Methods for the reliable induction of heterotopic ossification in the conditional Alk2Q207D mouse.

OBJECTIVES: Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice. METHODS: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury. RESULTS: In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 µg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 µg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice. CONCLUSIONS: These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

Skeletal fluorosis: don't miss the diagnosis!

Skeletal fluorosis is a rare toxic osteopathy characterized by massive bone fixation of fluoride. The disease occurs as an endemic problem in some parts of the world and is the result of prolonged ingestion or rarely by inhalation of high amounts of fluoride. Radiographic presentation is mainly characterized by bone changes with osteocondensation and later ossification of many ligaments and interosseous membranes. Skeletal fluorosis is not clinically obvious and can be confused with other rheumatologic disorders. Its severity lies in the development of skeletal deformities and neurological complications. Management of fluorosis generally focuses on symptom treatment.

Leptin accelerates the pathogenesis of heterotopic ossification in rat tendon tissues via mTORC1 signaling.

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.

[Bone morphogenetic proteins (BMP): clinical application for reconstruction of bone defects]. / Proteínas morfogenéticas óseas (BMP): aplicación clínica para reconstrucción de defectos en hueso.

Since the introduction of bone morphogenetic proteins, their use has become an invaluable ally for the treatment of bone defects. These proteins are potent growth factors, related to angiogenic and osteogenic activity. The osteoinductive capacity of recombinant bone morphogenetic protein (rhBMP) in the formation of bone and cartilage has been confirmed in in vitro studies and evaluated in clinical trials. To obtain a therapeutic effect, administration is systemic, by injection over the physiological dose. Among the disadvantages, ectopic bone formation or high morbidity in cases of spinal fusion is observed. In this review, the roles of bone morphogenetic proteins in bone repair and clinical applications are analyzed. These findings represent advances in the study of bone regeneration and application of growth factors for more predictable results.

Botulinum Toxin-induced Muscle Paralysis Inhibits Heterotopic Bone Formation.

BACKGROUND: Short-term muscle atrophy induced by botulinum toxin A (BTxA) has been observed to impair osteogenesis in a rat closed femur fracture model. However, it is unclear whether the underlying mechanism is a direct effect of BTxA on muscle-bone interactions or an indirect effect that is driven by skeletal unloading. Because skeletal trauma in the closed fracture model also leads to disuse atrophy, we sought to mitigate this confounding variable by examining BTxA effects on muscle-bone interactions in two complementary in vivo models in which osteogenesis is induced in the absence of skeletal unloading. The overall aim of this study was to identify a potential strategy to inhibit pathological bone formation and heterotopic ossification (HO). QUESTIONS/PURPOSES: (1) Does muscle paralysis inhibit periosteal osteogenesis induced by a transcortical defect? (2) Does muscle paralysis inhibit heterotopic bone formation stimulated by intramuscular bone morphogenetic protein (BMP) injection? METHODS: Focal osteogenesis was induced in the right hindlimb of mice through surgical initiation of a small transcortical defect in the tibia (fracture callus; n = 7/group) or intramuscular injection of BMP-2 (HO lesion; n = 6/group), both in the presence/absence of adjacent calf paralysis. High-resolution micro-CT images were obtained in all experimental groups 21 days postinduction and total volume (ie, perimeter of periosteal callus or HO lesion) and bone volume (calcified tissue within the total volume) were quantified as primary outcome measures. Finally, these outcome measures were compared to determine the effect of muscle paralysis on inhibition of local osteogenesis in both studies. RESULTS: After a transcortical defect, BTxA-treated mice showed profound inhibition of osteogenesis in the periosteal fracture callus 21 days postsurgery compared with saline-treated mice (total volume: 0.08 ± 0.06 versus 0.42 ± 0.11 mm(3), p < 0.001; bone volume: 0.07 ± 0.05 versus 0.32 ± 0.07 mm(3), p < 0.001). Similarly, BMP-2-induced HO formation was inhibited by adjacent muscle paralysis at the same time point (total volume: 1.42 ± 0.31 versus 3.42 ± 2.11 mm(3), p = 0.034; bone volume: 0.68 ± 0.18 versus 1.36 ± 0.79 mm(3), p = 0.045). CONCLUSIONS: Our data indicate that BTxA-induced neuromuscular inhibition mitigated osteogenesis associated with both a transcortical defect and BMP-2-induced HO. CLINICAL RELEVANCE: Focal neuromuscular inhibition represents a promising new approach that may lead to a new clinical intervention to mitigate trauma-induced HO, a healthcare challenge that is severely debilitating for civilian and war-wounded populations, is costly to both the patient and the healthcare system, and currently lacks effective treatments.

