Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling

In this study we investigated if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of β-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in β-catenin^Prx1ER conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (μCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the β-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through β-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in β-catenin^Prx1ER conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.     

Inhibition of beta‐catenin signaling by Pb leads to incomplete fracture healing

There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb‐exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb‐treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased β‐catenin levels. Reduction of β‐catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb‐exposed mice throughout fracture healing. β‐catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in β‐catenin signaling, suggesting the Wnt/β‐catenin as a pivotal pathway in the influence of Pb on fracture repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1397–1405, 2014.     

Comparison of Fracture Healing Among Different Inbred Mouse Strains

Quantitative trait locus analysis can be used to identify genes critically involved in biological processes. No such analysis has been applied to identifying genes that control bone fracture healing. To determine the feasibility of such an approach, healing of femur fractures was measured between C57BL/6, DBA/2, and C3H inbred strains of mice. Healing was assessed by radiography and histology and measured by histomorphometry and biomechanical testing. In all strains, radiographic bridging of the fracture was apparent after 3 weeks of healing. Histology showed that healing occurred through endochondral ossification in all strains. Histomorphometric measurements found more bone in the C57BL/6 fracture calluses 7 and 10 days after fracture. In contrast, more cartilage was present after 7 days in the C3H callus, which rapidly declined to levels less than those of C57BL/6 or DBA/2 mice by 14 days after fracture. An endochondral ossification index was calculated by multiplying the callus percent cartilage and bone areas as a measure of endochondral ossification. At 7 and 10 days after fracture, this value was higher in C57BL/6 mice. Using torsional mechanical testing, normalized structural and material properties of the C57BL/6 healing femurs were higher than values from the DBA/2 or C3H mice 4 weeks after fracture. The data indicate that fracture healing proceeds more rapidly in C57BL/6 mice and demonstrate that genetic variability significantly contributes to the process of bone regeneration. Large enough differences exist between C57BL/6 and DBA/2 or C3H mice to permit a quantitative trait locus analysis to identify genes controlling bone regeneration.     

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     

Fracture healing is accelerated in the absence of the adaptive immune system

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild‐type (WT) and recombination activating gene 1 knockout (RAG1^?/?) mice lacking the adaptive immune system. Healing was investigated using micro–computed tomography (µCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1^?/? mice compared to the WT group. µCT evaluation of RAG1^?/? specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1^?/? compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1^?/? mice than in the WT mice. Endochondral ossification was accelerated in the RAG1^?/? mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1^?/? mice. In contrast, expression of anti‐inflammatory interleukin 10 (IL‐10) was strongly upregulated in RAG1^?/? mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1^?/? mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti‐inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair. © 2011 American Society for Bone and Mineral Research.     

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.     

EP1−/− mice have enhanced osteoblast differentiation and accelerated fracture repair

As a downstream product of cyclooxygenase 2 (COX‐2), prostaglandin E₂ (PGE₂) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro–computed tomographic (µCT), and biomechanical measures. EP1^?/? mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1^?/? mice also had an earlier appearance of tartrate‐resistant acid phosphatase (TRAcP)–positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1^?/? mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE₂ receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing. © 2011 American Society for Bone and Mineral Research.     

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14 days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.     

Disruption of glucocorticoid signaling in chondrocytes delays metaphyseal fracture healing but does not affect normal cartilage and bone development

States of glucocorticoid excess are associated with defects in chondrocyte function. Most prominently there is a reduction in linear growth but delayed healing of fractures that require endochondral ossification to also occur. In contrast, little is known about the role of endogenous glucocorticoids in chondrocyte function. As glucocorticoids exert their cellular actions through the glucocorticoid receptor (GR), we aimed to elucidate the role of endogenous glucocorticoids in chondrocyte function in vivo through characterization of tamoxifen-inducible chondrocyte-specific GR knockout (chGRKO) mice in which the GR was deleted at various post-natal ages. Knee joint architecture, cartilage structure, growth plates, intervertebral discs, long bone length and bone micro-architecture were similar in chGRKO and control mice at all ages. Analysis of fracture healing in chGRKO and control mice demonstrated that in metaphyseal fractures, chGRKO mice formed a larger cartilaginous callus at 1 and 2 week post-surgery, as well as a smaller amount of well-mineralized bony callus at the fracture site 4 week post-surgery, when compared to control mice. In contrast, chondrocyte-specific GR knockout did not affect diaphyseal fracture healing. We conclude that endogenous GC signaling in chondrocytes plays an important role during metaphyseal fracture healing but is not essential for normal long bone growth.     

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing

Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFA^fl/fl) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFA^fl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFA^fl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFA^fl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.     

Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost^−/− mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14 days), as well as at 28 days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area.Normal endochondral ossification progressed in wild type and Sost^−/− mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost^−/− fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost^−/− calluses over wild type with greater bone volume at day 10 (221%, p < 0.01). The resultant Sost^−/− united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p < 0.05), and the strength of the fractured Sost^−/− tibiae was greater than that that of wild type fractured tibiae.In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.     

Shock wave application enhances pertussis toxin protein-sensitive bone formation of segmental femoral defect in rats.

