Therapy Of Bone Diseases Research Articles

Colloid, adhesive and release properties of nanoparticular ternary complexes between cationic and anionic polysaccharides and basic proteins like bone morphogenetic protein BMP-2

Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases.

Beta-adrenergic signaling affect osteoclastogenesis via osteocytic MLO-Y4 cells' RANKL production

The sympathetic nervous system play a pivotal role in bone remodeling through β-adrenoceptor (β-AR). However, it is not well documented whether the β-adrenoceptor pathway has the potential to influence osteocytes. In this study, cell viability, the expression of β-AR subtypes, enzymes of catecholamine synthesis or degradation, bone-related gene and protein in osteocytic MLO-Y4 cells were investigated by β-adrenergic stimulation. Isoproterenol (ISO) promoted RANKL to OPG expression in osteocytes, as well as osteoclasts formation in osteocytes-RAW264.7 cell co-cultures but not RAW264.7 cell monoculture. The ISO-stimulated effect was enhanced in β1-AR antagonist pretreatment, but was rescued by blocking β2-AR. The results indicate that β1-and β2-AR play reciprocal roles on MLO-Y4 cells in the regulation of osteoclastogenesis, and osteocyte β-adrenergic signaling might be a new valuable therapy for bone disease.

Bone demineralization is improved by ivacaftor in patients with cystic fibrosis carrying the p.Gly551Asp mutation

Low bone mineral density (BMD) is a common problem in adults with cystic fibrosis (CF), the etiology of which is multifactorial. In this study, we provide the first evidence that ivacaftor improves BMD in CF patients carrying the p.Gly551Asp mutation. Consistently, in vitro experiments with TNF-α-stimulated primary F508del-CFTR osteoblasts demonstrated that correction of p.Phe508del-CFTR markedly decreased RANKL protein production, a major factor of bone resorption. These clinical and fundamental observations suggest that rescue of mutated CFTR protein improves bone remodeling and support the link between CFTR and bone cell physiology. These findings represent a step forward in the development of potential new therapies for CF-related bone disease.

Pharmacologically Inactive Bisphosphonates as an Alternative Strategy for Targeting Osteoclasts: In Vivo Assessment of 5-Fluorodeoxyuridine-Alendronate in a Preclinical Model of Breast Cancer Bone Metastases.

Bisphosphonates have effects that are antiresorptive, antitumor, and antiapoptotic to osteoblasts and osteocytes, but an effective means of eliciting these multiple activities in the treatment of bone metastases has not been identified. Antimetabolite-bisphosphonate conjugates have potential for improved performance as a class of bone-specific antineoplastic drugs. The primary objective of the study was to determine whether an antimetabolite-bisphosphonate conjugate will preserve bone formation concomitant with antiresorptive and antitumor activity. 5-FdU-ale, a highly stable conjugate between the antimetabolite 5-fluoro-2'-deoxyuridine and the bisphosphonate alendronate, was tested for its therapeutic efficacy in a mouse model of MDA-MB231 breast cancer bone metastases. In vitro testing revealed osteoclasts to be highly sensitive to 5-FdU-ale. In contrast, osteoblasts had significantly reduced sensitivity. Tumor cells were resistant in vitro but in vivo tumor burden was nevertheless significantly reduced compared with untreated mice. Sensitivity to 5-FdU-ale was not mediated through inhibition of farnesyl diphosphate synthase activity, but cell cycle arrest was observed. Although serum tartrate-resistant acid phosphatase (TRAP) levels were greatly reduced by both drugs, there was no significant decrease in the serum bone formation marker osteocalcin with 5-FdU-ale treatment. In contrast, there was more than a fivefold decrease in serum osteocalcin levels with alendronate treatment (p < 0.001). This finding is supported by time-lapse micro-computed tomography analyses, which revealed bone formation volume to be on average 1.6-fold higher with 5-FdU-ale treatment compared with alendronate (p < 0.001). We conclude that 5-FdU-ale, which is a poor prenylation inhibitor but maintains potent antiresorptive activity, does not reduce bone formation and has cytostatic antitumor efficacy. These results document that conjugation of an antimetabolite with bisphosphonates offers flexibility in creating potent bone-targeting drugs with cytostatic, bone protection properties that show limited nephrotoxicity. This unique class of drugs may offer distinct advantages in the setting of targeted adjuvant therapy and chemoprevention of bone diseases. © 2016 American Society for Bone and Mineral Research.