Bone healing with oxytocin-loaded microporous ß-TCP bone substitute in ectopic bone formation model and critical-sized osseous defect of rat.

AIM: This study investigated the efficacy of the hypothalamic nonapeptide oxytocin (OT) by direct delivery to local defects using a microporous ß-tricalcium phosphate (TCP) as the carrier for the future applications as a method to achieve predictable bone regeneration of large osseous defects requiring sinus bone graft and guided bone regeneration procedures for implant placement. MATERIAL AND METHODS: Both the ectopic and new bone formation induced by the OT-loaded microporous ß-TCP powder was histomorphometrically compared with unloaded ß-TCP in a subcutaneous ectopic bone formation model and calvarial critical-sized defects (CSDs) in 45 rats. RESULTS: The OT-loaded ß-TCP clearly enhanced ectopic bone formation compared with the unloaded control group. A High initial OT dose (250 µg) significantly increased ectopic bone formation at an early healing time-point compared with a lower OT dose (50 µg). The OT-loaded samples displayed greater new bone formation in the rat calvarial CSDs. Extensive new bone formation was achieved in the calvarial CSDs with the higher OT dose. CONCLUSION: These results suggest that local OT delivery to bone substitute promotes new bone formation via an osteoinductive mode of action.

Voriconazole-associated soft tissue ossification: an undescribed cause of glenohumeral joint capsulitis.

Voriconazole-related periostitis has been increasingly described in the literature over the last several years as a recognizable disease entity, especially in lung transplant patients. This relationship should be considered when approaching immunosuppressed patients presenting with diffuse bone pain and imaging findings of periostitis. We present a case of voriconazole-associated periostitis, capsular and enthesial ossification and glenuhumeral capsulitis in a patient with a hematologic malignancy. To the authors' knowledge, soft tissue ossification associated with voriconazole has not been described in the radiology literature.

Hedgehog signaling inhibition by the small molecule smoothened inhibitor GDC-0449 in the bone forming prostate cancer xenograft MDA PCa 118b.

BACKGROUND: Hedgehog signaling is a stromal-mesenchymal pathway central to the development and homeostasis of both the prostate and the bone. Aberrant Hedgehog signaling activation has been associated with prostate cancer aggressiveness. We hypothesize that Hedgehog pathway is a candidate therapeutic target in advanced prostate cancer. We confirm increased Hedgehog signaling in advanced and bone metastatic castrate resistant prostate cancer and examine the pharmacodynamic effect of Smoothened inhibition by the novel reagent GDC-0449 in an experimental prostate cancer model. METHODS: Hedgehog signaling component expression was assessed in tissue microarrays of high grade locally advanced and bone metastatic disease. Male SCID mice subcutaneously injected with the bone forming xenograft MDA PCa 118b were treated with GDC-0449. Hedgehog signaling in the tumor microenvironment was assessed by proteomic and species specific RNA expression and compared between GDC-0449 treated and untreated animals. RESULTS: We observe Hedgehog signaling in high grade locally advanced and bone marrow infiltrating disease. Evidence of paracrine activation of Hedgehog signaling in the tumor xenograft, was provided by increased Sonic Hedgehog expression in human tumor epithelial cells, coupled with increased Gli1 and Patched1 expression in the murine stromal compartment, while normal murine stroma did not exhibit Hh signaling expression. GDC-0449 treatment attenuated Hh signaling as evidenced by reduced expression of Gli1 and Ptch1. Reduction in proliferation (Ki67) was observed with no change in tumor volume. CONCLUSIONS: GDC-0449 treatment is pharmacodynamically effective as evidenced by paracrine Hedgehog signaling inhibition and results in tumor cell proliferation reduction. Understanding these observations will inform the clinical development of therapy based on Hedgehog signaling inhibition.

Complications associated with posterior and transforaminal lumbar interbody fusion.

