Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantation in Tibial Fractures

Perrin, Simon; Julien, Anaïs; Lageneste, Oriane Duchamp de; Abou-Khalil, Rana; Colnot, Céline

doi:10.21769/bioprotoc.4107

Cited by 8 publications

(13 citation statements)

References 10 publications

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“…In vitro differentiation was performed as described. ( 31 ) Skeletal muscle from tamoxifen induced Bmpr1a control and Bmpr1a cKO mice were digested and Pdgfra ‐derived muMPs were sorted based on GFP+ expression. After cell sorting, Pdgfra ‐derived muMPs were expanded in six‐well plates for in vitro differentiation.…”

Section: Methodsmentioning

confidence: 99%

“…Primary cultures of periosteal cells (PCs) and bone marrow stromal cells (BMSCs) were performed as described. (10,31) Briefly, 4-week-old to 8-week-old mice were euthanized, and femurs and tibias were dissected to remove fat, adjacent skeletal muscles, and tendons. Epiphyses were then cut and removed.…”

Section: Non-stabilized Tibial Fracturementioning

confidence: 99%

“…Tissue and cell transplantations were performed as described. (2,10,31,32) Tibial fracture was induced in the host mice as described in 'Non-stabilized tibial fracture' section. For EDL muscle transplantation, EDL muscle was dissected from tendon to tendon and grafted adjacent to the fracture site.…”

Section: Tissue and Cell Transplantationmentioning

confidence: 99%

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Skeletal Stem/Progenitor Cells in Periosteum and Skeletal Muscle Share a Common Molecular Response to Bone Injury

Julien

Perrin

Martínez‐Sarrà

et al. 2020

Journal of Bone and Mineral Research

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Bone regeneration involves skeletal stem/progenitor cells (SSPCs) recruited from bone marrow, periosteum, and adjacent skeletal muscle. To achieve bone reconstitution after injury, a coordinated cellular and molecular response is required from these cell populations. Here, we show that SSPCs from periosteum and skeletal muscle are enriched in osteochondral progenitors, and more efficiently contribute to endochondral ossification during fracture repair as compared to bone‐marrow stromal cells. Single‐cell RNA sequencing (RNAseq) analyses of periosteal cells reveal the cellular heterogeneity of periosteum at steady state and in response to bone fracture. Upon fracture, both periosteal and skeletal muscle SSPCs transition from a stem/progenitor to a fibrogenic state prior to chondrogenesis. This common activation pattern in periosteum and skeletal muscle SSPCs is mediated by bone morphogenetic protein (BMP) signaling. Functionally, Bmpr1a gene inactivation in platelet‐derived growth factor receptor alpha (Pdgfra)‐derived SSPCs impairs bone healing and decreases SSPC proliferation, migration, and osteochondral differentiation. These results uncover a coordinated molecular program driving SSPC activation in periosteum and skeletal muscle toward endochondral ossification during bone regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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Section: Methodsmentioning

confidence: 99%

Section: Non-stabilized Tibial Fracturementioning

confidence: 99%

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Skeletal Stem/Progenitor Cells in Periosteum and Skeletal Muscle Share a Common Molecular Response to Bone Injury

Julien

Perrin

Martínez‐Sarrà

et al. 2020

Journal of Bone and Mineral Research

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“…Samples were processed as previously described 63 . Fractured tibias were collected and fixed in ice-cold 4% paraformaldehyde 24 hours, decalcified 2 weeks in 19% EDTA (EU00084, Euromedex), placed 24h in 30% sucrose (200-301-B, Euromedex) gradient and embedded in cryoprotectant.…”

Section: Methodsmentioning

confidence: 99%

Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma

Hachemi,

Perrin,

Ethel

et al. 2024

Preprint

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Musculoskeletal traumatic injuries (MTI) involve soft tissue lesions adjacent to a bone fracture leading to fibrous nonunion. The impact of MTI on the inflammatory response to fracture and on the immunomodulation of skeletal stem/progenitor cells (SSPCs) remains unknown. Here, we used single cell transcriptomic analyses to describe the immune cell dynamics after bone fracture and identified distinct macrophage subsets with successive pro-inflammatory, pro-repair and anti-inflammatory profiles. Concurrently, SSPCs transition via a pro- and anti-inflammatory fibrogenic phase of differentiation prior to osteochondrogenic differentiation. In a preclinical MTI mouse model, the injury response of immune cells and SSPCs is disrupted leading to a prolonged pro-inflammatory phase and delayed resolution of inflammation. Macrophage depletion improves bone regeneration in MTI demonstrating macrophage involvement in fibrous nonunion. Finally, pharmacological inhibition of macrophages using the CSF1R inhibitor Pexidartinib ameliorates healing. These findings reveal the coordinated immune response of macrophages and skeletal stem/progenitor cells as driver of bone healing and as a primary target for the treatment of trauma-associated fibrosis.

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“…Open non-stabilized tibial fractures were induced as previously described 44 . Mice were anesthetized with an intraperitoneal injection of Ketamine (50 mg/mL) and Medetomidine (1 mg/kg) and received a subcutaneous injection of Buprenorphine (0.1 mg/kg) for analgesia.…”

Section: Methodsmentioning

confidence: 99%

Single nuclei transcriptomics reveal the differentiation trajectories of periosteal skeletal/stem progenitor cells in bone regeneration

Perrin,

Ethel,

Bretegnier

et al. 2023

Preprint

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SummaryBone regeneration is a highly efficient process allowing bones to fully regenerate after fracture. Skeletal stem/progenitor cells (SSPCs) allowing bone regeneration are recruited from several sources, including the periosteum located at the outer surface of bones. Periosteal SSPCs are major actors of bone healing and display the unique property to form both bone and cartilage after fracture. Yet, the steps of periosteal SSPCs activation and differentiation leading to osteogenesis and chondrogenesis remain poorly understood. Here, we generated a single-nuclei atlas of the periosteum at steady-state and in response to bone fracture. We described the heterogeneity of the uninjured periosteum and identified a population of periosteal SSPCs expressing stemness markers, such asPi16andLy6a(Sca1). After fracture, we observed major changes in cell populations with a reduction of the SSPC population, a concomitant increase in injury-induced fibrogenic cells and immune cells, followed by the appearance of chondrocytes and osteoblasts. We show that periosteal SPPCs respond to injury via a unique differentiation trajectory. Cells first leave their stem/progenitor state to adopt an injury-activated fibrogenic fate, prior to undergoing either osteogenesis or chondrogenesis. Overall, our study provides a complete temporal topography of the fracture healing environment and the dynamic response of periosteal SSPC to injury, redefining our knowledge on the early steps of bone regeneration.

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Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantation in Tibial Fractures

Cited by 8 publications

References 10 publications

Skeletal Stem/Progenitor Cells in Periosteum and Skeletal Muscle Share a Common Molecular Response to Bone Injury

Skeletal Stem/Progenitor Cells in Periosteum and Skeletal Muscle Share a Common Molecular Response to Bone Injury

Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma

Single nuclei transcriptomics reveal the differentiation trajectories of periosteal skeletal/stem progenitor cells in bone regeneration

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