Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

Isolation, identification and osteogenic capability analysis of mesenchymal stem cells derived from different layers of human maxillary sinus membrane.

AIM: To discover the populations of mesenchymal stem cells (MSCs) derived from different layers of human maxillary sinus membrane (hMSM) and evaluate their osteogenic capability. MATERIALS AND METHODS: hMSM was isolated into a monolayer using the combined method of physical separation and enzymatic digestion. The localization of MSCs in hMSM was performed by immunohistological staining and other techniques. Lamina propria layer-derived MSCs (LMSCs) and periosteum layer-derived MSCs (PMSCs) from hMSM were expanded using the explant cell culture method and identified by multilineage differentiation assays, colony formation assay, flow cytometry and so on. The biological characteristics of LMSCs and PMSCs were compared using RNA sequencing, reverse transcription and quantitative polymerase chain reaction, immunofluorescence staining, transwell assay, western blotting and so forth. RESULTS: LMSCs and PMSCs from hMSMs were both CD73-, CD90- and CD105-positive, and CD34-, CD45- and HLA-DR-negative. LMSCs and PMSCs were identified as CD171+/CD90+ and CD171-/CD90+, respectively. LMSCs displayed stronger proliferation capability than PMSCs, and PMSCs presented stronger osteogenic differentiation capability than LMSCs. Moreover, PMSCs could recruit and promote osteogenic differentiation of LMSCs. CONCLUSIONS: This study identified and isolated two different types of MSCs from hMSMs. Both MSCs served as good potential candidates for bone regeneration.

Discovery of CTSK+ Periosteal Stem Cells Mediating Bone Repair in Orbital Reconstruction.

Purpose: The purpose of this study was to explore the role of cathepsin K positive (CTSK+) periosteal stem cells (PSCs) in orbital bone repair and to clarify the source of endogenous stem cells for orbital bone self-repair. Methods: Periosteum samples obtained by clinical orbital bone repair surgery were analyzed, after which immunofluorescence and immunohistochemical staining were used to detect the content of bone marrow-derived cells and CTSK+ PSCs in periosteum as well as the mobilization of PSCs. CTSK+ PSCs were characterized by flow cytometry. Transcriptome sequencing was used to compare the transcriptomic characteristics of CTSK+ PSCs and bone marrow mesenchymal stem cells (BMSCs). Results: The orbital periosteum contained CTSK+CD200+ cell lineage, including CD200+CD105- PSCs and CD200+CD105+ progenitor cells. CTSK and osteocalcin (OCN) colocalized in the inner layer of the orbital periosteum, suggesting the osteogenic differentiation potential of CTSK+ PSCs. CTSK expression was much higher in periosteum after mobilization. Immunofluorescence showed low amounts of scattered CD31+ and CD45+ cells in the orbital periosteum. The stem cell characteristics of CTSK+ PSCs were verified by multidirectional differentiation. Flow cytometry found CD200+CD105- CTSK+ PSCs and CD200variantCD105+ progenitor cells. Transcriptome sequencing of CTSK+ PSCs and BMSCs found 3613 differential genes with significant differences. Gene Ontology (GO) analysis showed the differences between the two types of stem cells, revealing that PSCs were more suitable for intramembranous osteogenesis. Conclusions: CTSK+ PSCs may be endogenous stem cells for orbital bone repair. They are mobilized after orbital fracture and have unique features suitable for intramembranous osteogenesis, completely different from BMSCs.

Mechanical Properties of Human Concentrated Growth Factor (CGF) Membrane and the CGF Graft with Bone Morphogenetic Protein-2 (BMP-2) onto Periosteum of the Skull of Nude Mice.

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm3) or the CGF/BMP-2 (1.0 µg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery.

Influence of Human Jaw Periosteal Cells Seeded ß-Tricalcium Phosphate Scaffolds on Blood Coagulation.

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.

Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization.

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.

Isolation and Culture of Periosteum-Derived Progenitor Cells from Mice.

This chapter describes the methods of isolation of mouse periosteal progenitor cells. There are three basic methods utilized. The bone grafting method was developed utilizing the fracture healing process to expand the progenitor populations. Bone capping methods requires enzymatic digestion and purification of cells from the native periosteum, while the Egression/Explant method requires the least manipulation with placement of cortical bone fragments with attached periosteum in a culture dish. Various cell surface antibodies have been employed over the years to characterize periosteum derived progenitor cells, but the most consistent minimal criteria was recommended by the International Society for Cellular Therapy. Confirmation of the multipotent status of these isolated cells can be achieved by differentiation into the three basic mesodermal lineages in vitro.

Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.  Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

TGF-ß/Smad2 signalling regulates enchondral bone formation of Gli1+ periosteal cells during fracture healing.

OBJECTIVES: Most bone fracture heals through enchondral bone formation that relies on the involvement of periosteal progenitor cells. However, the identity of periosteal progenitor cells and the regulatory mechanism of their proliferation and differentiation remain unclear. The aim of this study was to investigate whether Gli1-CreERT2 can identify a population of murine periosteal progenitor cells and the role of TGF-ß signalling in periosteal progenitor cells on fracture healing. MATERIALS AND METHODS: Double heterozygous Gli1-CreERT2 ;Rosa26-tdTomatoflox/wt mice were sacrificed at different time points for tracing the fate of Gli1+ cells in both intact and fracture bone. Gli1-CreERT2 -mediated Tgfbr2 knockout (Gli1-CreERT2 ;Tgfbr2flox/flox ) mice were subjected to fracture surgery. At 4, 7, 10, 14 and 21 days post-surgery, tibia samples were harvested for tissue analyses including µCT, histology, real-time PCR and immunofluorescence staining. RESULTS: Through cell lineage-tracing experiments, we have revealed that Gli1-CreER T2 can be used to identify a subpopulation of periosteal progenitor cells in vivo that persistently reside in periosteum and contribute to osteochondral elements during fracture repair. During the healing process, TGF-ß signalling is continually activated in the reparative Gli1+ periosteal cells. Conditional knockout of Tgfbr2 in these cells leads to a delayed and impaired enchondral bone formation, at least partially due to the reduced proliferation and chondrogenic and osteogenic differentiation of Gli1+ periosteal cells. CONCLUSIONS: TGF-ß signalling plays an essential role on fracture repair via regulating enchondral bone formation process of Gli1+ periosteal cells.

Dual neutralisation of IL-17F and IL-17A with bimekizumab blocks inflammation-driven osteogenic differentiation of human periosteal cells.

OBJECTIVES: Interleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankylosing spondylitis (AS) with several IL-17 blockers currently clinically approved. Despite this, the role of IL-17 in bone pathology is poorly understood. This study aimed to investigate IL-17 signalling in the context of pathological bone formation. METHODS: A biomimetic human periosteum-derived cell (hPDC) model of osteogenic differentiation was used in combination with recombinant IL-17 cytokines, T-cell supernatants or serum from patients with AS. IL-17A, IL-17F and bimekizumab monoclonal antibodies were used to block IL-17 cytokine action. RESULTS: Recombinant IL-17A and IL-17F are pro-osteogenic with respect to hPDC differentiation. T helper 17 or γδ-T cell supernatants also potently stimulated in vitro bone formation, which was blocked deeper by dual inhibition of IL-17A and IL-17F than by neutralisation of IL-17A or IL-17F individually. Osteogenic blockade may be due to an increase in expression of the Wnt antagonist DKK1. Interestingly, osteocommitment was also induced by serum obtained from patients with AS, which was also abrogated by dual neutralisation of IL-17A and IL-17F. CONCLUSIONS: These data show for the first time that IL-17A and IL-17F enhance in vitro osteogenic differentiation and bone formation from hPDCs, inhibition of which may offer an attractive therapeutic strategy to prevent pathological bone formation.

Resveratrol can enhance osteogenic differentiation and mitochondrial biogenesis from human periosteum-derived mesenchymal stem cells.

