Block Bone Resorption Research Articles

Molecular and functional mapping of Plekhm1-Rab7 interaction in osteoclasts.

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases.

Novel Small Molecule Inhibitor of Osteoclast Differentiation

Osteoporosis results in over a million bone fractures each year despite current therapeutics that inhibit bone resorption. The need for better anti-osteoporotic therapeutics is clear. Recently, extracellular vesicles (EVs) containing RANK that are released by osteoclasts and serve as novel regulators of bone remodeling were identified. These RANK-containing EVs both block bone resorption by osteoclasts and stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. Small molecules with the same activity could prove to be a new class of therapeutic for treatment of bone disease. To seek such small molecules, a virtual screen identified small molecules that were predicted to bind RANKL in the same location as RANK, and an initial cell culture screen showed eighteen of the candidates reduced osteoclast formation in vitro at a concentration of 100 μM. In this project, one candidate, 3-Nitro-4-phosphobenzoic acid (NPA), was examined in greater detail. NPA does-dependently reduced recombinant RANKL-stimulated differentiation of RAW 264.7 cells into osteoclast-like cells with an IC50 of 38 μM. In calcitriol-stimulated mouse marrow, primary cultures that produce both osteoblasts and osteoclasts, NPA (50 μM) reduced osteoclast formation by 88%, while alkaline phosphatase positive osteoblast numbers were higher. These data show that NPA, in cell culture, is able to simultaneously inhibit osteoclast differentiation and promote osteoblast formation. Thus, NPA is a candidate to be a lead molecule for novel dual-function, small molecule, therapeutic agents to treat osteoporosis.

Analysis of osteoblast, osteoclast levels and radiographic patterns in the healing process of bone fractures (preliminary research)

Objectives: The healing process of a bone fracture goes through many phases. The hard callus phase was critical where the original structure was conducted. The hard callus growth depends on osteoblasts and osteoclasts active, and this condition can be analyzed on the radiograph. This study aimed to examine the analysis of bone fracture healing between osteoblasts and osteoclast numbers and radiographic patterns.
 Materials and Methods: The study used 12 male Wistar rats with an incomplete fracture in the right femur made by a dental tapered bur with 0.3 mm in length and 0.2 mm in depth. Digital radiographic examinations were carried out on days 0, 5, 10, 17, and 25 after fracturing in a lateral position. Furthermore, a radiographic analysis was performed using Image-J to obtain changes in the value of length and depth in the healing area. The research was conducted to find the radiopaque and radiolucent patterns and the number of osteoblasts and osteoclasts.
 Results: This study resulted in a change in the radiograph pattern. Callus formation resulted in fracture areas with a smaller distance from day 0 to day 25. The bone healing process begins with granulation tissue formation, followed by the gradual replacement of the connective tissue and bone. This process is comparable to the increase in osteoblasts up to day 25, which blocks bone resorption. Osteoclasts regulate bone resorption, and their number increases after 10 and 17 days to replace bone formation. Osteoclasts decline after 25 days because osteoblasts inhibit them, which control bone formation.
 Conclusion: The conclusions were obtained there are changes in the radiograph pattern. The radiopaque increased while the radiolucent decreased; the osteoclast pattern tended to be stable and lowered while the osteoblasts increased during the fracture healing process. The correlation of all the factors is very closely related.

POLR2A blocks osteoclastic bone resorption and protects against osteoporosis by interacting with CREB1.

Bone-resorbing osteoclasts significantly contribute to osteoporosis, and understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss. Here, we report that POLR2A is upregulated during osteoclastogenesis. Functional analyses showed that the inhibition of POLR2A decreased osteoclastogenesis, whereas the overexpression of POLR2A had completely opposite effects in vitro. Notably, the osteoclast-specific deletion of POLR2A blocks bone resorption in vivo. Furthermore, POLR2A loss-of-function suppresses estrogen deficiency-induced bone resorption. Mechanistically, POLR2A regulates the assembly of CREB1 on the regulatory elements of its target genes. Collectively, using genetic, pharmacological, and disease mouse models, we have identified a previously undescribed protein that interacts with CREB1 to regulate osteoclastic bone resorption.

