Characterization of Heterotopic Ossification Using Radiographic Imaging: Evidence for a Paradigm Shift

Heterotopic ossification (HO) is the growth of extra-skeletal bone which occurs following trauma, burns, and in patients with genetic bone morphogenetic protein (BMP) receptor mutations. The clinical and laboratory evaluation of HO is dependent on radiographic imaging to identify and characterize these lesions. Here we show that despite its inadequacies, plain film radiography and single modality microCT continue to serve as a primary method of HO imaging in nearly 30% of published in vivo literature. Furthermore, we demonstrate that detailed microCT analysis is superior to plain film and single modality microCT radiography specifically in the evaluation of HO formed through three representative models due to its ability to 1) define structural relationships between growing extra-skeletal bone and normal, anatomic bone, 2) provide accurate quantification and growth rate based on volume of the space-occupying lesion, thereby facilitating assessments of therapeutic intervention, 3) identify HO at earlier times allowing for evaluation of early intervention, and 4) characterization of metrics of bone physiology including porosity, tissue mineral density, and cortical and trabecular volume. Examination of our trauma model using microCT demonstrated two separate areas of HO based on anatomic location and relationship with surrounding, normal bone structures. Additionally, microCT allows HO growth rate to be evaluated to characterize HO progression. Taken together, these data demonstrate the need for a paradigm shift in the evaluation of HO towards microCT as a standard tool for imaging.     

BMP and mTOR signaling in heterotopic ossification: Does their crosstalk provide therapeutic opportunities?

Heterotopic ossification (HO) refers to the pathological formation of ectopic bone in soft tissues, it occurs following severe trauma or in patients with a rare genetic disorder known as fibrodysplasia ossificans progressiva. The pathological process of HO formation is a two-step mechanism: inflammation and destruction of connective tissues, followed by bone formation. The latter is further subdivided into three stages: fibroproliferation/angiogenesis, chondrogenesis, and osteogenesis. Currently, therapeutic options for HO are limited. New potential therapeutics will most likely arise from a more detailed understanding of the signaling pathways implicated in each stage of ectopic bone formation and molecular targets that may be effective at both the early and late stages of HO. Bone morphogenetic protein (BMP) signaling is believed to play a key role in the overall HO process. Recently, the mammalian target of rapamycin (mTOR) signaling pathway has received attention as a critical pathway for chondrogenesis, osteogenesis, and HO. Inhibition of mTOR signaling has been shown to block trauma-induced and genetic HO. Intriguingly, recent studies have revealed crosstalk between mTOR and BMP signaling. Moreover, mTOR has emerged as a factor involved in the early hypoxic and inflammatory stages of HO. We will summarize the current knowledge of the roles of mTOR and BMP signaling in HO, with a particular focus on the crosstalk between mTOR and BMP signaling. We also discuss the activation of AMP activated protein kinase (AMPK) by the most widely used drug for type 2 diabetes, metformin, which exerts a dual negative regulatory effect on mTOR and BMP signaling, suggesting that metformin is a promising drug treatment for HO. The discovery of an mTOR-BMP signaling network may be a potential molecular mechanism of HO and may represent a novel therapeutic target for the pharmacological control of HO.     

Sensory nerve induced inflammation contributes to heterotopic ossification

Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1(-/-) ), BMP2-mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation.     

Berberine and aspirin prevent traumatic heterotopic ossification by inhibition of BMP signalling pathway and osteogenic differentiation

Heterotopic ossification (HO) is a pathological process that often occurs in soft tissues following severe trauma. There is no effective therapy for HO. The BMP signalling pathway plays an essential role in the pathogenesis of HO. Our previous study showed that AMPK negatively regulates the BMP signalling pathway and osteogenic differentiation. The present study aims to study the effect of two AMPK activators berberine and aspirin on osteogenic differentiation and HO induced by traumatic injury. The effects of two AMPK activators, berberine and aspirin, on BMP signalling and osteogenic differentiation were measured by western blot, ALP and Alizarin red S staining in C3H10T1/2 cells. A mouse model with Achilles tenotomy was employed to assess the effects of berberine and aspirin on HO using μCT and histological analysis. First, our study showed that berberine and aspirin inhibited phosphorylation of Smad1/5 induced by BMP6 and the inhibition was attributed to the down-regulation of ALK2 expression. Second, the combination of berberine and aspirin yielded more potent effects on BMP signalling. Third, we further found that there was an additive effect of berberine and aspirin combination on osteogenic differentiation. Finally, we found that berberine and aspirin blocked trauma-induced ectopic bone formation in mice, which may be through suppression of phosphorylation of Smad1/5 in injured tissues. Collectively, these findings indicate that berberine and aspirin inhibit osteogenic differentiation in C3H10T1/2 cells and traumatic HO in mice, possibly through the down-regulation of the BMP signalling pathway. Our study sheds a light on prevention and treatment of traumatic HO using AMPK pharmacological activators berberine and aspirin.     

