The role of endothelial‐mesenchymal transition in heterotopic ossification

Heterotopic ossification (HO) is a process by which bone forms in soft tissues, in response to injury, inflammation, or genetic disease. This usually occurs by initial cartilage formation, followed by endochondral ossification. A rare disease called fibrodysplasia ossificans progressiva (FOP) allows this mechanism to be induced by a combination of genetic mutation and acute inflammatory responses. FOP patients experience progressive HO throughout their lifetime and form an ectopic skeleton. Recent studies on FOP have suggested that heterotopic cartilage and bone is of endothelial origin. Vascular endothelial cells differentiate into skeletal cells through a mesenchymal stem cell intermediate that is generated by endothelial‐mesenchymal transition (EndMT). Local inflammatory signals and/or other changes in the tissue microenvironment mediate the differentiation of endothelial‐derived mesenchymal stem cells into chondrocytes and osteoblasts to induce HO. We discuss the current evidence for the endothelial contribution to heterotopic bone formation. © 2012 American Society for Bone and Mineral Research.     

VEGF improves, whereas sFlt1 inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis.

We studied the interaction between VEGF and BMP2 during bone formation and bone healing. Results indicate that VEGF antagonist inhibited BMP2-elicited bone formation, whereas the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing through modulation of angiogenesis. INTRODUCTION: Angiogenesis is closely associated with bone formation during normal bone development and is important for the bone formation elicited by BMP4. However, it remains unknown whether vascular endothelial growth factor (VEGF) also interacts with other BMPs, especially BMP2, in bone formation and bone healing. MATERIALS AND METHODS: For this study, mouse muscle-derived stem cells were transduced to express BMP2, VEGF, or VEGF antagonist (sFlt1). We studied the angiogenic process during endochondral bone formation elicited by BMP2, a prototypical osteogenic BMP. Using radiographic and histologic analyses, we also evaluated the interaction between VEGF and BMP2 during bone formation and bone healing. RESULTS: Our results indicate that BMP2-elicited bone formation comprises two phases of angiogenesis, with an early phase occurring before the appearance of hypertrophic cartilage, followed by a late phase coupled with the appearance of hypertrophic cartilage. Our finding that the administration of sFlt1, a specific antagonist of VEGF, significantly inhibited BMP2-induced bone formation and the associated angiogenesis indicates that endogenous VEGF activity is important for bone formation. Furthermore, we found that the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing by improving angiogenesis, which in turn led to accelerated cartilage resorption and enhanced mineralized bone formation. Our findings also indicate that the ratio between VEGF and BMP2 influences their synergistic interaction, with a higher proportion of VEGF leading to decreased synergism. Our study also revealed unique VEGF-BMP2 interactions that differ from the VEGF-BMP4 interactions that we have described previously. CONCLUSIONS: This study, along with previously published work, shows that VEGF interacts synergistically with both BMP4 and BMP2 but elicits substantially different effects with these two BMPs.     

BMP‐9‐induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment

Heterotopic ossification (HO) is defined as the formation of bone inside soft tissue. Symptoms include joint stiffness, swelling, and pain. Apart from the inherited form, the common traumatic form generally occurs at sites of injury in damaged muscles and is often associated with brain injury. We investigated bone morphogenetic protein 9 (BMP‐9), which possesses a strong osteoinductive capacity, for its involvement in muscle HO physiopathology. We found that BMP‐9 had an osteoinductive influence on mouse muscle resident stromal cells by increasing their alkaline phosphatase activity and bone‐specific marker expression. Interestingly, BMP‐9 induced HO only in damaged muscle, whereas BMP‐2 promoted HO in skeletal muscle regardless of its state. The addition of the soluble form of the ALK1 protein (the BMP‐9 receptor) significantly inhibited the osteoinductive potential of BMP‐9 in cells and HO in damaged muscles. BMP‐9 thus should be considered a candidate for involvement in HO physiopathology, with its activity depending on the skeletal muscle microenvironment. © 2011 American Society for Bone and Mineral Research.     