Bisphosphonates in common pediatric and adult bone sarcomas

The therapeutic strategies proposed currently for bone sarcomas are based on neo-adjuvant chemotherapy, delayed en-bloc wide resection, and adjuvant chemotherapy. Unfortunately, bone sarcomas are characterized by high rates of poor drug response, with a high risk of drug resistance, local recurrence and/or a high propensity for induced metastases. The pathogenesis of bone sarcomas is strongly associated with dysregulation of local bone remodeling and increased osteolysis that plays a part in tumor development. In this context, bisphosphonates (BPs) have been proposed as a single agent or in combination with conventional drugs to block bone resorption and the vicious cycle established between bone and sarcoma cells. Pre-clinical in vitro studies revealed the potential "anti-tumor" activities of nitrogen-bisphosphonates (N-BPs). In pre-clinical models, N-BPs reduced significantly primary tumor growth in osteosarcoma and Ewing sarcoma, and the installation of lung metastases. In chondrosarcoma, N-BPs reduced the recurrence of local tumors after intralesional curettage, and increased overall survival. In pediatric and adult osteosarcoma patients, N-BPs have been assessed in combination with conventional chemotherapy and surgery in randomized phase 3 studies with no improvement in clinical outcome. The lack of benefit may potentially be explained by the biological impact of N-BPs on macrophage differentiation/recruitment which may alter CD8+-T lymphocyte infiltration. Thanks to their considerable affinity for the mineralized extracellular matrix, BPs are an excellent platform for drug delivery in malignant bone sites with reduced systemic toxicity, which opens up new opportunities for their future use.

Suitability and limitations of mesenchymal stem cells to elucidate human bone illness.

Functional impairment of mesenchymal stem cells (MSCs), osteoblast progenitor cells, has been proposed to be a pathological mechanism contributing to bone disorders, such as osteoporosis (the most common bone disease) and other rare inherited skeletal dysplasias. Pathological bone loss can be caused not only by an enhanced bone resorption activity but also by hampered osteogenic differentiation of MSCs. The majority of the current treatment options counteract bone loss, and therefore bone fragility by blocking bone resorption. These so-called antiresorptive treatments, in spite of being effective at reducing fracture risk, cannot be administered for extended periods due to security concerns. Therefore, there is a real need to develop osteoanabolic therapies to promote bone formation. Human MSCs emerge as a suitable tool to study the etiology of bone disorders at the cellular level as well as to be used for cell therapy purposes for bone diseases. This review will focus on the most relevant findings using human MSCs as an in vitro cell model to unravel pathological bone mechanisms and the application and outcomes of human MSCs in cell therapy clinical trials for bone disease.

Boosting mesenchymal stem cells regenerative activities on biopolymers-calcium phosphate functionalized collagen membrane

The regeneration of bone-soft tissue interface, using functional membranes, remains challenging and can be promoted by improving mesenchymal stem cells (MSCs) paracrine function. Herein, a collagen membrane, used as guided bone regeneration membrane, was functionalized by calcium phosphate, chitosan and hyaluronic acid hybrid coating by simultaneous spray of interacting species process. Composed of brushite, octacalcium phosphate and hydroxyapatite, the hybrid coating increased the membrane stiffness by 50%. After 7 days of MSCs culture on the hybrid coated polymeric membrane, biological studies were marked by a lack of osteoblastic commitment. However, MSCs showed an enhanced proliferation along with the secretion of cytokines and growth factors that could block bone resorption and favour endothelial cell recruitment without exacerbating polynuclear neutrophils infiltration. These data shed light on the great potential of inorganic/organic coated collagen membranes as an alternative bioactive factor-like platform to improve MSCs regenerative capacity, in particular to support bone tissue vascularization and to modulate inflammatory infiltrates.