Posterior lumbar interbody fusion and transforaminal lumbar interbody fusion are commonly performed to obtain a 360° arthrodesis through a posterior-only approach. These techniques are currently used in the management of spondylolisthesis, degenerative scoliosis, pseudarthrosis, recurrent disk herniation, and chronic low back pain with associated degenerative disk disease. Several adverse events have been described, including intraoperative neurologic injury, implant migration or subsidence, dural tears, infection, heterotopic ossification, BMP-related radiculitis, and osteolysis. Although the use of newer materials (eg, bone morphogenetic proteins) and procedures (eg, minimally invasive surgery) is on the rise, they are associated with unique concerns. Understanding the potential adverse events and steps that can be taken to prevent, detect, and manage complications is critical in patient counseling and perioperative decision making.

Behaviour of human physeal chondro-progenitorcells in early growth plate injury response in vitro.

PURPOSE: The aim of this study was to investigate the proliferation and differentiation behaviour of a defined cell population gained from the human growth plate, namely, chondro-progenitorcells (CPCs), in the initial inflammatory phase of growth plate injury response in vitro. METHODS: Growth plate cells were sorted via FACS and differentiated along adipogenic and osteogenic lineage to confirm their progenitor features. To mimic the inflammatory phase of injury response at the growth plate they were treated with IL-1ß and exposed to cyclic mechanical loading. A BrdU assay was used to investigate CPC proliferation. CPC differentiation behaviour was analysed by RT-PCR. RESULTS: CPCs (CD45-, CD34-, CD73+, CD90+, and CD105+) showed a successful differentiation along adipogenic and osteogenic lineage. Under conditions simulating the inflammatory phase of injury response at the growth plate in vitro CPCs differentiated towards hypertrophy while chondrogenesis and ossification were inhibited. Proliferation was not significantly altered. CONCLUSION: This study showed that CPCs can be isolated from the human growth plate and expanded in vitro. In the first phase of injury response at the growth plate these cells differentiate towards hypertrophy. As longitudinal growth is obtained by chondrocyte proliferation and volume increase during hypertrophy this maturation might be the first step towards post-traumatic growth disorders such as unwanted premature ossification of the growth plate.

Recombinant human bone morphogenetic protein 2-induced heterotopic ossification of the retroperitoneum, psoas muscle, pelvis and abdominal wall following lumbar spinal fusion.

A 45-year-old man presented with vertebral collapse at L5 as an initial manifestation of multiple myeloma and underwent spinal fusion surgery using recombinant human bone morphogenetic protein-2 (rhBMP-2). Subsequent computed tomography (CT) scans and X-rays revealed heterotopic ossification of the left psoas muscle, pelvis, and anterior abdominal wall. While the occurrence of heterotopic ossification has previously been reported when rhBMP-2 has been used for spinal fusion surgery, this case demonstrates that it can occur to a much greater degree than previously seen.

[Study of the expression level of zinc finger factor osterix associated with fluoride bone injury].

OBJECTIVE: To observe the expression of zinc finger transcription factor Osterix (Osx) in different groups of floride (F) exposure in order to investigate its role in the cause of fluoride bone injuries and ossification around bone. METHODS: Western blot and fluorescence real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expressions of Osx mRNA and its protein in fibroblast, and the concentrations of F was 0.01, 0.1, 10 mg/L in experimental groups, and the control was 0 mg/L. RESULTS: The levels of Osx protein in F exposure groups increased at first and then decreased with the levels of F exposure, in which a significant increases were found after fibroblasts were exposed to F for about 24-48 h in all experimental groups (P < 0.05). In comparition with the 0 h F exposure group, the trend of Osx was the same, the results of RT-PCR indicated that the levels of Osx mRNA significantly increased in the group of 24-48 h F exposure groups (P < 0.05), overall, the trends of Osx mRNA level were similar to the trend of Osx protein, which went up at first and then went down in each group. CONCLUSION: The expressions of Osx factor in fibroblast could be promoted by fluoride, which could play an important role in the process of the transformation from fibroblasts to osteoblast induced by fluoride, and in the development of fluoride bone injuries and ossification around bone.

Heterotopic ossification after local steroid injection.

Pachydermodactyly (PDD) is a rare, benign disease associated with progressive swelling of the periarticular soft tissue of phalangeal hand joints typically treated with local steroid injections. We present a case of a 37-year-old man with PDD treated with local steroid injections. He later developed heterotopic ossification and para-articular calcifications in the injection sites. Heterotopic ossification is not associated with PDD nor is it a recognised complication of local steroid injections. This is the first case in literature of heterotopic ossification occurring after local steroid injection and brings to attention a new potential complication of a widely performed procedure.

Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration.

We report chondrocyte phenotype and ectopic ossification in a collagenase-induced patellar tendon injury model. Collagenase or saline was injected intratendinously in one limb. The patella tendon was harvested for assessment at different times. There was an increase in cellularity, vascularity, and loss of matrix organization with time after collagenase injection. The tendon did not heal histologically until week 32. Ectopic mineralization as indicated by von Kossa staining started from week 8. Tendon calcification was mediated by endochondral ossification, as shown by expression of type X collagen. viva CT imaging and polarization microscopy showed characteristic bony porous structures and collagen fiber arrangement, respectively, in the calcific regions. Marrow-like cells and blood vessels were observed inside calcific deposits. Chondrocyte-like cells as indicated by morphology, expression of type II collagen, and sox 9 were seen around and embedded inside the calcific deposits. Fibroblast-like cells expressed type II collagen and sox 9 at earlier times, suggesting that erroneous differentiation of healing tendon fibroblasts may account for failed healing and ossification in collagenase-induced tendon degeneration. Because this animal model replicates key histopathological changes in calcific tendinopathy, it can be used as a model for the study of its pathogenesis at the patellar tendon.

Morphological changes of injected calcium phosphate cement in osteoporotic compressed vertebral bodies.

SUMMARY: This study was undertaken to investigate the radiologic and clinical outcomes of vertebroplasty with calcium phosphate (CaP) cement in patients with osteoporotic vertebral compression fractures. The morphological changes of injected CaP cement in osteoporotic compressed vertebral bodies were variable and unpredictable. We suggest that the practice of vertebroplasty using CaP should be reconsidered. INTRODUCTION: Recently, CaP, an osteoconductive filler material, has been used in the treatment of osteoporotic compression fractures. However, the clinical results of CaP-cement-augmented vertebrae are still not well established. The purpose of this study is to assess the clinical results of vertebroplasty with CaP by evaluating the morphological changes of CaP cement in compressed vertebral bodies. METHODS: Fourteen patients have been followed for more than 2 years after vertebroplasty. The following parameters were reviewed: age, sex, T score, compliance with osteoporosis medications, visual analog scale score, compression ratio, subsequent compression fractures, and any morphological changes in the filler material. RESULTS: The morphological changes of injected CaP included reabsorption, condensation, bone formation (osteogenesis), fracture of the CaP solid hump, and heterotopic ossification. Out of 14 patients, 11 (78.6%) developed progression of the compression of the CaP-augmented vertebral bodies after vertebroplasty. CONCLUSIONS: The morphological changes of the injected CaP cement in the vertebral bodies were variable and unpredictable. The compression of the CaP-augmented vertebrae progressed continuously for 2 years or more. The findings of this study suggest that vertebroplasty using CaP cement should be reconsidered.

Heterotopic osteogenesis by murine demineralized incisors at lesions sites induced by concanavalin a in mice.

The aim of this study was to examine the effects of Concanavalin A (Con A) administration on the early (preosteogenic) and late stages of osteogenesis induced by implantation of demineralized murine incisors into syngeneic mice. Local administration of Con A resulted in an increased yield of demineralized incisor-induced bone when injected during the preosteogenic stage of induction. This effect was not observed when Con A was injected after heterotopic osteogenesis had been established. This suggests that Con A recruits osteoprogenitor cells, but does not stimulate differentiated chondroblasts and osteoblasts.

Induction of cementogenesis and periodontal ligament regeneration by recombinant human transforming growth factor-beta3 in Matrigel with rectus abdominis responding cells.