Location‐dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9

Extremity amputation or traumatic injury can often lead to the formation of heterotopic ossification (HO). Studies to induce HO in rat muscle using cell‐based gene therapy show that this process appears to be location dependent. In the present study, HO was induced in mice and rats through injection of immunologically matched cells transduced with either a replication‐defective adenovirus possessing bone morphogenetic protein 2 (BMP2) or an empty adenovirus vector (control). Injection in rat near the skeletal bone resulted in HO, whereas cells injected into the same muscle group but distal from the bone did not result in bone formation. When cells were injected in the same limb at both locations at the same time, HO was formed at both sites. Characterization of the bone formation in rats versus mice demonstrated that different sources of osteogenic progenitors were involved, which may account for the location dependent bone formation observed in the rat. Further experimentation has shown that a potential reason for this difference may be the inability of rat to activate matrix metalloproteinase 9 (MMP9), an essential protease in mice necessary for recruitment of progenitors. Inhibition of active MMP9 in mice led to a significant decrease in HO. The studies reported here provide insight into the mechanisms and pathways leading to bone formation in different animals and species. It appears that not all animal models are appropriate for testing HO therapies, and our studies also challenge the conventional wisdom that larger animal models are better for testing treatments affecting bone. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 34:1894–1904, 2016.     

Inhibition of ectopic bone formation by a selective retinoic acid receptor α‐agonist: A new therapy for heterotopic ossification?

Heterotopic ossification (HO) consists of formation of ectopic cartilage followed by endochondral bone and is triggered by major surgeries, large wounds, and other conditions. Current therapies, including low‐dose irradiation, are not always effective and do not target the skeletogenic process directly. Because chondrogenesis requires a decrease of nuclear retinoic acid receptor α (RARα) action, we reasoned that pharmacologic activation of this receptor pathway should inhibit HO. Thus, we selected the synthetic retinoid NRX195183, a potent and highly selective RARα‐agonist, and found that it did inhibit chondrogenesis in mouse limb micromass cultures. We established a mouse HO model consisting of subcutaneous implantation of Matrigel mixed with rhBMP‐2. Control mice receiving daily oral doses of vehicle (peanut oil) or retinol (a natural nonactive retinoid precursor) developed large HO‐like masses by days 9–12 that displayed abundant cartilage, endochondral bone, vessels, and marrow. In contrast, formation of HO‐like masses was markedly reduced in companion mice receiving daily oral doses of α‐agonist. These ectopic masses contained sharply reduced amounts of cartilage and bone, blood vessels, and TRAP‐positive osteoclasts, and expressed markedly lower levels of master chondrogenic genes including Sox9, cartilage genes such as collagen XI and X, and osteogenic genes including Runx2. The data provide proof‐of‐principle evidence that a pharmacological strategy involving a selective RARα‐agonist can indeed counteract an ectopic skeletal‐formation process effectively and efficiently, and could thus represent a novel preventive treatment for HO. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:271–277, 2010     

Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation.

After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage. INTRODUCTION: The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation. MATERIALS AND METHODS: We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle. RESULTS: Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation. CONCLUSIONS: Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.     

Action of IL‐1β during fracture healing

After bone injury, developmental processes such as endochondral and intramembranous ossification are recapitulated as the skeleton regenerates. In contrast to development, skeletal healing involves inflammation. During the early stages of healing a variety of inflammatory cells infiltrate the injured site, debride the wound, and stimulate the repair process. Little is known about the inflammatory process during bone repair. In this work, we examined the effect of a pro‐inflammatory cytokine, Interleukin‐1 beta (IL‐1β), on osteoblast and stem cell differentiation and on intramembranous and endochondral ossification, because IL‐1β exerts effects on skeletal homeostasis and is upregulated in response to fracture. We determined that IL‐1β stimulated proliferation of osteoblasts and production of mineralized bone matrix, but suppressed proliferation and inhibited differentiation of bone marrow derived MSCs. We next performed loss‐ and gain‐of‐function experiments to determine if altering IL‐1β signaling affects fracture healing. We did not detect any differences in callus, cartilage, and bone matrix production during healing of nonstabilized or stabilized fractures in mice that lacked the IL‐1β receptor compared to wild‐type animals. We observed subtle alterations in the healing process after administering IL‐1β during the early phases of repair. At day 10 after injury, the ratio of cartilage to callus was increased, and by day 14, the proportion of cartilage to total callus and to bone was reduced. These changes could reflect a slight acceleration of endochondral ossification, or direct effects on cartilage and bone formation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:778–784, 2010     

Genetic and pharmacological inhibition of retinoic acid receptor γ function promotes endochondral bone formation

The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ‐null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ‐null mice. The antagonist‐induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle‐treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ‐null mice, and increases in pSmad1/5/8 levels and Id1‐luc activity were observed in RARγ antagonist‐treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096–1105, 2017.