Artesunate, an Anti-Malaria Agent, Attenuates Experimental Osteoarthritis by Inhibiting Bone Resorption and CD31hiEmcnhi Vessel Formation in Subchondral Bone.

Osteoarthritis (OA) is a common and debilitating joint disease worldwide without interventions available to reverse its progression. Artesunate (ART), an anti-malaria agent, possesses diverse biological activities, including the inhibition of osteoclastogenesis and angiogenesis in various cells, but its role in subchondral bone during OA progression is not known. Here, we explored the curative effects of ART on the pathogenesis of OA in anterior cruciate ligament transection (ACLT) mice models. We found that ART attenuated articular cartilage degeneration, defined by lowered histologic scoring of OA and retarded calcification of the cartilage zone. Moreover, ART improved the expression of lubricin and aggrecan and reduced the expression of collagen X (Col X) and matrix metalloproteinase-13 (MMP-13). In parallel, ART normalized abnormal subchondral bone remodeling by maintaining bone volume fraction (BV/TV) and subchondral bone plate thickness (SBP Th) and reducing trabecular pattern factor (Tb.pf) compared to the vehicle-treated mice. Our results indicated that ART suppressed osteoclastic bone resorption through regulating RANKL-OPG system, restored coupled bone remodeling by indirectly inhibiting TGF-β/Smad2/3 signaling. Additionally, ART abrogated CD31hiEmcnhi vessel formation via downregulating the expression of vascular endothelial growth factor (VEGF) and angiogenin-1 in subchondral bone. In conclusion, ART attenuates ACLT-induced OA by blocking bone resorption and CD31hiEmcnhi vessel formation in subchondral bone, indicating that this may be a new therapeutic alternative for OA.

Blueberry juice protects osteocytes and bone precursor cells against oxidative stress partly through SIRT1

Oxidative stress and abnormal osteocyte apoptosis are often related to dysregulation of bone turnover and chronic bone loss, and so fruit and vegetables with high antioxidant potential may play an important role in the prevention and/or management of osteoporosis. Osteocytes are the main regulators of bone remodelling. For the first time, we demonstrate here that blueberry juice (BJ), obtained from Vaccinium myrtillus, rich in polyphenols, shows antioxidant and antiosteoclastogenic properties in MLO‐Y4 osteocytes. We report that BJ prevents oxidative stress‐induced apoptosis and reverses the increase in receptor activator of nuclear factor κB ligand and sclerostin expression, crucial factors for osteoclast activation and bone resorption. BJ is also able to prevent oxidative stress‐induced cell cytotoxicity in bone marrow mesenchymal stromal cells (MSCs), which are considered to be an important tool for cell therapy in bone disorders. No significant difference in preventing these events was observed between BJ and blueberry dry extract containing equal amounts of total soluble polyphenols. We have also shown that blueberry acts as both an antioxidant and an activator of sirtuin type 1, a class III histone deacetylase involved in cell death regulation and considered a molecular target for blocking bone resorption without affecting osteoclast survival. Overall, these novel data obtained in osteocytes and MSCs may help us clarify the mechanisms by which blueberry counteracts oxidative stress‐induced damage in bone remodelling and osteogenesis at the cellular and molecular level. Our findings are consistent with the reported beneficial effects of blueberry on bone tissue reported in animal studies, which suggest that blueberry may be a useful supplement for the prevention and/or management of osteoporosis and osteogenic process.

Specialized Roles for Actin in Osteoclasts: Unanswered Questions and Therapeutic Opportunities.