Modulation of bone morphogenetic protein antagonists to stimulate clinical osteogenesis

Bone morphogenetic proteins (BMPs) are important for the development and functioning of a wide variety of tissues and organ systems. Their ability to induce bone formation has been harnessed for clinical application. Specifically, local application of BMPs into fractures and fusions has shown some efficacy in inducing bone formation. However, clinical success has not been as robust as might be expected from the results obtained using animal models. This difference may be due to a number of mechanisms regulating BMP activity in vivo. One class of major regulators is the extracellular antagonist (e.g. Noggin, Gremlin, DAN), the dysfunction of which has been shown to result in ectopic bone formation in animal models and human disease. We hypothesize that local application of BMPs at high concentrations induces increased production of BMP antagonists, thereby limiting BMP activity and clinical efficacy. Therapies blocking the function of BMP antagonists should therefore result in enhanced BMP activity and increased bone formation. Furthermore, titrated systemic regulation of BMP antagonist may potentially reverse osteoporosis. Our collective experience with the clinical use of BMP illustrates the importance of understanding mechanisms of endogenous antagonism and regulation in the exogenous application of a protein as a therapeutic.     

Cell-based gene therapy for repair of critical size defects in the rat fibula

More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.     

Inhibition of IL-17 prevents the progression of traumatic heterotopic ossification

Traumatic heterotopic ossification (HO) is the abnormal formation of bone in soft tissues as a consequence of injury. However, the pathological mechanisms leading to traumatic HO remain unknown. Here, we report that aberrant expression of IL-17 promotes traumatic HO formation by activating β-catenin signalling in mouse model. We found that elevated IL-17 and β-catenin levels are correlated with a high degree of HO formation in specimens from patients and HO animals. We also show that IL-17 initiates and promotes HO progression in mice. Local injection of an IL-17 neutralizing antibody attenuates ectopic bone formation in a traumatic mouse model. IL-17 enhances the osteoblastic differentiation of mesenchymal stem cells (MSCs) by activating β-catenin signalling. Moreover, inhibition of IL-17R or β-catenin signalling by neutralizing antibodies or drugs prevents the osteogenic differentiation of isolated MSCs and decreases HO formation in mouse models. Together, our study identifies a novel role for active IL-17 as the inducer and promoter of ectopic bone formation and suggests that IL-17 inhibition might be a potential therapeutic target in traumatic HO.     

Substance P signaling mediates BMP-dependent heterotopic ossification

Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP(+) sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibit injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

Involvement of endogenous bone morphogenetic protein (BMP) 2 and BMP6 in bone formation

Although accumulated evidence has shown the bone anabolic effects of bone morphogenetic proteins (BMPs) that were exogenously applied in vitro and in vivo, the roles of endogenous BMPs during bone formation remain to be clarified. This study initially investigated expression patterns of BMPs in the mouse long bone and found that BMP2 and BMP6 were the main subtypes expressed in hypertrophic chondrocytes that induce endochondral bone formation. We then examined the involvement of the combination of these BMPs in bone formation in vivo by generating the compound-deficient mice (Bmp2+/-;Bmp6-/-). Under physiological conditions, these mice exhibited moderate growth retardation compared with the wild-type (WT) littermates during the observation period up to 52 weeks of age. Both the fetal and adult compound-deficient mice showed a reduction in the trabecular bone volume with suppressed bone formation, but normal bone resorption, whereas the single deficient mice (Bmp2+/- or Bmp6-/-) did not. When a fracture was created at the femoral midshaft and the bone healing was analyzed, the endochondral bone formation, but not intramembranous bone formation, was impaired by the compound deficiency. In the cultures of bone marrow cells, however, there was no difference in osteogenic differentiation between WT and compound-deficient cells in the presence or absence of the exogenous BMP2. We thus concluded that endogenous BMP2 and BMP6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions.     