BACKGROUND: Osteoporosis is a metabolic bone disorder that leads to low bone mass and microstructural deterioration of bone tissue and increases bone fractures. Resveratrol, a natural polyphenol compound, has pleiotropic effects including anti-oxidative, anti-aging, and anti-cancer effects. Resveratrol also has roles in increasing osteogenesis and in upregulating mitochondrial biogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is still unclear that resveratrol can enhance osteogenic differentiation or mitochondrial biogenesis of periosteum-derived MSCs (PO-MSCs), which play key roles in bone tissue maintenance and fracture healing. Thus, in order to test a possible preventive or therapeutic effect of resveratrol on osteoporosis, this study investigated the effects of resveratrol treatments on osteogenic differentiation and mitochondrial biogenesis of PO-MSCs. METHODS: The optimal doses of resveratrol treatment on PO-MSCs were determined by cell proliferation and viability assays. Osteogenic differentiation of PO-MSCs under resveratrol treatment was assessed by alkaline phosphatase activities (ALP, an early biomarker of osteogenesis) as well as by extracellular calcium deposit levels (a late biomarker). Mitochondrial biogenesis during osteogenic differentiation of PO-MSCs was measured by quantifying both mitochondrial mass and mitochondrial DNA (mtDNA) contents. RESULTS: Resveratrol treatments above 10 µM seem to have negative effects on cell proliferation and viability of PO-MSCs. Resveratrol treatment (at 5 µM) on PO-MSCs during osteogenic differentiation increased both ALP activities and calcium deposits compared to untreated control groups, demonstrating an enhancing effect of resveratrol on osteogenesis. In addition, resveratrol treatment (at 5 µM) during osteogenic differentiation of PO-MSCs increased both mitochondrial mass and mtDNA copy numbers, indicating that resveratrol can bolster mitochondrial biogenesis in the process of PO-MSC osteogenic differentiation. CONCLUSION: Taken together, the findings of this study describe the roles of resveratrol in promoting osteogenesis and mitochondrial biogenesis of human PO-MSCs suggesting a possible application of resveratrol as a supplement for osteoporosis and/or osteoporotic fractures.

DDR2, a discoidin domain receptor, is a marker of periosteal osteoblast and osteoblast progenitors.

INTRODUCTION: The periosteum has a bilayered structure that surrounds cortical bone. The outer layer is rich in connective tissue and fibroblasts, while the inner layer in contact with the cortical surface of the bone predominantly consists of osteoblasts and osteoblast progenitors. The identification of cell-specific surface markers of the bilayered structure of the periosteum is important for the purpose of tissue regeneration. MATERIALS AND METHODS: We investigated the expression of the discoidin domain tyrosine kinase receptor DDR2, fibroblast specific protein-1 (FSP-1) and alkaline phosphatase (ALP) in the periosteum of cortical bone by immunohistochemistry. Osteogenic differentiation was compared between DDR2- and FSP-1-expressing cells flow-sorted from the periosteum. RESULTS: We showed that DDR2 predominantly labeled osteogenic cells residing in the inner layer of the periosteum and that Pearson's coefficient of colocalization indicated a significant correlation with the expression of ALP. The mineralization of DDR2-expressing osteogenic cells isolated from the periosteum was significantly induced. In contrast, FSP-1 predominantly labeled the outer layer of periosteal fibroblasts, and Pearson's coefficient of colocalization indicated that FSP-1 was poorly correlated with the expression of DDR2 and ALP. FSP-1-expressing periosteal fibroblasts did not exhibit osteogenic differentiation for the induction of bone mineralization. CONCLUSION: DDR2 is a novel potential cell surface marker for identifying and isolating osteoblasts and osteoblast progenitors within the periosteum that can be used for musculoskeletal regenerative therapies.

Skeletal stem cells: insights into maintaining and regenerating the skeleton.

Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance and repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with inconsistent characteristics. However, recent in vivo tracking experiments have revealed the presence of SSCs not only within the bone marrow but also within the periosteum and growth plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to lineage potential. It has also been revealed that, during digit tip regeneration and in some non-mammalian vertebrates, the dedifferentiation of osteoblasts may contribute to skeletal regeneration. Here, we examine how these research findings have furthered our understanding of the diversity and plasticity of SSCs that mediate skeletal maintenance and repair.

Response of human periosteal cells to degradation products of zinc and its alloy.

Zinc (Zn) and its alloys are proposed as promising resorbable materials for osteosynthesis implants. Detailed studies should be undertaken to clarify their properties in terms of degradability, biocompatibility and osteoinductivity. Degradation products of Zn alloys might affect directly adjacent cellular and tissue responses. Periosteal stem cells are responsible for participating in intramembranous ossification during fracture healing. The present study aims at examining possible effects emanating from Zn or Zn-4Ag (wt%) alloy degradation products on cell viability and osteogenic differentiation of a human immortalized cranial periosteal cell line (TAg cells). Therefore, a modified extraction method was used to investigate the degradation behavior of Zn and Zn-4Ag alloys under cell culture conditions. Compared with pure Zn, Zn-4Ag alloy showed almost fourfold higher degradation rates under cell culture conditions, while the associated degradation products had no adverse effects on cell viability. Osteogenic induction of TAg cells revealed that high concentration extracts significantly reduced calcium deposition of TAg cells, while low concentration extracts enhanced calcium deposition, indicating a dose-dependent effect of Zn ions. Our results give evidence that the observed cytotoxicity effects were determined by the released degradation products of Zn and Zn-4Ag alloys, rather than by degradation rates calculated by weight loss. Extracellular Zn ion concentration was found to modulate osteogenic differentiation of TAg cells. These findings provide significant implications and guidance for the development of Zn-based alloys with an optimized degradation behavior for Zn-based osteosynthesis implants.

Quantitative proteomics analysis of deer antlerogenic periosteal cells reveals potential bioactive factors in velvet antlers.

Velvet antlers (VA) have been used as medicines and nutraceuticals for over 2000 years. Meanwhile, deer antlers are the only mammalian organs that can fully regenerate after annual shedding. The antler formation and regeneration rely on the stem cells resident in antlerogenic periosteum (AP), transplantation of which can induce ectopic antler formation. Here, a comprehensive quantitative proteomic analysis of antlerogenic periosteal cells (AP cells), compared with the adjacent facial periosteal cells (FP cells), was carried out, from both extracellular and intracellular perspectives. In this study, the stable isotope labeling by amino acids in cell culture (SILAC) was applied to ensure the precision of quantification. Then, the protein equalization strategy and reverse-phase liquid chromatography (RPLC) separation in high pH were utilized to improve the depth of proteome profiling. Proteomics analysis of the conditioned media (CM) from AP and FP cells showed that significantly over-expressed extracellular proteins in AP cells were involved in cell proliferation, angiogenesis and neurogenesis. Combining the extracellular and intracellular proteomes, we found several potential secreted proteins might regulate antler formation and regeneration, such as SFRP4 and LUM. These results provide new insight into the underlying mechanism of antler formation and regeneration.

Inhibition of the Prostaglandin EP-1 Receptor in Periosteum Progenitor Cells Enhances Osteoblast Differentiation and Fracture Repair.

Fracture healing is a complex and integrated process that involves mesenchymal progenitor cell (MPC) recruitment, proliferation and differentiation that eventually results in bone regeneration. Prostaglandin E2 (PGE2) is an important regulator of bone metabolism and has an anabolic effect on fracture healing. Prior work from our laboratory showed EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation. We also showed that bone marrow MSCs from EP1-/- mice exhibit increased osteoblastic differentiation in vitro. In this study we investigate the changes in the periosteal derived MPCs (PDMPCs), which are crucial for fracture repair, upon EP1 deletion. EP1-/- PDMPCs exhibit increased numbers of total (CFU-F) and osteoblastic colonies (CFU-O) as well as enhanced osteoblastic and chondrogenic differentiation. Moreover, we tested the possible therapeutic application of a specific EP1 receptor antagonist to accelerate fracture repair. Our findings showed that EP1 antagonist administration to wild type mice in the early stages of repair similarly resulted in enhanced CFU-F, CFU-O, and osteoblast differentiation in PDMPCs and resulted in enhanced fracture callus formation at 10 days post fracture and increased bone volume and improved biomechanical healing of femur fractures at 21 days post fracture.

αSMA Osteoprogenitor Cells Contribute to the Increase in Osteoblast Numbers in Response to Mechanical Loading.

Bone is a dynamic tissue that site-specifically adapts to the load that it experiences. In response to increasing load, the cortical bone area is increased, mainly through enhanced periosteal bone formation. This increase in area is associated with an increase in the number of bone-forming osteoblasts; however, the origin of the cells involved remains unclear. Alpha-smooth muscle actin (αSMA) is a marker of early osteoprogenitor cells in the periosteum, and we hypothesized that the new osteoblasts that are activated by loading could originate from αSMA-expressing cells. Therefore, we used an in vivo fate-mapping approach in an established axial loading model to investigate the role of αSMA-expressing cells in the load-induced increase in osteoblasts. Histomorphometric analysis was applied to measure the number of cells of different origin on the periosteal surface in the most load-responsive region of the mouse tibia. A single loading session failed to increase the number of periosteal αSMA-expressing cells and osteoblasts. However, in response to multiple episodes of loading, the caudal, but not the cranial, periosteal surface was lined with an increased number of osteoblasts originating from αSMA-expressing cells 5 days after the initial loading session. The proportion of osteoblasts derived from αSMA-labeled progenitors increased by 70% (p < 0.05), and the proportion of αSMA-labeled cells that had differentiated into osteoblasts was doubled. We conclude that αSMA-expressing osteoprogenitors can differentiate and contribute to the increase in periosteal osteoblasts induced by mechanical loading in a site-specific manner.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the in vivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible for lifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration in vivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.