BACKGROUND AND OBJECTIVE: In primates and in primates only, the transforming growth factor-b proteins induce endochondral bone formation. Transforming growth factor-b3 also induces periodontal tissue regeneration. Two regenerative treatments using human recombinant transforming growth factor-b3 were examined after implantation in mandibular furcation defects of the nonhuman primate, Papio ursinus. MATERIAL AND METHODS: Class III furcation defects were surgically created bilaterally in the mandibular first and second molars of two adult Chacma baboons (P. ursinus). Different doses of recombinant transforming growth factor-beta3 reconstituted with Matrigel matrix were implanted in the rectus abdominis muscle to induce heterotopic ossicles for subsequent transplantation to selected furcation defects. Twenty days after heterotopic implantation, periodontal defects were re-exposed, further debrided and implanted with minced fragments of induced heterotopic ossicles. Contralateral class III furcation defects were implanted directly with recombinant transforming growth factor-beta3 in Matrigel matrix with the addition of minced fragments of autogenous rectus abdominis muscle. Treated quadrants were not subjected to oral hygiene procedures so as to study the effect of the direct application of the recombinant morphogen in Matrigel on periodontal healing. Histomorphometric analyses on undecalcified sections cut from specimen blocks harvested on day 60 measured the area of newly formed alveolar bone and the coronal extension of the newly formed cementum along the exposed root surfaces. RESULTS: Morphometric analyses showed greater alveolar bone regeneration and cementogenesis in furcation defects implanted directly with 75 microg of transforming growth factor-beta3 in Matrigel matrix with the addition of minced muscle tissue. CONCLUSION: Matrigel matrix is an optimal delivery system for the osteogenic proteins of the transforming growth factor-beta superfamily, including the mammalian transforming growth factor-beta3 isoform. The addition of minced fragments of rectus abdominis muscle provides responding stem cells for further tissue induction and morphogenesis by the transforming growth factor-beta3 protein.

Complications of recombinant human BMP-2 for treating complex tibial plateau fractures: a preliminary report.

UNLABELLED: Bone morphogenic proteins (BMPs) are potent osteoinductive agents. Their use in fracture surgery is still being studied and the clinical indications are evolving. Heterotopic bone after BMP use in spine surgery is a known complication. While some literature describes the ability of BMP to enhance fracture healing, few articles describe complications of BMP. In tibial plateau fractures, after elevating the cartilage en mass, a subchondral void may be created in these fractures. Structural support provided by bone void-filling agents can be augmented with osteoinduction achieved by BMP. We asked whether heterotopic bone formation would occur more frequently with BMP-2 when used in tibial plateau fractures and whether BMP-2 enhanced the ability to maintain surgically restored subchondral bone integrity. Heterotopic bone developed more frequently in patients receiving BMP (10 of 17) than in patients not receiving BMP (one of 23). Four patients receiving BMP and no patients not receiving BMP underwent removal of heterotopic bone. Maintenance of subchondral bone integrity was similar without and with the use of BMP. BMP is a potent osteoinductive agent; however, when used for an off-label indication in periarticular situations, complications such as heterotopic bone are common and increase reoperation rates. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Synthetic alginate is a carrier of OP-1 for bone induction.

Bone morphogenetic proteins (BMPs) can induce bone formation in vivo when combined with appropriate carriers. Several materials, including animal collagens and synthetic polymers, have been evaluated as carriers for BMPs. We examined alginate, an approved biomaterial for human use, as a carrier for BMP-7. In a mouse model of ectopic bone formation, the following four carriers for recombinant human OP-1 (BMP-7) were tested: alginate crosslinked by divalent cations (DC alginate), alginate crosslinked by covalent bonds (CB alginate), Type I atelocollagen, and poly-D,L-lactic acid-polyethyleneglycol block copolymer (PLA-PEG). Discs of carrier materials (5-mm diameter) containing OP-1 (3-30 microg) were implanted beneath the fascia of the back muscles in six mice per group. These discs were recovered 3 weeks after implantation and subjected to radiographic and histologic studies. Ectopic bone formation occurred in a dose-dependent manner after the implantation of DC alginate, atelocollagen, and PLA-PEG, but occurred only at the highest dose implanted with CB alginate. Bone formation with DC alginate/OP-1 composites was equivalent to that with atelocollagen/OP-1 composites. Our data suggest DC alginate, a material free of animal products that is already approved by the FDA and other authorities, is a safe and potent carrier for OP-1. This carrier may also be applicable to various other situations in the orthopaedic field.