Comparative study of (Asp)7-CHOL-modified liposome prepared using pre-insertion and post-insertion methods for bone targeting in vivo

Specific delivery of drugs to bone tissue is very challenging due to the architecture and structure of bone tissue. A seven-repeat sequence of aspartate, a representative bone-targeting oligopeptide, is preferentially used for targeted therapy for bone diseases. In this study, Asp7-cholesterol((Asp)7-CHOL) was synthesized and (Asp)7-CHOL-modified liposome loaded with doxorubicin (DOX) was successfully prepared using both pre-insertion (pre-L) and post-insertion (post-L) methods. The formulation was optimized according to particle size, zeta potential and the drug-loading efficiency of the liposome. In addition, the bone affinity of the (Asp)7-CHOL-modified liposome was evaluated using a hydroxyapatite (HA) absorption method. The results suggested that (Asp)7-CHOL-modified liposome show excellent HA absorption; pre-L showed slightly higher HA binding than post-L. However, post-L had a higher DOX entrapment efficiency than pre-L. In vivo imaging further demonstrated that pre-L showed a higher bone-targeting efficiency than post-L, which was consistent with in vitro results. In all, (Asp)7-CHOL-modified liposome showed excellent bone-targeting activity, suggesting their potential for use as a drug delivery system for bone disease-targeted therapies.

Targeting the SH3 domain of human osteoclast-stimulating factor with rationally designed peptoid inhibitors.

Human osteoclast-stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption. The protein contains a modular Src homology 3 (SH3) domain that mediates the intermolecular recognition and interaction of hOSF with its biological partners. Here, we proposed targeting the hOSF SH3 domain to disrupt hOSF-partner interactions for bone disease therapy by using SH3 inhibitors. In the procedure, the primary sequences of three known hOSF-interacting proteins (c-Src, SMN and Sam68) were parsed, from which totally 31 octapeptide segments that contain the core SH3-binding motif PXXP were extracted, and their binding behavior to hOSF SH3 domain was investigated at structural level using a biomolecular modeling protocol. Several SH3-binding candidates were identified theoretically and then determined to have high or moderate affinity for the domain using fluorescence spectroscopy assays. One potent peptide (425) APPARPVK(432) (Kd = 3.2 μM), which corresponds to the residues 425-432 of Sam68 protein, was used as template to derive N substitution of peptides (peptoids). Considering that proline is the only endogenous N-substituted amino acid that plays a critical role in SH3-peptide binding, the substitution was addressed at the two key proline residues (Pro427 and Pro430) of the template peptide with nine N-substituted amino acid types. By systematically evaluating the structural and energetic effects of different N-substituted amino acids presenting at the two proline sites on peptide binding, we rationally designed five peptoid inhibitors and then determined in vitro their binding affinity to hOSF SH3 domain. Consequently, two designed peptoids APPAR(N-Clp)VK and APPAR(N-Ffa)VK with Pro430 replaced by N-Clp and N-Ffa were confirmed to have increased (Kd = 0.87 μM) and comparable (Kd = 2.9 μM) affinities relative to the template, respectively. In addition, we also found that the Pro427 residue plays an essential role in restricting peptide/peptoid conformations to polyproline II (PPII) helix as the basic requirement of SH3 binding so that the residue cannot be modified. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells.

: The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies for tissue engineering, bone disease, obesity, and metabolic-disorders.