     

Insulin‐like growth factor 2 (IGF‐2) potentiates BMP‐9‐induced osteogenic differentiation and bone formation

Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self‐renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP‐9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin‐like growth factor 2 (IGF‐2) on BMP‐9‐induced bone formation. We have found that endogenous IGF‐2 expression is low in MSCs. Expression of IGF‐2 can potentiate BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF‐2 has been shown to augment BMP‐9‐induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF‐2 enhances BMP‐9‐induced endochondral ossification, whereas IGF‐2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF‐2 effect on BMP‐9‐induced ALP activity and matrix mineralization. Mechanistically, IGF‐2 is further shown to enhance the BMP‐9‐induced BMPR‐Smad reporter activity and Smad1/5/8 nuclear translocation. PI3‐kinase (PI3K) inhibitor LY294002 abolishes the IGF‐2 potentiation effect on BMP‐9‐mediated osteogenic signaling and can directly inhibit BMP‐9 activity. These results demonstrate that BMP‐9 crosstalks with IGF‐2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP‐9 and IGF‐2 may be explored as an effective bone‐regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture. © 2010 American Society for Bone and Mineral Research.     

Cartilage-specific β-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development

The WNT/β-catenin signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. Although the importance of β-catenin signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate β-catenin in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for β-catenin in committed growth-plate chondrocytes has been less robust. To identify the specific role of cartilage-derived β-catenin in regulating cartilage and bone development, we studied chondrocyte-specific gain- and loss-of-function genetic mouse models using the tamoxifen-inducible Col2Cre(ERT2) transgene in combination with β-catenin(fx(exon3)/wt) or β-catenin(fx/fx) floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage-specific β-catenin signaling promotes chondrocyte maturation, possibly involving a bone morphogenic protein 2 (BMP2)-mediated mechanism. Second, cartilage-specific β-catenin facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced matrix metalloproteinase (MMP) expression at sites of cartilage degradation, and potentially by enhancing Indian hedgehog (IHH) signaling activity to recruit vascular tissues. Finally, cartilage-specific β-catenin signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage-derived β-catenin signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation.     

Crosstalk between substance P and calcitonin gene‐related peptide during heterotopic ossification in murine Achilles tendon

Heterotopic ossification (HO) is abnormal bone formation within soft tissue, usually predisposed by neurogenic or musculoskeletal trauma. Inflammation resulting from trauma is considered to be the main trigger for HO by eliciting changes within the injury site, including elevation of bone morphogenetic proteins (BMPs). Recent research, however, has also associated changes in sensory neuropeptide expression with HO. Substance P (SP) and calcitonin gene‐related peptide (CGRP) are two of those neuropeptides that have been implicated with various aspects of HO, including regulation of inflammation and BMP signaling. Despite discoveries associating SP and CGRP with soft tissue HO, it remains unclear whether SP and CGRP have a direct role in the induction of HO. Here, we investigated the effect of SP and CGRP in vivo with the aid of inkjet‐based biopatterning technology to controllably deliver these neuropeptides onto a murine Achilles tendon. While we did not observe any significant effect with CGRP, SP alone promoted HO in vivo with increased expression of BMP2. Remarkably, when SP and CGRP were delivered together, CGRP counteracted the effect of SP and essentially blocked SP‐induced HO. This report contributes to the understanding of the complex problem of HO pathophysiology and warrants more study to better elucidate the interplay between SP and CGRP in the induction of HO. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1444–1455, 2018.

     

Prefabrication of a Vascularized Bone Graft With Beta Tricalcium Phosphate Using an In Vivo Bioreactor

We aimed to introduce an in vivo bioreactor‐vascular pedicle threaded through the central portion of a scaffold in which a vascularized bone graft was prefabricated using adenoviral human BMP‐2 gene (AdBMP2)‐modified bone marrow mesenchymal stem cells (BMSCs), beta tricalcium phosphate (β‐TCP), a vessel bundle, and muscularis membrane(group A). As controls, Adβgal‐BMSCs/β‐TCP granules, vessel bundle, and the muscularis membrane (group B); BMSCs/β‐TCP granules, vessel bundle, and muscularis membrane (group C); and β‐TCP granules, vessel bundle, and muscularis membrane (group D) were prepared. Formation of bone tissue and a vascular network was assessed by microangiography and histological methods 4 weeks after prefabrication. New cartilage and bone tissue in the space between β‐TCP granules (mainly endochondral bone) were confirmed by histology, and a de novo vascular network circulating from the vessel bundle through newly formed bone tissue was observed in group A. Formation of bone or cartilage was not observed in the control groups. We concluded that the in vivo bioreactor is a promising method for prefabrication of vascularized functional bone.     

Soluble VEGFR1 reverses BMP2 inhibition of intramembranous ossification during healing of cortical bone defects

BMP2 is widely used for promotion of bone repair and regeneration. However, bone formation induced by BMP2 is quite variable. Bone forming progenitor cells in different locations appear to respond to BMP2 in different ways, and repair outcomes can vary as a consequence of modulating effects by other factors. In this study, we have examined the effects of VEGF on BMP2‐induced repair of a cortical bone defect, a 1 mm diameter drill hole, in the proximal tibia of mice. Treatment of the defect with either a bolus of PBS or soluble VEGFR1 (sVEGFR1), a decoy receptor for VEGF, had the same effects on bone formation via intramembranous ossification in the defect and cartilage formation and injured periosteum, during the healing process. In contrast, treatment with BMP2 inhibited intramembranous bone formation in the defect while it promoted cartilage and endochondral bone formation in the injured periosteum compared with mice treated with PBS or sVEGFR1. The inhibitory effect of BMP2 on bone formation was unlikely due to increased osteoclast activity and decreased invasion of blood vessels in the defect. Most importantly, co‐delivery of BMP2 and sVEGFR1 reversed the inhibition of intramembranous bone formation by BMP2. Furthermore, the decreased accumulation of collagen and production of bone matrix proteins in the defect of groups with BMP2 treatment could also be prevented by co‐delivery of BMP2 and sVEGFR1. Our data indicate that introducing a VEGF‐binding protein, such as sVEGFR1, to reduce levels of extracellular VEGF, may enhance the effects of BMP2 on intramembranous bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1461–1469, 2017.

     

Effects of blockade of endogenous Gi signaling in Tie2‐expressing cells on bone formation in a mouse model of heterotopic ossification

Available evidence indicates that some Tie2‐expressing (Tie2⁺) cells serve as multipotent progenitors that have robust BMP‐dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein G_i is required for cell motility, we hypothesized that blockade of endogenous G_i signaling in Tie2⁺ cell populations would prevent HO formation. Blockade of G_i signaling in Tie2⁺ cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2⁺/PTX⁺). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2⁺ cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2⁺/GFP⁺ mice, GFP⁺ cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of G_i signaling in Tie2⁺ cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of G_i protein coupled receptors in Tie2⁺ cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1212–1217, 2015.     

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.     

Long‐term administration of AMD3100, an antagonist of SDF‐1/CXCR4 signaling,alters fracture repair

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF‐1 (CXCL12) is considered a master regulator of CXCR4‐positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF‐1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF‐1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro‐computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF‐1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1853–1859, 2012     

Bone Formation During Distraction Osteogenesis Is Dependent on Both VEGFR1 and VEGFR2 Signaling

Introduction : Distraction osteogenesis (DO) is characterized by the induction of highly vascularized new bone formation through an intramembranous process largely devoid of the formation of cartilage. Materials and Methods : To test the hypothesis that DO is strictly dependent on vascualrization, we inhibited vascular endothelial growth factor (VEGF) activity by antibody blockade of both receptors VEGFR1 (Flt‐1) and VEGFR2 (Flk‐1) or only VEGFR2 (Flk‐1) in a previously developed murine tibia DO model. During normal DO, VEGFR1 (Flt‐1), VEGFR2 (Flk‐1), VEGFR3 (Flt4) and all four VEGF ligand (A, B, C, and D) mRNAs are induced. Results : The expression of mRNA for the receptors generally paralleled those of the ligands during the period of active distraction. Bone formation, as assessed by μCT, showed a significant decrease with the double antibody treatment and a smaller decrease with single antibody treatment. Vessel volume, number, and connectivity showed progressive and significant inhibition in all of these of parameters between the single and double antibody blockade. Molecular analysis showed significant inhibition in skeletal cell development with the single and double antibody blockade of both VEGFR1 and 2. Interestingly, the single antibody treatment led to selective early development of chondrogenesis, whereas the double antibody treatment led to a failure of both osteogenesis and chondrogenesis. Conclusions : Both VEGFR1 and VEGFR2 are functionally essential in blood vessel and bone formation during DO and are needed to promote osteogenic over chondrogenic lineage progression.