Osteoclasts are cells of the hematopoietic lineage that are specialized to resorb bone. In osteoclasts, the actin cytoskeleton engages in at least two unusual activities that are required for resorption. First, microfilaments form a dynamic and structurally elaborate actin ring. Second, microfilaments bind vacuolar H+-ATPase (V-ATPase) and are involved in forming the V-ATPase-rich ruffled plasma membrane. The current review examines these two specialized functions with emphasis on the identification of new therapeutic opportunities. The actin ring is composed of substructures called podosomes that are interwoven to form a cohesive superstructure. Studies examining the regulation of the formation of actin rings and its constituent proteins are reviewed. Areas where there are gaps in the knowledge are highlighted. Microfilaments directly interact with the V-ATPase through an actin binding site in the B2-subunit of V-ATPase. This binding interaction is required for ruffled membrane formation. Recent studies show that an inhibitor of the interaction blocks bone resorption in pre-clinical animal models, including a model of post-menopausal osteoporosis. Because the unusual actin-based resorption complex is unique to osteoclasts and essential for bone resorption, it is likely that deeper understanding of its underlying mechanisms will lead to new approaches to treat bone disease.

The Bromodomain Inhibitor N-Methyl pyrrolidone Prevents Osteoporosis and BMP-Triggered Sclerostin Expression in Osteocytes.

(1) Background: In an adult skeleton, bone is constantly renewed in a cycle of bone resorption, followed by bone formation. This coupling process, called bone remodeling, adjusts the quality and quantity of bone to the local needs. It is generally accepted that osteoporosis develops when bone resorption surpasses bone formation. Osteoclasts and osteoblasts, bone resorbing and bone forming cells respectively, are the major target in osteoporosis treatment. Inside bone and forming a complex network, the third and most abundant cells, the osteocytes, have long remained a mystery. Osteocytes are responsible for mechano-sensation and -transduction. Increased expression of the osteocyte-derived bone inhibitor sclerostin has been linked to estrogen deficiency-induced osteoporosis and is therefore a promising target for osteoporosis management. (2) Methods: Recently we showed in vitro and in vivo that NMP (N-Methyl-2-pyrrolidone) is a bioactive drug enhancing the BMP-2 (Bone Morphogenetic Protein 2) induced effect on bone formation while blocking bone resorption. Here we tested the effect of NMP on the expression of osteocyte-derived sclerostin. (3) Results: We found that NMP significantly decreased sclerostin mRNA and protein levels. In an animal model of osteoporosis, NMP prevented the estrogen deficiency-induced increased expression of sclerostin. (4) Conclusions: These results support the potential of NMP as a novel therapeutic compound for osteoporosis management, since it preserves bone by a direct interference with osteoblasts and osteoclasts and an indirect one via a decrease in sclerostin expression by osteocytes.

The Hippo pathway in normal development and cancer.

The Hippo pathway is a central regulator of organ size and tissue homeostasis. Hippo kinases and adaptor proteins mediate the phosphorylation and inactivation of YAP and TAZ, two closely related transcription co-activators. The Hippo pathway responds to a variety of extracellular and intracellular signals, spanning from cell-cell contact and mechanical cues to ligands of G-protein-coupled receptors and metabolic avenues. In some instances, YAP/TAZ activation is tuned by forces that bypass the Hippo kinase module, adding further complexity to the biology of the pathway. Over the past two decades, the Hippo pathway has increasingly been connected with developmental processes and tissue repair, being intimately tied to the function of tissue-specific progenitor cells. Pervasive activation of YAP/TAZ has been recognized in a multitude of human tumors and connected with the acquisition of malignant traits, including resistance to anticancer therapies, distant dissemination and maintenance of cancer stem cells. On this ground, Hippo-related biomarkers are increasingly investigated in translational studies striving to identify prognostic and predictive factors. In addition, the dependency of many tumors on YAP/TAZ may be exploited for therapeutic purposes. Albeit no direct inhibitors are currently available, drug repositioning approaches provided hints that YAP/TAZ inhibition can be achieved with old drugs, such as cholesterol-lowering agents or compounds blocking bone resorption.

