Alexander S. Spiro

Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL‐1) and IL‐18, two cytokines of the IL‐1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL‐33, another IL‐1 family member, has not been addressed yet. Since we observed that the expression of IL‐33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL‐33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1‐deficient mice, which lack the IL‐33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL‐33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL‐33 completely abolished the generation of TRACP+ multinucleated osteoclasts, even in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF). Although our molecular studies revealed that IL‐33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL‐33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL‐33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL‐33 as an inhibitor of bone resorption.

BMP-7-induced ectopic bone formation and fracture healing is impaired by systemic NSAID application in C57BL/6-mice.

Nonsteroidal antiinflammatory drugs (NSAIDs) are known to potentially impair the fracture healing process. The aim of the present study was to determine if the impairment of bone healing by systemic NSAID application is, at least in part, due to an interaction of NSAIDs with the bone anabolic BMP‐7 pathway. Therefore, we first analyzed fracture healing in control and diclofenac‐treated mice, where we not only found a significant impairment of fracture healing due to diclofenac treatment as assessed by biomechanical testing and µCT imaging, but also found high coexpression of bone morphogenetic protein‐7 (BMP‐7) and cyclooxygenase‐2 (COX‐2) within the fracture callus of both groups. To experimentally address the possible interaction between BMP‐7 and COX‐2, we then induced ectopic bone formation in control (n = 10) and diclofenac‐treated mice (n = 10) by application of BMP‐7 (recombinant human OP‐1, rhOP‐1) into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact‐radiography, µCT, histology, and histomorphometry. Diclofenac application decreased the trabecular number and bone mass in the ectopic bone nodules significantly due to reduced osteoblast number and activity. These data demonstrate that the bone anabolic effect of BMP‐7 and fracture healing is impaired by diclofenac application, and suggest that the potential negative impact of NSAIDs on fracture healing is, at least in part, due to interference with BMP‐7 signaling.

Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.

Combined treatment of congenital pseudarthrosis of the tibia, including recombinant human bone morphogenetic protein-2: A CASE SERIES

The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) for the treatment of congenital pseudarthrosis of the tibia has been investigated in only one previous study, with promising results. The aim of this study was to determine whether rhBMP-2 might improve the outcome of this disorder. We reviewed the medical records of five patients with a mean age of 7.4 years (2.3 to 21) with congenital pseudarthrosis of the tibia who had been treated with rhBMP-2 and intramedullary rodding. Ilizarov external fixation was also used in four of these patients. Radiological union of the pseudarthrosis was evident in all of them at a mean of 3.5 months (3.2 to 4) post-operatively. The Ilizarov device was removed after a mean of 4.2 months (3.0 to 5.3). These results indicate that treatment of congenital pseudarthrosis of the tibia using rhBMP-2 in combination with intramedullary stabilisation and Ilizarov external fixation may improve the initial rate of union and reduce the time to union. Further studies with more patients and longer follow-up are necessary to determine whether this surgial procedure may significantly enhance the outcome of congenital pseudarthrosis of the tibia, considering the refracture rate (two of five patients) in this small case series.

Temporary screw epiphyseodesis of the distal tibia: a therapeutic option for ankle valgus in patients with hereditary multiple exostosis.

Background Ankle valgus in multiple hereditary exostoses (HME) is frequently seen and may result in activity related pain, deformity, or even early degenerative arthritis. Standard treatment protocols do not exist. The effect of medial screw hemiepiphysedesis of the distal tibia to correct ankle valgus is evaluated in this patient population. Methods A retrospective review of 9 patients with HME and ankle valgus deformity (15 affected extremities) who were treated by a screw hemiepiphyseodesis of the medial physis of the distal tibia is presented. Mean age at time of operation was 11.8±1.6 years (range: 9.6 to 14.7 y). All patients underwent clinical and radiological evaluation. To define the magnitude of ankle valgus we evaluated tibial tilt angle and Malhotra stations preoperatively, at time of screw removal and/or at time of the most recent clinical follow-up. Results In 11 extremities the screws were removed after an average time of 22.6±8.0 months (range: 12 to 35 mo) and all patients were examined after a mean time of 36.7±18.7 months after hemiepiphyseodesis (range: 13 to 69 mo). The mean preoperative tibiotalar tilt was 14.3±4.4 degrees (range: 9 to 22 degrees). At time of screw removal the tibiotalar tilt was normalized to 0.4±1.7 degrees (range: −2 to 4 degrees), according to a mean rate of correction of 0.58±0.23 degrees by the month (range: 0.23±0.92 degrees). With follow up 4 ankles demonstrated a change in the fibular station. None of the patients overcorrected into a severe varus, one patient developed a recurrent ankle valgus. Conclusions The temporary screw hemiepiphyseodesis of the distal tibial physis is an effective, technically simple, and minimally invasive technique to correct ankle valgus deformity in children with HME. Level of Evidence IV.

