound healing pathways are activated and a hematoma is formed at the area of the injury. Inflammatory cells and activated platelets soon infiltrate the hematoma and start secreting cytokines that can stimulate angiogenesis and initiate cellular events associated with the later stages of fracture healing [4]. Before the inflammation stage subsides, the repair process is initiated. An early indication of skeletal repair is the appearance of a chondrocyte-derived cartilage template that bridges and temporarily stabilizes the fractured bone fragments (soft callus). The cartilaginous callus serves as a template for formation of the hard bony callus by osteoblasts. Eventually the cartilage is eliminated from the callus that is composed of woven bone. Finally a remodeling process, dominated by osteoblasts, osteocytes and osteoclasts, returns the newly formed bone to its original bone configuration.

Despite the fact that the phases of bone healing have been well characterized, the cellular origins and molecular pathways underlying bone healing are somewhat unclear. Several possible sources of skeletal progenitor cells for bone healing have been reported [5] including endosteum [6], [7], periosteum [7], [8], [9], [10], bone marrow [8], [11], [12], vascular walls [13] and adjacent soft tissue [14]. Of these, a pivotal role for periosteum-derived progenitor cells in bone healing has been confirmed in several in vitro and in vivo studies, though it is not clear if this is a general property of periosteal cells, or a property restricted to distinct osteochondroprogenitors within this tissue [15].

In contrast to bone repair, the cellular mechanisms underlying bone development during embryogenesis have been well documented. Here, the SRY (Sex Determining Region Y)-Box9 (Sox9) transcription factor plays an essential role in determining skeletal progenitor cells' fate prior to overt chondrocyte and osteoblast development [16]. Thereafter, this osteochondroprogenitor cell population segregates into Sox9⁺ chondrocyte progenitors and Sox9⁻, Runx2/Sp7⁺ osteoblast progenitors that deposit cartilage and bone, respectively [16], [17]. Sox9 is necessary for establishing skeletal elements in the cranial, axial and appendicular systems [18], [19], [20]. In addition, Sox9 is sufficient to initiate chondrogenic programs when activated in mesenchymal stem cells, embryonic stem cells and even fibroblasts [21], [22], [23], [24].

Fracture healing has been characterized by many as the postnatal analogue of embryonic skeletal development, since many of the molecular mechanisms that control differentiation and growth during embryogenesis recur during fracture repair [25]. Since Sox9 defines osteochondroprogenitor cells during skeletogenesis and a similar differentiation program is likely shared between skeletal development and adult long bone repair, we hypothesized that Sox9 might play a major role in adult long bone repair. In this study, we demonstrate that an osteochondroprogenitor cell population positive for Sox9 resides in the periosteum of adult long bones and that upon activation by fracture stimulation, these osteochondroprogenitor cells direct fracture repair, differentiating into chondrocytes, osteoblasts and osteocytes.