The Skeletal Phenotype in Neurofibromatosis Type 1 - Structural Defects, Molecular Mechanisms and Therapeutic Approaches

Patients with Neurofibromatosis Type 1 (NF1) develop subcutaneous benign tumors and dysfunction of multiple organs. About 30% of NF1 patients are affected by skeletal manifestations such as osteopenia, kyphoscoliosis, tibia bowing, or pseudarthrosis of the tibia. NF1 is caused by autosomal dominant mutation of the NF1 gene encoding the protein neurofibromin a regulator of the RAS/MAPK/ERK pathway. During the last decade pre-clinical studies provided multiple insights into the molecular mechanisms of the NF1 associated skeletal phenotype. They also allowed analysis of bone structural defects in tissue specific knockout mice and their comparison with the changes observed in patient samples. Importantly, preclinical models allowed exploration of the potential therapeutic approaches. In Prx1Cre;Nf1flox/flox and Col1a1Cre;Nf1flox/flox mice we demonstrated that loss of neurofibromin leads to multi scale defects in cortical bone: i) increased marco-porosity, ii) increased micro-porosity (osteocyte lacunae), iii) diminished mineralization, and iv) reduced organic matrix maturation. These changes significantly weaken overall mechanical strength of bone tissue in murine models. In NF1 patient bone samples similar changes were observed which may result in fractures. Inhibition of the RAS/MAPK/ ERK pathway with Lovastatin, Trametinib, PD198306, P98059, and PD0325901 normalized bone healing in Nf1 knockout mouse models. Downregulation of the RAS/MAPK/ERK pathway restored normal osteoblast differentiation/function and prevented accumulation of fibroblasts within the bone fracture site. Another approach tested in mouse models was to stimulate osteoblastic bone formation with bone morphogenic protein 2 or 7 (BMP-2, BMP-7) and block bone resorption with bisphosphonates. Recently Asfotase-α, replacing alkaline phosphatase (ALP) function specifically in bone tissue, was used to restore normal bone mass in inducible Tet-Off based Osx-Cre;Nf1flox/flox mice. In summary, neurofibromin controls development of the skeletal system by regulating the RAS/MAPK/ ERK pathway in chondrocytes, pre-osteoblasts, osteoblasts, and osteocytes. Preclinical model studies, exploring therapeutic approaches based on targeting RAS/MAPK/ERK pathway and ALP activity yield promising results which will likely instruct future clinical trial designs.

Regulation of Osteoblast Function in Myeloma Bone Disease By Semaphorin 4D

Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D.We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease.After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression.Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. DisclosuresRoodman:Amgen: Consultancy.

Peri-Implant Mucositis and Peri-Implantitis: A Current Understanding of Their Diagnosis, Clinical Implications, and a Report of Treatment Using a Combined Therapy Approach.

Abstract The clinical condition involving dental implants, characterized by soft tissue inflammation, bleeding, suppuration and rapid bone loss is widely known as peri implantitis. The main objective in the treatment of peri-implantis is to arrest the progression of the disease and at the same time to keep the dental implant in the mouth solving the inflammations signs of bleeding and pain. The aim of this paper is to highlight the effectiveness of the use of titanium brush and antimicrobial photodynamic therapy (aPDT) to decontaminate the implant surface, in association with regenerative procedures by means of autologous bone and demineralized bovine bone mineral (DBBM) in the treatment of peri-implantitis defects.