Therapy for bone metastasis from different cancers ⁎

Abstract The bone is the most common target organ of cancer metastasis. Bone metastasis leads to considerable morbidity due to skeletal-related events (SREs). These include bone pain, hypercalcemia, pathologic fractures, and compression of the spinal cord. Cancers such as those of the lung, breast, prostate, and kidney are more likely to cause SREs than other cancer types. Additionally, some blood cancers, including multiple myeloma and lymphoma, frequently cause SREs. In this article, we review the conventional therapies for metastatic bone disease, including drug therapy, radiotherapy, and surgery. Among osteoclast-targeting agents, bisphosphonates and nuclear factor kappa-B ligand inhibitors are the most widely used agents to prevent cancer-related bone loss. Unsealed radioisotopes are also considered promising in cancer therapy. Currently, iodine-131, strontium-89, and radium-223 are available for the treatment of bone metastasis. However, the treatments for blood cancers with SREs are different from those of other cancers. In those cases, new classes of agents including proteasome inhibitors, immunomodulatory drugs, monoclonal antibodies, and histone deacetylase inhibitors have shown remarkable efficacy. We also discuss the potential development of new therapies for these diseases.

Strontium folate loaded biohybrid scaffolds seeded with dental pulp stem cells induce in vivo bone regeneration in critical sized defects.

Strontium folate (SrFO) is a recently developed bone promoting agent with interest in medical and pharmaceutical fields due to its improved features in comparison to current strontium based therapies for osteoporosis and other bone diseases. In this work SrFO derivative was synthesized and loaded into biohybrid scaffolds obtained through lyophilisation of semi-interpenetrating networks of chitosan polyethylene glycol dimethacrylate and beta tri-calcium phosphate (βTCP) fabricated using free radical polymerization. The scaffolds were seeded with pluripotent stem cells obtained from human dental pulp and their potential to regenerate bone tissues were assessed using a critical sized defect model of calvaria in rats and compared with those obtained without SrFO. The results obtained both in vitro and in vivo demonstrated excellent cyto-compatibility with resorption of scaffolds in 4-6 weeks and a total regeneration of the defect, with a more rapid and dense bone formation in the group with SrFO. Thus, the use of stem cells sourced from human dental pulp in combination with SrFO are very promising systems for their application in compromised osseous tissue regeneration.

Chondrogenic potential and anti-senescence effect of hypoxia on canine adipose mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the ability to differentiate into multi-lineage cells, which confers great promise for use in regenerative medicine. In this study, canine adipose MSCs (cAD-MSCs) were isolated from canine adipose tissue. These cells clearly represented stemness (Oct4, Sox2, and Nanog) and differentiation potential into the mesoderm (adipocytes, chondrocytes, and osteoblasts) at early passages. The aim of this study was to evaluate the effects of hypoxia on the differentiation potential into mesoderm, and the expression of anti-apoptotic genes associated with cell survival for the optimal culturing of MSCs. We observed that the proliferation of the cAD-MSCs meaningfully increased when cultured under hypoxic condition than in normoxic condition, during 7 consecutive passages. Also, we found that hypoxia strongly expressed anti-senescence related genes such as HDAC1 (histone deacetylase 1), DNMT1 (DNA (cytosine-5)-methyltransferase 1), Bcl-2 (inhibitor of apoptosis), TERT (telomerase reverse transcriptase), LDHA (lactate dehydrogenase A), SLC2A1 (glucose transporter), and DKC1 (telomere holoenzyme complex) and differentiation potential of cAD-MSCs into chondrocytes, than seen under the normoxic culture conditions. We also examined the multipotency of hypoxic conditioned MSCs using quantitative real-time RT-PCR. We found that the expression levels of stemness genes such as Oct-4, Nanog, and Sox-2 were increased in hypoxic condition when compared to the normoxic condition. Collectively, these results suggest that hypoxic conditions have the ability to induce proliferation of MSCs and augment their chondrogenic potential. This study suggests that cell proliferation of cAD-MSC under hypoxia could be beneficial, when considering these cells for cell therapies of canine bone diseases.

Effects of dendritic core-shell glycoarchitectures on primary mesenchymal stem cells and osteoblasts obtained from different human donors.