Extracorporeal shock waves (ESWs) elicit a dose-dependent effect on the healing of segmental femoral defects in rats. After ESW treatment, the segmental defect underwent progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Along with the intensive bone formation, there was a persistent increase in TGF-beta1 and BMP-2 expression. Pretreatment with pertussis toxin reduced ESW-promoted callus formation and gap healing, which presumably suggests that Gi proteins mediate osteogenic signaling. INTRODUCTION: Extracorporeal shock waves (ESWs) have previously been used to promote bone repair. In our previous report, we found that ESWs promoted osteogenic differentiation of mesenchymal cells through membrane perturbation and activation of Ras protein. In this report, we show that ESWs elicit a dose-dependent effect on the healing of segmental defects and that Gi proteins play an important role in mediating ESW stimulation. MATERIALS AND METHODS: Rats with segmental femoral defects were subjected to ESW treatment at different energy flux densities (EFD) and impulses. Bone mass (mineral density and calcium content), osteogenic activities (bone alkaline phosphatase activity and osteocalcin content), and immunohistochemistry were assessed. RESULTS: An optimal ESW energy (500 impulses at 0.16 mJ/mm2 EFD) stimulated complete bone healing without complications. ESW-augmented healing was characterized by significant increases (p < 0.01) in callus size, bone mineral density, and bone tissue formation. With exposure to ESW, alkaline phosphatase activity and osteocalcin production in calluses were found to be significantly enhanced (p < 0.05). After ESW treatment, the histological changes we noted included progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Intensive bone formation was associated with a persistent increase in transforming growth factor-beta 1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) expression, suggesting both growth factors were active in ESW-promoted bone formation. We also found that pertussis toxin, an inhibitor of membrane-bound Gi proteins, significantly reduced (p < 0.01) ESW promotion of callus formation and fracture healing. CONCLUSION: ESW treatments enhanced bone formation and the healing of segmental femoral defects in rats. It also seems likely that TGF-beta1 and BMP-2 are important osteogenic factors for ESW promotion of fracture healing, presumably through Gi protein-mediated osteogenic signaling.     

The role of morphogens in endochondral ossification

Summary The formation of bone occurs normally by one of two developmental processes: intramembranous or endochondral ossification. Endochondral ossification occurs in the morphogenesis of the limb buds and growth plates, and in the regeneration of bone following injury (fracture callus). Two classes of diffusible morphogen-like molecules (MLMs) involved in limb development are the bone morphogenetic proteins (BMPs) and retinoic acid (RA). These MLMs are associated, respectively, with the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) of the primitive limb bud. They function as potent regulators of pattern formation and are involved in tissue proliferation and differentiation. The presence of endochondral ossification in fracture callus suggests a role for MLMs in that process as well. To date, virtually nothing is known about the role of morphogens in the regeneration of bone (fracture healing). In this article, we review the current knowledge of MLMs in bone formation and propose a theory on their role in fracture healing. We hypothesize that MLMs involved in fracture healing may also express spatial and temporal information. A more complete understanding of the role of morphogens in both limb development and fracture healing is of major importance to practicing orthopedists and their patients.     

Zoledronic acid suppresses callus remodeling but enhances callus strength in an osteoporotic rat model of fracture healing

Mini-abstractIn this study, we demonstrated that the use of zoledronic acid does not impair fracture healing, but results in superior callus size and resistance at the fracture site, which could be the consequence of a lower rate of bone turnover due to its anti-catabolic effect.ObjectiveTo investigate the effect of inhibition of bone remodeling by the bisphosphonate, zoledronic acid, on callus properties in an osteoporotic rat model of fracture healing.MethodsOvariectomized (OVX) rats were randomly divided into four treatment groups (n = 24 per group): saline control (CNT); and three systemic zoledronic acid-injected groups (0.1 mg/kg), administered 1 day (ZOLD1), 1 week (ZOLW1), and 2 weeks (ZOLW2) after fracture. Rats were killed at either 6 or 12 weeks postoperatively. Postmortem analyses included radiography, microcomputed tomography, histology, histomorphometry, biomechanical tests, and nanoindentation tests.ResultsTreatment with zoledronic acid led to a significant increase in trabecular bone volume within the callus, as well as in callus resistance, compared to those in the saline control rats; delayed administration (ZOLW2) reduced intrinsic material properties, including ultimate stress and elastic modulus, and microarchitecture parameters, including bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and connectivity density (Conn.D), compared with ZOLD1 at 12 weeks after surgery. OVX had a negative effect on the progression of endochondral ossification at 6 weeks. Zoledronic acid administration at an early stage following fracture may bind to early callus, and thus not affect subsequent callus formation and endochondral ossification, while delayed administration (ZOLW2) mildly suppresses bony callus remodeling.ConclusionThe superior results obtained with zoledronic acid (ZOLD1, ZOLW1, and ZOLW2) compared to CNT in terms of callus size and resistance could be the consequence of a lower rate of bone turnover at the fracture site due to the anti-catabolic effect of zoledronic acid. Mild suppression of callus remodeling by delayed administration did not impair the initial phase of the fracture healing process.     

A novel mouse model to study fracture healing of the proximal femur

The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone. However, only a few experimental models exist to study metaphyseal bone healing in mice. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4-mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing.