Chuanyong Lu

Bone morphogenetic protein 2 stimulates endochondral ossification by regulating periosteal cell fate during bone repair

Bone repair depends on the coordinated action of numerous growth factors and cytokines to stimulate new skeletal tissue formation. Among all the growth factors involved in bone repair, Bone Morphogenetic Proteins (BMPs) are the only molecules now used therapeutically to enhance healing. Although BMPs are known as strong bone inducers, their role in initiating skeletal repair is not entirely elucidated. The aim of this study was to define the role of BMP2 during the early stages of bone regeneration and more specifically in regulating the fate of skeletal progenitors. During healing of non-stabilized fractures via endochondral ossification, exogenous BMP2 increased the deposition and resorption of cartilage and bone, which was correlated with a stimulation of osteoclastogenesis but not angiogenesis in the early phase of repair. During healing of stabilized fractures, which normally occurs via intramembranous ossification, exogenous BMP2 induced cartilage formation suggesting a role in regulating cell fate decisions. Specifically, the periosteum was found to be a target of exogenous BMP2 as shown by activation of the BMP pathway in this tissue. Using cell lineage analyses, we further show that BMP2 can direct cell differentiation towards the chondrogenic lineage within the periosteum but not the endosteum, indicating that skeletal progenitors within periosteum and endosteum respond differently to BMP signals. In conclusion, BMP2 plays an important role in the early stages of repair by recruiting local sources of skeletal progenitors within periosteum and endosteum and by determining their differentiation towards the chondrogenic and osteogenic lineages.

Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development.

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh-/- chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh-/- skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh-/- cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh-/- cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh-/- cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage - comprising osteoblast precursors and endothelial cells - as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh-/- embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

Multiple roles for CCR2 during fracture healing

SUMMARY Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory cells to sites of injury occurs in response to specific signaling pathways. The CC chemokine receptor type 2 (CCR2) is crucial for recruiting macrophages, as well as regulating osteoclast function. In this study, we examined fracture healing in Ccr2−/− mice. We first demonstrated that the expression of Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2−/− mice compared with wild-type controls at 3 days after injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after injury in mutant mice. At day 14, Ccr2−/− mice had less bone in their calluses. At day 21, Ccr2−/− mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2−/− osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2−/− mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.

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.

Effect of age on vascularization during fracture repair

Age affects fracture repair; however, the underlying mechanisms are not well understood. The goal of this study was to assess the effects that age has on vascularization during fracture healing. Tibial fractures were created in juvenile (4‐week‐old), middle‐aged (6‐month‐old), and elderly (18‐month‐old) mice. The length density and surface density of blood vessels within fracture calluses were analyzed using stereology at 7 days after fracture. The expression of molecules that regulate vascular invasion of the fracture callus was also compared among the three age groups by immunohistochemistry and in situ hybridization. At 7 days after fracture, juvenile mice had a higher surface density of blood vessels compared to the middle‐aged and elderly. Hypoxia‐inducible factor‐1α protein and transcripts of vascular endothelial growth factor were detected at 3 days postinjury in juvenile but not middle‐aged and elderly mice. Stronger Mmp‐9 and ‐13 expression was detected in fracture calluses at day 7 in the juvenile compared to the middle‐aged and elderly mice. At 21 days postfracture, expression of both Mmps was more robust in the elderly than juvenile and middle‐aged animals. These data indicate that age affects vascularization during fracture repair, and the changes we observed are directly correlated with altered expression of biochemical factors that regulate the process of angiogenesis. However, whether the increased vascularization is the cause or result of accelerated bone repair in juvenile animals remains unknown. Nonetheless, our results indicate that enhancing vascularization during fracture repair in the elderly may provide unique therapeutic opportunities.

Immunolocalization of BMPs, BMP antagonists, receptors, and effectors during fracture repair

Bone morphogenetic proteins (BMPs) are potent bone inducers used clinically to enhance fracture repair. BMPs have been shown to be produced in the fracture callus; however, the comparative expression of BMPs and BMP signaling components has only been partially examined at the cellular level. The aim of the present study was to establish a detailed spatiotemporal localization of BMPs and BMP signaling components in mouse models of stabilized and nonstabilized fractures. During healing of nonstabilized fractures, which occurs via endochondral ossification, BMP2, 3, 4, 5, and 8, noggin, BMPRIA, BMPRII, and pSmad 1/5/8 were immunolocalized in the activated periosteum as early as 3 days after fracture. BMP2, 4, 5, 6, 7, and 8 and noggin were also found in isolated inflammatory cells within granulation tissue during the early stages of repair, but not BMP receptors and effectors. During the soft callus phase of repair, all BMPs and BMP signaling components were detected in chondrocytes with various intensities of staining depending on the stage of chondrocyte differentiation and their location in the callus. The strongest staining was observed in hypertrophic chondrocytes with decreased intensity during the hard callus phase of repair. All BMPs and components of the BMP pathway were detected in osteoblasts and osteocytes within new bone, with strongest intensity of immunoreaction reported during the early soft callus phase followed by decreasing intensity during the hard callus phase of repair. Most components of the BMP pathway were also detected in endothelial cells associated with new bone. In stabilized fractures that heal strictly via intramembranous ossification, BMPs and BMP antagonists were detected in isolated inflammatory cells and BMP signaling components were not detectable in osteoblasts or osteocytes within new bone. In conclusion, the BMP signaling pathway is primarily activated during fracture healing via endochondral ossification, suggesting that this pathway may influence the mode of healing during the recruitment of skeletal progenitors.

The role of oxygen during fracture healing

Oxygen affects the activity of multiple skeletogenic cells and is involved in many processes that are important for fracture healing. However, the role of oxygen in fracture healing has not been fully studied. Here we systematically examine the effects of oxygen tension on fracture healing and test the ability of hyperoxia to rescue healing defects in a mouse model of ischemic fracture healing. Mice with tibia fracture were housed in custom-built gas chambers and groups breathed a constant atmosphere of 13% oxygen (hypoxia), 21% oxygen (normoxia), or 50% oxygen (hyperoxia). The influx of inflammatory cells to the fracture site, stem cell differentiation, tissue vascularization, and fracture healing were analyzed. In addition, the efficacy of hyperoxia (50% oxygen) as a treatment regimen for fracture nonunion was tested. Hypoxic animals had decreased tissue vascularity, decreased bone formation, and delayed callus remodeling. Hyperoxia increased tissue vascularization, altered fracture healing in un-complicated fractures, and improved bone repair in ischemia-induced delayed fracture union. However, neither hypoxia nor hyperoxia significantly altered chondrogenesis or osteogenesis during early stages of fracture healing, and infiltration of macrophages and neutrophils was not affected by environmental oxygen after bone injury. In conclusion, our results indicate that environmental oxygen levels affect tissue vascularization and fracture healing, and that providing oxygen when fractures are accompanied by ischemia may be beneficial.

Rejuvenation of the inflammatory system stimulates fracture repair in aged mice

Age significantly reduces the regenerative capacity of the skeleton, but the underlying causes are unknown. Here, we tested whether the functional status of inflammatory cells contributes to delayed healing in aged animals. We created chimeric mice by bone marrow transplantation after lethal irradiation. In this model, chondrocytes and osteoblasts in the regenerate are derived exclusively from host cells while inflammatory cells are derived from the donor. Using this model, the inflammatory system of middle‐aged mice (12 month old) was replaced by transplanted bone marrow from juvenile mice (4 weeks old), or age‐matched controls. We found that the middle‐aged mice receiving juvenile bone marrow had larger calluses and more bone formation during early stages and faster callus remodeling at late stages of fracture healing, indicating that inflammatory cells derived from the juvenile bone marrow accelerated bone repair in the middle‐aged animals. In contrast, transplanting bone marrow from middle‐aged mice to juvenile mice did not alter the process of fracture healing in juvenile mice. Thus, the roles of inflammatory cells in fracture healing may be age‐related, suggesting the possibility of enhancing fracture healing in aged animals by manipulating the inflammatory system.

MMP9 regulates the cellular response to inflammation after skeletal injury

Like other tissue injuries, bone fracture triggers an inflammatory response, which plays an important role in skeletal repair. Inflammation is believed to have both positive and negative effects on bone repair, but the underlying cellular mechanisms are not well understood. To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. These results suggested that the inflammatory response could influence skeletal cell differentiation after fracture. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, and which we previously showed is a potential regulator of cell fate decisions during fracture repair. Mmp9(-/-) mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9(-/-) compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9(-/-) mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9(-/-) stroma and periosteum. Following transplantation, Mmp9(-/-) mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Moreover, Mmp9(-/-) periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9(-/-) hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9(-/-) mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. Taken together, these results show that MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation.

Mechanical Stability Affects Angiogenesis during Early Fracture Healing

Objectives: The goal of this study was to determine to what extent mechanical stability affects vascular repair during fracture healing. Methods: Stabilized and nonstabilized tibia fractures were created in adult mice. Fracture tissues were collected at multiple time points during early fracture healing. Vasculature in fractured limbs was visualized by immunohistochemistry with an anti-PECAM-1 antibody on tissue sections and then quantified with stereology. Oxygen tension, vascular endothelial growth factor expression, and lactate accumulation at the fracture site were measured. Gene expression was compared between stabilized and nonstabilized fractures by microarray analysis. Results: We found that new blood vessel formation was robust by 3 days after fracture. Quantitative analysis showed that nonstabilized fractures had higher length density and surface density than stabilized fractures at 3 days after injury, suggesting that nonstabilized fractures were more vascularized. Oximetry analysis did not detect a significant difference in oxygen tension at the fracture site between stabilized and nonstabilized fractures during the first 3 days after injury. Further microarray analysis was performed to determine the effects of mechanical stability on the expression of angiogenic factors. No significant difference in the expression of vascular endothelial growth factors and other angiogenic factors was detected between stabilized and nonstabilized fractures. Conclusions: Mechanical instability promotes angiogenesis during early fracture healing and further research is required to determine the underlying mechanisms.