The biological impact of novel nano-scaled drug delivery vehicles in highly topical therapies of bone diseases have to be investigated in vitro before starting in vivo trials. Highly desired features for these materials are a good cellular uptake, large transport capacity for drugs and a good bio-compatibility. Essentially the latter has to be addressed as first point on the agenda. We present a study on the biological interaction of maltose-modified poly(ethyleneimine) (PEI-Mal) on primary human mesenchymal stem cell, harvested from reaming debris (rdMSC) and osteoblasts obtained from four different male donors. PEI-Mal-nanoparticles with two different molecular weights of the PEI core (5000 g/mol for PEI-5k-Mal-B and 25,000 g/mol for PEI-25k-Mal-B) have been administered to both cell lines. As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 μg/ml to 1 mg/ml and periods of 24 h up to 28 days. Studies conducted by different methods of microscopy as light microscopy, fluorescence microscopy, transmission-electron-microscopy and quantitative assays (LDH and DC-protein) indicate as well a good cellular uptake of the nanoparticles as a particle- and concentration-dependent impact on the cellular macro- and micro-structure of the rdMSC samples. In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B. At higher concentrations PEI-25k-Mal-B is toxic and induces a directly observable mitochondrial damage. The alkaline phosphatase assay (ALP), has been conducted to check on the possible influence of nanoparticles on the differentiation capabilities of rdMSC to osteoblasts. In addition the production of mineralized matrix has been shown by von-Kossa stained samples. No influence of the nanoparticles on the ALP per cell has been detected. Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples. To summarize, while featuring a good cellular uptake, PEI-5k-Mal-B induces only minimal adverse effects and features clearly superior biocompatibility compared to the larger PEI-25k-Mal-B.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0128-y) contains supplementary material, which is available to authorized users.

Antiresorptive therapy in bone disease: a physician's perspective

Antiresorptive therapy in bone disease: a physician's perspective

Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway.

Osteoblasts play critical roles in bone formation. Our previous study showed that chitosan nanofibers can stimulate osteoblast proliferation and maturation. This translational study used an animal model of bone defects to evaluate the effects of chitosan nanofiber scaffolds on bone healing and the possible mechanisms. In this study, we produced uniform chitosan nanofibers with fiber diameters of approximately 200 nm. A bone defect was surgically created in the proximal femurs of male C57LB/6 mice, and then the left femur was implanted with chitosan nanofiber scaffolds for 21 days and compared with the right femur, which served as a control. Histological analyses revealed that implantation of chitosan nanofiber scaffolds did not lead to hepatotoxicity or nephrotoxicity. Instead, imaging analyses by X-ray transmission and microcomputed tomography showed that implantation of chitosan nanofiber scaffolds improved bone healing compared with the control group. In parallel, microcomputed tomography and bone histomorphometric assays further demonstrated augmentation of the production of new trabecular bone in the chitosan nanofiber-treated group. Furthermore, implantation of chitosan nanofiber scaffolds led to a significant increase in the trabecular bone thickness but a reduction in the trabecular parameter factor. As to the mechanisms, analysis by confocal microscopy showed that implantation of chitosan nanofiber scaffolds increased levels of Runt-related transcription factor 2 (Runx2), a key transcription factor that regulates osteogenesis, in the bone defect sites. Successively, amounts of alkaline phosphatase and osteocalcin, two typical biomarkers that can simulate bone maturation, were augmented following implantation of chitosan nanofiber scaffolds. Taken together, this translational study showed a beneficial effect of chitosan nanofiber scaffolds on bone healing through stimulating trabecular bone production due to upregulation of Runx2-mediated alkaline phosphatase and osteocalcin gene expressions. Our results suggest the potential of chitosan nanofiber scaffolds for therapy of bone diseases, including bone defects and bone fractures.