EFFECT OF LOCATION AND BONE GRAFT REMODELING ON RESULTS OF BRISTOW-LATARJET PROCEDURE

Introduction. Operation Bristow-Latarjet proved itself as one of the most effective and predictable surgical treatments. despite its widespread use, there are various complications associated with improper installation of the bone block and the violation of its remodeling.Objective: To obtain new data on the effect of location and remodeling of bone graft block on functional outcome and stability of the shoulder joint in patients with recurrent anterior instability after the operation Bristow-latarjet.Material and methods. The material for the study served as the analysis of results of treatment of 64 patients with posttraumatic recurrent anterior shoulder dislocation who underwent Bristow-latarjet operation. postoperatively, assessed a provision and the degree of bone remodeling unit according to computed tomography in the sagittal, axial slices, and through 3d modeling. To evaluate the functional outcome scale were used western Ontario Shoulder Index (wOSI) and Rowe scale.Results. At the level of the articular surface (congruent or flattening) in the axial plane were 89% bone blocks, too medially or laterally arranged 9% and 2% grafts, respectively. On sagittal cT images in the middle third of the articular surface of the scapula was located 28% of the bone blocks at the bottom 60%, in the upper third of 12%. Analysis of the dependence of the results of treatment of graft positioning showed that patients with excellent and good summary on the scale WOSI and Rowe, had a correct location of the bone block in the middle and lower third of the articular process of the blade. It can be assumed that excessive lateralized or medialized bone block position in the axial plane of a more profound effect on the outcome than cranial displacement of the latter with the sagittal plane. Bony union of the graft was found by CT in 74% of cases, soft tissue 26%, the degree of resorption of the graft revealed 0-1 84% 2-3 degree in 26% of cases. In the last periods of the investigation summary measure according to the scales WOSI and Rowe after Bristow surgery was 80±5 and 450±50 points, after Latarjet surgery 80±5 and 430±60 points. (p0,05).Conclusion. Positioning bone graft block affected the results of operations Bristow-Latarjet. Patients with excellent and good summary on the scale WOSI and Rowe had a correct location of the bone block relative to the articular surface of the scapula. The best overall performance on scales wOSI and Rowe were observed in patients with the fusion of the bone graft and block bone resorption does not exceed 0-1 degree compared with patients having soft tissue fusion and resorption 2-3 degree.

Molecular Mechanisms of Bone Metastasis: Which Targets Came from the Bench to the Bedside?

Bone metastases ultimately result from a complex interaction between cancer cells and bone microenvironment. However, prior to the colonization of the bone, cancer cells must succeed through a series of steps that will allow them to detach from the primary tumor, enter into circulation, recognize and adhere to specific endothelium, and overcome dormancy. We now know that as important as the metastatic cascade, tumor cells prime the secondary organ microenvironment prior to their arrival, reflecting the existence of specific metastasis-initiating cells in the primary tumor and circulating osteotropic factors. The deep comprehension of the molecular mechanisms of bone metastases may allow the future development of specific anti-tumoral therapies, but so far the approved and effective therapies for bone metastatic disease are mostly based in bone-targeted agents, like bisphosphonates, denosumab and, for prostate cancer, radium-223. Bisphosphonates and denosumab have proven to be effective in blocking bone resorption and decreasing morbidity; furthermore, in the adjuvant setting, these agents can decrease bone relapse after breast cancer surgery in postmenopausal women. In this review, we will present and discuss some examples of applied knowledge from the bench to the bed side in the field of bone metastasis.

New anti-resorptives and antibody mediated anti-resorptive therapy.

The ageing population and an increase in both the incidence and prevalence of cancer pose a healthcare challenge, some of which is borne by the orthopaedic community in the form of osteoporotic fractures and metastatic bone disease. In recent years there has been an increasing understanding of the pathways involved in bone metabolism relevant to osteoporosis and metastases in bone. Newer therapies may aid the management of these problems. One group of drugs, the antibody mediated anti-resorptive therapies (AMARTs) use antibodies to block bone resorption pathways. This review seeks to present a synopsis of the guidelines, pharmacology and potential pathophysiology of AMARTs and other new anti-resorptive drugs. We evaluate the literature relating to AMARTs and new anti-resorptives with special attention on those approved for use in clinical practice. Denosumab, a monoclonal antibody against Receptor Activator for Nuclear Factor Kappa-B Ligand. It is the first AMART approved by the National Institute for Health and Clinical Excellence and the US Food and Drug Administration. Other novel anti-resorptives awaiting approval for clinical use include Odanacatib. Denosumab is indicated for the treatment of osteoporosis and prevention of the complications of bone metastases. Recent evidence suggests, however, that denosumab may have an adverse event profile similar to bisphosphonates, including atypical femoral fractures. It is, therefore, essential that orthopaedic surgeons are conversant with these medications and their safe usage. Denosumab has important orthopaedic indications and has been shown to significantly reduce patient morbidity in osteoporosis and metastatic bone disease.