Alterations in skeletal and mineral metabolism following thermal injuries

Severe burns and other chronic inflammatory diseases are associated with altered skeletal metabolism that result in an increased incidence of osteopenia. In thermally injured children and adults there is a dramatic decrease in bone formation accompanied with an increase or maintenance of bone resorption. Children also exhibit a growth delay and subsequently fail to reach a predicted stature. Animal models, including the thermal injury mouse model, are being used to understand the mechanisms behind the uncoupling of bone formation and resorption that occurs following a major burn. The model has numerous commonalities with the human condition such as reduced bone formation, increased bone resorption, and decreased endochondral growth. The mechanisms that modulate calcium and skeletal metabolism following a thermal injury are complex and likely involve a number of endocrine, cytokine, and immune factors. Specifically, the potential roles of glucocorticoids, growth hormone, insulin-like growth factor-1, parathyroid hormone, interleukin-1 and -6, and tumor necrosis factor alpha are addressed. Subsequent to the increased survival rate of burn victims, there has been a heightened focus on therapeutic interventions that prevent or decrease the impact of thermal injuries on the skeletal system. These include exercise programs, exogenous recombinant human growth hormone, insulin, and oxandrolone.     

Anaplerotic Accumulation of Tricarboxylic Acid Cycle Intermediates as Well as Changes in Other Key Metabolites During Heterotopic Ossification

Heterotopic ossification (HO) is the de novo formation of bone that occurs in soft tissue, through recruitment, expansion, and differentiation of multiple cells types including transient brown adipocytes, osteoblasts, chondrocytes, mast cells, and platelets to name a few. Much evidence is accumulating that suggests changes in metabolism may be required to accomplish this bone formation. Recent work using a mouse model of heterotopic bone formation reliant on delivery of adenovirus‐transduced cells expressing low levels of BMP2 showed the immediate expansion of a unique brown adipocyte‐like cell. These cells are undergoing robust uncoupled oxidative phosphorylation to a level such that oxygen in the microenvironment is dramatically lowered creating areas of hypoxia. It is unclear how these oxygen changes ultimately affect metabolism and bone formation. To identify the processes and changes occurring over the course of bone formation, HO was established in the mice, and tissues isolated at early and late times were subjected to a global metabolomic screen. Results show that there are significant changes in both glucose levels, as well as TCA cycle intermediates. Additionally, metabolites necessary for oxidation of stored lipids were also found to be significantly elevated. The complete results of this screen are presented here, and provide a unique picture of the metabolic changes occurring during heterotopic bone formation. J. Cell. Biochem. 117: 1044–1053, 2016. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Effect of mechanical loading on heterotopic ossification in cervical total disc replacement: a three-dimensional finite element analysis

The development of heterotopic ossification (HO) is considered one of the major complications following cervical total disc replacement (TDR). Even though previous studies have identified clinical and biomechanical conditions that may stimulate HO, the mechanism of HO formation has not been fully elucidated. The objective of this study is to investigate whether mechanical loading is a biomechanical condition that plays a substantial role to decide the HO formation. A finite element model of TDR on the C5–C6 was developed, and HO formation was predicted by simulating a bone adaptation process under various physiological mechanical loadings. The distributions of strain energy on vertebrae were assessed after HO formation. For the compressive force, most of the HO formation occurred on the vertebral endplates uncovered by the implant footplate which was similar to the Type 1 HO. For the anteriorly directed shear force, the HO was predominantly formed in the anterior parts of both the upper and lower vertebrae as the Type 2 HO. For both the flexion and extension moments, the HO shapes were similar to those for the shear force. The total strain energy was reduced after HO formation for all loading conditions. Two distinct types of HO were predicted based on mechanically induced bone adaptation processes, and our findings were consistent with those of previous clinical studies. HO formation might have a role in compensating for the non-uniform strain energy distribution which is one of the mechanical parameters related to the bone remodeling after cervical TDR.     

Elastin‐like‐polypeptide based fusion proteins for osteogenic factor delivery in bone healing

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029–1037, 2016     

The inhibition of skeletal changes in a rat burn model with a local muscle flap

The effects of early wound closure using a local muscle flap on the development of periosteal new bone formation in a rat burn model were studied. Following a full-thickness burn to one hind limb, periosteal new bone formation along the tibial diaphysis was measured by the use of the fluorochrome agent calcein and an image-analysis system. Prostaglandin E levels, a known inflammatory mediator, from the bone beneath the burn also were measured. Periosteal new bone formation was inhibited by 50 percent in animals that had debridement and wound closure with a gastrocnemius muscle flap and skin graft on postburn day 2 compared to untreated controls or animals closed with skin grafts only. There was a trend toward reduced prostaglandin E measurements from tibial sections in the early closure group compared to untreated controls. This study demonstrates that early wound closure using a local muscle flap inhibits the periosteal new bone formation which is possibly associated with the inflammation in a rat burn model.     

Bone morphogenetic protein (BMP)1-3 enhances bone repair

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFβ, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E₁ osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair.     

In vitro and in vivo osteogenic activity of licochalcone A

We investigated the in vitro and in vivo osteogenic activity of licochalcone A. At low concentrations, licochalcone A stimulated the differentiation of mouse pre-osteoblastic MC3T3-E1 subclone 4 (MC4) cells and enhanced the bone morphogenetic protein (BMP)-2-induced stimulation of mouse bi-potential mesenchymal precursor C2C12 cells to commit to the osteoblast differentiation pathway. This osteogenic activity of licochalcone A was accompanied by the activation of extracellular-signal regulated kinase (ERK). The involvement of ERK was confirmed in a pharmacologic inhibition study. Additionally, noggin (a BMP antagonist) inhibited the osteogenic activity of licochalcone A in C2C12 cells. Licochalcone A also enhanced the BMP-2-stimulated expression of various BMP mRNAs. This suggested that the osteogenic action of licochalcone A in C2C12 cells could be dependent on BMP signaling and/or expression. We then tested the in vivo osteogenic activity of licochalcone A in two independent animal models. Licochalcone A accelerated the rate of skeletal development in zebrafish and enhanced woven bone formation over the periosteum of mouse calvarial bones. In summary, licochalcone A induced osteoblast differentiation with ERK activation in both MC4 and C2C12 cells and it exhibited in vivo osteogenic activity in zebrafish skeletal development and mouse calvarial bone formation. The dual action of licochalcone A in stimulating bone formation and inhibiting bone resorption, as described in a previous study, might be beneficial in treating bone-related disorders.     

Lactacystin, a proteasome inhibitor, enhances BMP-induced osteoblastic differentiation by increasing active Smads

Proteasome inhibitors enhance bone formation and osteoblastic differentiation in vivo and in vitro. In the present study, we examined whether the molecular mechanisms of lactacystin, one of many proteasome inhibitors, stimulated the osteoblastic differentiation of C2C12 cells that is induced by bone morphogenetic proteins (BMPs). Pretreatment with lactacystin enhanced the alkaline phosphatase (ALP) activity induced by BMP2, BMP4 or BMP7, but lactacystin did not induce ALP in the absence of BMPs. In addition, lactacystin-stimulated BMP2 induced mRNA expression of ALP, type I collagen, osteonectin, osteocalcin, Id1, Osterix, and Runx2. Lactacystin maintained BMP2-induced phosphorylation of Smad1/5/8 and increased the length of time that these Smads were bound to target DNA. Moreover, lactacystin prevented BMP receptor-induced Smad degradation. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by lactacystin was also observed in primary osteoblasts. These findings suggest that pretreatment with lactacystin accelerates BMP-induced osteoblastic differentiation by increasing the levels of phosphorylated Smads, which are maintained because BMP receptor-induced degradation is inhibited. We propose that optimized stimulation by proteasome inhibitors in a clinical setting may facilitate autogenous or BMP-induced bone formation in areas of defective bone.     

Antisense targeting of TGF-beta1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-beta1 on bone formation remains elusive. In particular, the role of autocrine TGF-beta1 on human osteoblasts is largely unknown. Here we show the effect of TGF-beta1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-beta1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-beta1. Notably, TGF-beta1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-beta1. Moreover, TGF-beta1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-beta1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.