Daily intake of β-cryptoxanthin prevents bone loss by preferential disturbance of osteoclastic activation in ovariectomized mice

Although β-cryptoxanthin, a xanthophyll carotenoid, has been shown to exert an anabolic effect on bone calcification, little attention has been paid thus far to the precise mechanism of bone remodeling. Daily oral administration of β-cryptoxanthin significantly inhibited osteoclastic activation as well as reduction of bone volume in ovariectomized mice. In vitro studies revealed that β-cryptoxanthin inhibited differentiation and maturation of osteoclasts by repression of the nuclear factor-κB-dependent transcriptional pathway. Our results suggest that supplementation with β-cryptoxanthin would be beneficial for prophylaxis and for therapy of metabolic bone diseases associated with abnormal osteoclast activation.

Effects of Tanshinone IIA on osteogenic differentiation of mouse bone marrow mesenchymal stem cells.

Tanshinone IIA (TSA) is a lipophilic diterpene purified from the Chinese herb Danshen, which exhibits potent antioxidant and anti-inflammatory properties. Effect of TSA remains largely uninvestigated on the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), which are widely used in cell-based therapy of bone diseases. In the present study, both ALP activity at day 7 and calcium content at day 24 were upregulated during the osteogenesis of mouse BM-MSCs treated with TSA (1 and 5μM), demonstrating that it promoted the osteogenesis at both early and late stages. We found that TSA promoted osteogenesis and inhibited osteoclastogenesis, evident by RT-PCR analysis of osteogenic marker gene expressions. However, osteogenesis was inhibited by TSA at 20μM. We further revealed that TSA (1 and 5μM) upregulated BMP and Wnt signaling. Co-treatment with Wnt inhibitor DKK-1 or BMP inhibitor noggin significantly decreased the TSA-promoted osteogenesis, indicating that upregulation of BMP and Wnt signaling plays a significant role and contributes to the TSA-promoted osteogenesis. Of clinical interest, our study suggests TSA as a promising therapeutic strategy during implantation of BM-MSCs for a more effective treatment of bone diseases.

Cafestol has a weaker inhibitory effect on osteoclastogenesis than kahweol and promotes osteoblast differentiation.

Bone homeostasis is regulated by a balance between osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-mediated bone formation. Thus, developing a compound that simultaneously inhibits OCL function and promotes OBL function would be useful as a new medical therapy for bone diseases. Here, we examined the effects of cafestol, a coffee diterpene, on the differentiation of OCLs and OBLs. Cafestol prevented OCL formation in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Interestingly, the viability of OCLs treated with 10-50 µM cafestol was significantly higher than that of untreated cells. At the molecular level, cafestol markedly decreased RANKL-induced phosphorylation of extracellular signal-regulated kinase (Erk) and inhibitor of nuclear factor kappa B alpha (IκBα). Compared to kahweol, another coffee-specific diterpene, the inhibitory effects of cafestol were milder on OCL differentiation, and cafestol and kahweol showed different characteristics in induction of the phase ΙΙ antioxidant enzymes and sensitivities in nuclear factor-erythroid 2-related factor 2 (Nrf2)-deficient BMMs. In addition to inhibiting OCLs, cafestol enhanced the differentiation of osteoblastic cells by increasing the mRNA levels of differentiation markers. Thus, cafestol inhibits OCL differentiation and promotes OBL differentiation, suggesting that cafestol may be a novel agent for bone diseases.

A semi-mechanistic model of bone mineral density and bone turnover based on a circular model of bone remodeling.

Development of novel therapies for bone diseases can benefit from mathematical models that predict drug effect on bone remodeling biomarkers. Therefore, a bone cycle model (BCM) was developed that takes into consideration the concept of the basic multicellular unit and the dynamic equilibrium of bone remodeling. The model is a closed form cyclical model with four compartments representing resorption, formation, primary mineralization, and secondary mineralization. Equations describing the time course of bone turnover biomarkers were developed using the flow rate of bone cycle units (BCU) between the compartments or the amount of BCU in each compartment. A disease progression model representing bone loss in osteoporosis, a vitamin D and calcium supplementation (placebo) model, and a drug model for antiresorptive treatments were added to the model. Initial model parameter values were derived from published bone turnover data. The BCM accurately described biomarker-time profiles in postmenopausal women receiving either placebo or bisphosphonate treatment. The slow continual increase in bone mineral density (BMD) observed after 1year of treatment was accurately described when changes in bone turnover were combined with increases in mineralization. For this purpose, the secondary mineralization compartment was replaced by three catenary chain compartments representing increasing mineral content. The refined BCM satisfactorily predicted biomarker profiles after long-term (10-year) bisphosphonate treatment. Furthermore, the model successfully described individual bone turnover markers and BMD results following treatment with denosumab in postmenopausal women. Analyses with this model could be used to optimize dosing regimens and to predict effects of novel osteoporotic treatments.