A novel approach to inhibit bone resorption: exosite inhibitors against cathepsin K.

Cathepsin K (CatK) is a major drug target for the treatment of osteoporosis. Potent active site-directed inhibitors have been developed and showed variable success in clinical trials. These inhibitors block the entire activity of CatK and thus may interfere with other pathways. The present study investigates the antiresorptive effect of an exosite inhibitor that selectively inhibits only the therapeutically relevant collagenase activity of CatK. Human osteoclasts and fibroblasts were used to analyse the effect of the exosite inhibitor, ortho-dihydrotanshinone (DHT1), and the active site inhibitor, odanacatib (ODN), on bone resorption and TGF-ß1 degradation. Cell cultures, Western blot, light and scanning electron microscopy as well as energy dispersive X-ray spectroscopy, molecular modelling and enzymatic assays were used to evaluate the inhibitors. DHT1 selectively inhibited the collagenase activity of CatK, without affecting the viability of osteoclasts. Both inhibitors abolished the formation of resorption trenches, with DHT1 having a slightly higher IC50 value than ODN. Maximal reductions of other resorption parameters by DHT1 and ODN were comparable, respectively 41% and 33% for total resorption surface, 46% and 48% for resorption depths, and 83% and 61% for C-terminal telopetide fragment (CTX) release. DHT1 did not affect the turnover of fibrosis-associated TGF-ß1 in fibroblasts, whereas 500 nM ODN was inhibitory. Our study shows that an exosite inhibitor of CatK can specifically block bone resorption without interfering with other pathways.

NO/GMP as mediators of estrogen effects in bone

Background Osteoporosis is a major health problem, especially in post-menopausal women. Current osteoporosis therapies have major drawbacks (e.g., estrogens increase breast cancer risks), and reduce fractures by only ~20-30%. Most osteoporosis drugs block bone resorption, and there is an urgent need to develop anabolic therapies that can stimulate bone formation. We previously showed that the NO/cGMP/PKG pathway mediates pro-proliferative effects of mechanical stimulation in osteoblasts and anti-apoptotic effects of estrogens in osteocytes [1,2]. These findings lead us to hypothesize that cGMP-elevating agents may have boneprotective effects. Results We tested cinaciguat, a prototype soluble guanylate cyclase stimulator, and nitrosylcobinamide (NO-Cbi), a novel NO-donor with anti-oxidant properties, in a mouse model of estrogen deficiency-induced osteoporosis [3]. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat, NO-Cbi, or low-dose estrogen replacement. Micro-CT analyses of tibiae showed that all three pharmacological interventions significantly improved trabecular bone architecture in ovariectomized animals, with similar effect sizes. Cinaciguat and NO-Cbi reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol, and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. As previously reported, ovariectomy dramatically increased osteoclast numbers, and this effect was completely reversed by estradiol. NO-Cbi significantly decreased the number of osteoclasts in ovariectomized mice, whereas cinaciguat had only modest effects, suggesting cGMP-independent effects of NO-Cbi in osteoclasts. Conclusion We conclude that estrogen deficiency represents a state of relative NO and cGMP deficiency, and that NO-dependent or NO-independent guanylate cyclase stimulation may represent a novel, anabolic treatment strategy for post-menopausal osteoporosis. These data confirm an important role of NO/cGMP signaling in bone biology.