Short-Term Application of Dexamethasone Enhances Bone Morphogenetic Protein-7-Induced Ectopic Bone Formation In Vivo

BACKGROUND Long-term administration of glucocorticoids may lead to bone loss and osteoporosis as reported in previous experimental and clinical studies. On the other hand, several in vitro studies have demonstrated that dexamethasone treatment induces proliferation and differentiation of human and murine osteoblast precursors. Thereby, a positive interaction of dexamethasone with the osteoinductive bone morphogenetic proteins (BMPs) is reported in vitro, but in vivo studies are still missing. Thus, the aim of this study was to determine whether short-term application of dexamethasone may improve BMP-7-induced bone formation in vivo. METHODS Ectopic bone formation was induced in control and dexamethasone-treated mice by application of BMP-7 into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact radiography, microcomputed tomography, and histomorphometry. Furthermore, mice were subjected to histomorphometric analyses of their lumbar vertebrae and proximal tibiae to assess the systemic effect of short-term dexamethasone treatment on bone metabolism. RESULTS Dexamethasone application significantly increased the bone volume and osteoblast number of the ectopic bone nodules compared with untreated controls. Histomorphometric analyses of the lumbar vertebrae and proximal tibiae revealed no significant differences between the control and dexamethasone-treated mice. CONCLUSIONS This study demonstrates that BMP-7-induced ectopic bone formation is significantly enhanced by systemic short-term application of dexamethasone. These in vivo data confirm the results of previous in vitro studies and could be of interest for further studies with the intention to improve BMP-induced bone formation by short-term application of dexamethasone.

Osteolytic prostate cancer cells induce the expression of specific cytokines in bone-forming osteoblasts through a Stat3/5-dependent mechanism

Prostate cancer primarily metastasizes to bone, and the interaction of cancer cells with bone cells results in a local activation of bone formation and/or bone resorption. Since the cellular and molecular mechanisms underlying the development of these tumor-induced osteoblastic or osteolytic lesions are still poorly understood, we have compared the effects of two prostate cancer cell lines, osteoblastic MDA-PCa-2b cells and osteolytic PC-3 cells, on bone-forming osteoblasts. Using Affymetrix Gene Chip hybridization followed by qRT-PCR confirmation we were able to identify specific genes, including Smpd3 and Dmp1, whose expression is significantly reduced upon treatment with PC-3-conditioned medium. Moreover, we observed that PC-3-conditioned medium led to a marked induction of several cytokine genes, including Cxcl5, Cxcl12 and Tnfsf11, the latter one encoding for the osteoclast differentiation factor Rankl. Likewise, when we analyzed the effects of MDA-PCa-2b- and PC-3-conditioned medium on signal transduction in osteoblasts we did not only observe opposite effects on the canonical Wnt signalling pathway, but also a specific induction of Erk and Stat phosphorylation by PC-3-conditioned medium. Most importantly, the induction of Cxcl5, Cxcl12 and Tnfsf11 in osteoblasts by PC-3-conditioned medium was abrogated by the Stat3/5 inhibitor piceatannol, whereas the selective blockade of Stat1 and Erk activation had no effect. Together with the finding, that activated Stat3 in osteoblasts was detectable in bone biopsies from patients with osteolytic metastases, our data suggest that the Stat3/5-dependent activation of cytokine expression in osteoblasts may have a significant impact on cancer cell migration and proliferation, but also on osteoclast activation.

p65-Dependent production of interleukin-1β by osteolytic prostate cancer cells causes an induction of chemokine expression in osteoblasts

Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.