Cordycepin protected against the TNF-α-induced inhibition of osteogenic differentiation of human adipose-derived mesenchymal stem cells

Cordycepin, 3'-deoxyadenosine, is an effective component isolated from the rare Chinese caterpillar fungus Cordyceps militaris. It exerts potent anti-inflammatory actions in different cell and animal models. However, its action remains unclear on the TNF-α-induced inhibition of osteogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). In the present study, we demonstrated that cordycepin induced cell death at 20 and 40 μg/mL. Interestingly, 10 μg/mL cordycepin abrogated the cell death induced by 20 ng/mL TNF-α. Meanwhile, cordycepin exhibited a dose-dependent regulation of the osteogenesis of human ADMSCs: it promoted the differentiation at 10 μg/mL, whereas inhibited differentiation at 40 μg/mL. Furthermore, we discovered that 10 μg/mL cordycepin protected against the TNF-α (induced inhibition of osteogenic differentiation of human ADMSCs. It was also revealed that 10 μg/mL cordycepin restored Runx2 and Osx mRNA levels, which were significantly inhibited by TNF-αduring osteogenesis. At the same time, we found that 10 μg/mL cordycepin suppressed TNF-α-activated NF-κB signaling, by inhibiting IκBα phosphorylation and subsequent p65 release and translocation into the cell nucleus. Of clinical interest, the present study revealed mechanisms involved in inflammatory cytokine-inhibited osteogenesis, and it highlights the potential of cordycepin to promote the osteogenesis of human ADMSCs in cell-based therapy for inflammatory bone diseases.

Multiple Myeloma-Related Bone Disease: State-of-Art and Next Future Treatments

SUMMARY Multiple myeloma (MM) is a plasma cell malignancy associated with the development of life-threatening and/or severe osteolytic lesions, which significantly worsen the quality of life of affected patients. MM-related bone disease (BD) is the result of an overwhelming osteoclastic activity, while osteoblast-mediated bone formation is inhibited. Bisphosphonates are still the mainstay of therapy for BD. However, these drugs are associated with mid long-term sequelae. In this work, we review the pathogenesis and currently available therapies of MM-related BD. We describe the most recent and promising findings that may translate in changing the clinical practice in the next future.

Inhibition of RANKL-dependent cellular fusion in pre-osteoclasts by amiloride and a NHE10-specific monoclonal antibody.

The functions of Na(+) /H(+) exchangers (NHEs) during osteoclastic differentiation were investigated using the NHE inhibitor amiloride and a monoclonal antibody (MAb). Compared with sRANKL-stimulated control cells, amiloride decreased the number of large TRAP-positive osteoclast cells (OCs) with ≥10 nuclei and increased the number of small TRAP-positive OCs with ≤10 nuclei during sRANKL-dependent osteoclastic differentiation of RAW264.7 cells. NHE10 mRNA expression and OC differentiation markers were increased by sRANKL stimulation in dose- and time-dependent manners. NHEs 1-9 mRNA expression was not increased by sRANKL stimulation. Similar to amiloride, a rat anti-mouse NHE10 MAb (clone 6B11) decreased the number of large TRAP-positive OCs, but increased the number of small TRAP-positive OCs. These findings suggested that inhibition of NHEs by amiloride or an anti-NHE10 MAb prevented sRANKL-promoted cellular fusion. The anti-NHE10 MAb has the potential for use as an effective inhibitor of bone resorption for targeted bone disease therapy.