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Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.

ABSTRACT: This study compared fracture repair stabilized by intramedullary pin (IMP) or external fixation (EF) in GFP reporter mice. A modified IMP was used as control while EF utilized six needles inserted transversely through the tibia and into a segment of a syringe barrel. X-rays taken at days 0, 14, and 35 showed that IMP resulted in significant three-dimensional deformity with a large callus while EF showed minimal deformity and callus formation. Cryohistological analysis of IMP at day 14 confirmed a large ColX-RFPchry+ callus surrounded by woven bone (Col3.6-GFPcyan) and TRAP+ osteoclasts with mature bone (hOC-GFPtpz) at the base. By day 35, cartilaginous components had been resorbed and an outer cortical shell (OCS) showed evidence of inward modeling. In contrast, the EF at day 14 showed no evidence of cartilage formation. Instead, periosteal-derived osteoblasts (Col3.6-GFPcyan) entered the fracture cleft and formed woven bone that spanned the marrow space. By day 35, mature bone had formed that was contiguous with the opposing cortical bone. Fracture site stability greatly affects the cellular response during repair and must be considered in the preclinical models that test therapies for improving fracture healing.

SUBMITTER: Hagiwara Y

PROVIDER: S-EPMC4891973 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.

Hagiwara Yusuke Y Dyment Nathaniel A NA Jiang Xi X Jiang Ping Huang H Ackert-Bicknell Cheryl C Adams Douglas J DJ Rowe David W DW

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20150513 7

This study compared fracture repair stabilized by intramedullary pin (IMP) or external fixation (EF) in GFP reporter mice. A modified IMP was used as control while EF utilized six needles inserted transversely through the tibia and into a segment of a syringe barrel. X-rays taken at days 0, 14, and 35 showed that IMP resulted in significant three-dimensional deformity with a large callus while EF showed minimal deformity and callus formation. Cryohistological analysis of IMP at day 14 confirmed ...[more]

PMID: 25639792

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Project description:In response to bone fracture, periosteal progenitor cells proliferate, expand, and differentiate to form cartilage and bone in the fracture callus. These cellular functions require the coordinated activation of multiple transcriptional programs, and the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) regulate osteochondroprogenitor activation during endochondral bone development. However, recent observations raise important distinctions between the signaling mechanisms used to control bone morphogenesis and repair. Here, we tested the hypothesis that YAP and TAZ regulate osteochondroprogenitor activation during endochondral bone fracture healing in mice. Constitutive YAP and/or TAZ deletion from Osterix-expressing cells impaired both cartilage callus formation and subsequent mineralization. However, this could be explained either by direct defects in osteochondroprogenitor differentiation after fracture or by developmental deficiencies in the progenitor cell pool before fracture. Consistent with the second possibility, we found that developmental YAP/TAZ deletion produced long bones with impaired periosteal thickness and cellularity. Therefore, to remove the contributions of developmental history, we next generated adult onset-inducible knockout mice (using Osx-CretetOff ) in which YAP and TAZ were deleted before fracture but after normal development. Adult onset-induced YAP/TAZ deletion had no effect on cartilaginous callus formation but impaired bone formation at 14 days post-fracture (dpf). Earlier, at 4 dpf, adult onset-induced YAP/TAZ deletion impaired the proliferation and expansion of osteoblast precursor cells located in the shoulder of the callus. Further, activated periosteal cells isolated from this region at 4 dpf exhibited impaired osteogenic differentiation in vitro upon YAP/TAZ deletion. Finally, confirming the effects on osteoblast function in vivo, adult onset-induced YAP/TAZ deletion impaired bone formation in the callus shoulder at 7 dpf before the initiation of endochondral ossification. Together, these data show that YAP and TAZ promote the expansion and differentiation of periosteal osteoblast precursors to accelerate bone fracture healing. © 2020 American Society for Bone and Mineral Research (ASBMR).

Project description:IntroductionThe 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.MethodsTo 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.ResultsIn 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.ConclusionA 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.

Project description:Atrophic nonunion represents an extremely challenging clinical dilemma for both physicians and fracture patients alike, but its underlying mechanisms are still largely unknown. Here, we established a mouse model that recapitulates clinical atrophic nonunion through the administration of focal radiation to the long bone midshaft 2 weeks before a closed, semistabilized, transverse fracture. Strikingly, fractures in previously irradiated bone showed no bony bridging with a 100% nonunion rate. Radiation triggered distinct repair responses, separated by the fracture line: a less robust callus formation at the proximal side (close to the knee) and bony atrophy at the distal side (close to the ankle) characterized by sustained fibrotic cells and type I collagen-rich matrix. These fibrotic cells, similar to human nonunion samples, lacked osteogenic and chondrogenic differentiation and exhibited impaired blood vessel infiltration. Mechanistically, focal radiation reduced the numbers of periosteal mesenchymal progenitors and blood vessels and blunted injury-induced proliferation of mesenchymal progenitors shortly after fracture, with greater damage particularly at the distal side. In culture, radiation drastically suppressed proliferation of periosteal mesenchymal progenitors. Radiation did not affect hypoxia-induced periosteal cell chondrogenesis but greatly reduced osteogenic differentiation. Lineage tracing using multiple reporter mouse models revealed that mesenchymal progenitors within the bone marrow or along the periosteal bone surface did not contribute to nonunion fibrosis. Therefore, we conclude that atrophic nonunion fractures are caused by severe damage to the periosteal mesenchymal progenitors and are accompanied by an extraskeletal, fibro-cellular response. In addition, we present this radiation-induced periosteal damage model as a new, clinically relevant tool to study the biologic basis of therapies for atrophic nonunion. © 2018 American Society for Bone and Mineral Research.

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Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.

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Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.

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