Anterior Femoral Epiphysiodesis for the Treatment of Fixed Knee Flexion Deformity in Spina Bifida Patients

Background Fixed knee flexion deformity is a common problem in spina bifida patients. Owing to interference with ambulation, this deformity may lead to anterior knee pain and progressive crouch gait. If conservative treatment fails, surgical procedures including supracondylar femoral extension osteotomy, joint distraction, and posterior capsulotomy have to be considered. Potential complications of these procedures are fractures, neurovascular lesions, knee instability, and recurrent deformity with continued growth. As fixed knee flexion deformity in spina bifida patients frequently occurs bilaterally, this results in higher perioperative and postoperative risks and prolonged recovery, making these relatively invasive methods less appealing. In the skeletal immature patient, anterior femoral stapling is a feasible method to treat fixed knee flexion deformity as could be shown in an earlier study. In this study, anterior femoral stapling was performed in a series of patients with diverse etiologies, and the overall success rate was then calculated. The aim of this study was to determine whether anterior femoral epiphysiodesis may improve fixed knee flexion deformity in a group of spina bifida patients. Methods Ten spina bifida patients with bilateral fixed knee flexion deformity (20 knees) were treated by anterior femoral epiphysiodesis through stapling. None of the patients received further knee procedures. The mean preoperative fixed knee flexion deformity was 20.3±9.9 degrees (range: 10 to 40 degrees). Clinical and radiographic follow-up examinations were performed every 3 to 6 months after surgery. Results All patients except 1 experienced significant improvement with a mean correction rate of 0.9±0.5 degrees per month (range: 0.2 to 1.9 degrees). The residual flexion deformity averaged 5.3±5.3 degrees (range: 0 to 15 degrees; P<0.001). Conclusions Anterior femoral epiphysiodesis through stapling is an effective and safe method for the treatment of fixed knee flexion deformity in growing children and adolescents with spina bifida. Level of Evidence Therapeutic Study, Level IV.

High incidence of osteochondral lesions after open reduction and internal fixation of displaced ankle fractures: Medium-term follow-up of 100 cases.

BACKGROUND The incidence of osteochondral lesions (OCLs) in association with displaced ankle fractures has only been examined in two previous studies. In both studies magnetic resonance imaging (MRI) was performed prior to open reduction and internal fixation (ORIF). Because MRI may overdiagnose or overestimate the extent of OCLs in an acute trauma setting the aim of this study was to determine the incidence of OCLs after ORIF of displaced ankle fractures using MRI at medium-term follow-up, and to analyse if the severity of fracture or the clinical outcome correlates with the incidence of OCLs. PATIENTS AND METHODS Following institutional review board approval a total of 100 patients (mean age, 41.3 years; range, 17.9-64.3 years) with a displaced ankle fracture who had undergone ORIF according to the AO principles were included in this study. The American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot score was used to quantify the clinical outcome and MR images were evaluated for OCLs of the talus and distal tibia after a mean of 34.5 months (range, 17.5-54.1 months). RESULTS OCLs were found in 40.4% of the patients. Logistic regression revealed a significant correlation between the severity of fracture and the incidence of OCLs. Patients with a trimalleolar fracture (p=0.04) or an ankle fracture dislocation (p=0.003) had a significantly higher risk for developing an OCL compared to those with a type B fracture. Logistic regression also demonstrated a significant correlation between the clinical outcome (AOFAS score) and the incidence of OCLs (p=0.01). The risk for developing an OCL increases up to 5.6% when the AOFAS score decreases by one point. CONCLUSION OCLs were frequently found in association with acute ankle fractures at medium-term follow-up, and the severity of fracture was associated with an increased number of OCLs. Considering the disadvantages of MRI including the high cost and limited availability, the results of this study may help to explain why anatomic surgical realignment of displaced ankle fractures may still be associated with poor clinical outcomes.