Co-Culture of Bone Marrow Mesenchymal Stem Cell (BMSC) and Osteoclasts Regulates Cell Differentiation and Alleviates Osteoporosis by Up-Regulating miR-211

Bone marrow mesenchymal stem cells (BMSCs) can release a large amount of exosomes (EXO) during bone remodeling by osteoclasts. EXO contains miRNA-211, which has a variety of biological effects. However, little is known about whether miR-211 from BMSC-EXO affects the surrounding cells. Therefore, we aim to study the role of miRNA-211 derived from BMSC-EXO in regulating osteoclasts differentiation. Macrophage colony stimulating factor (M-CSF) and nuclear factor kappa B receptor activator (RANKL) were used to stimulate bone marrow macrophages (BMM) to obtain osteoclasts, which were treated with BMSC-EXO or LPS followed by analysis of osteoclast-related genes expression by PCR, ROS release by flow cytometry, actin ring formation by immunofluorescence, and osteoclast differentiation by anti-tartrate acid phosphatase (TRAP) staining. Finally, an in vivo experiment was conducted to verify BMSC-EXO’s effect on osteoporosis. BMSC-EXO significantly inhibited RNAKL-induced osteoclast differentiation of BMMs. During osteoclasts formation, BMSC-EXO inhibited ROS production induced by RANKL and the subsequent activation of NF-κB signaling pathway induced by ROS. In addition, BMSC-EXO significantly down-regulated the osteoclast genes including nuclear factor, cytoplasmic 1 (NFATc1), C-fos, tartrate-resistant acid phosphatase (TRAP) and osteoclast-associated immunoglobulin-like receptor (OSCAR) in activated T cells. BMSC-EXO inhibited ROS release by promoting miR-211 expression, thereby inhibiting the NF-κB signaling and ultimately participating in osteoclasts differentiation. In LPS-induced mouse osteoporosis models, BMSC-EXO inhibited LPS-induced bone loss and exerted a protective effect. In conclusion, microRNA-211 derived from BMSC-EXO can regulate osteoclasts differentiation, suggesting that it might be used as a potential approach for treating osteoporosis.

Collagen-binding integrin alpha11beta1 contributes to joint destruction in arthritic hTNFtg mice

Background: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) undergo a tumor-like transformation, wherein they develop an aggressive phenotype that is characterized by increased adhesion to components of cartilage extracellular matrix (ECM) and that contributes extensively to joint destruction. The collagen-binding integrin alpha11beta1 was previously shown to be involved in similar processes in cancer-associated fibroblasts mediating tumorigenicity and metastasis in certain tumors. Therefore, this study aimed to study the role of integrin alpha11beta1 in RA and to characterize the effects of alpha11beta1 deficiency on the disease course and severity in arthritic hTNFtg mice. Methods: The expression levels of integrin alpha11beta1 were analyzed by immunohistochemistry, immunofluorescence, and western blot analysis in synovial samples and FLS of patients with RA and osteoarthritis (OA) as well as in samples from wild type (wt) and arthritic hTNFtg mice. Furthermore, the subcellular expression of integrin alpha11beta1 was investigated in co-culture experiments with cartilage explants and analyzed by transmission electron microscopy. To investigate the effects of integrin alpha11beta1 deficiency, itga11-/- mice were interbred with hTNFtg mice and disease severity was assessed by clinical scoring of grip strength and paw swelling over the disease course. Hind paws of 12-weeks-old mice of all genotypes were analyzed by uCT imaging followed by stainings of paraffin-embedded tissue sections with Toluidine-blue and tartrate-resistant acid phosphatase (TRAP) to evaluate established parameters of joint destruction such as inflammation area, cartilage destaining, FLS attachment to the cartilage surface, and bone damage. Results: Expression levels of integrin alpha11beta1 were clearly elevated in synovial tissues and FLS from RA patients and hTNFtg mice, compared to the controls derived from OA patients and wt mice. Interestingly, this expression was shown to be particularly localized in focal adhesions of the FLS. As revealed by transmission electron microscopy, integrin alpha11beta1 expression was particularly evident in areas of direct cellular contact with the ECM of cartilage. Evaluations of clinical scorings and histomorphological analyses demonstrated that itga11-/-hTNFtg displayed alleviated clinical symptoms, higher bone volume, less cartilage destruction, and reduced FLS attachment to the cartilage in comparison to hTNFtg mice. Conclusions: The collagen-binding integrin alpha11beta1 is upregulated in the context of RA and its deficiency in mice with an inflammatory hTNFtg background leads to a significant reduction in the arthritic phenotype which makes integrin alpha11beta1 an interesting target for therapeutical intervention.

Tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis by modulating β-catenin signaling

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited therapeutic options. Tartrate-resistant acid phosphatase 5 (ACP5) performs a variety of functions. However, its role in IPF remains unclear. Here, we demonstrate that the levels of ACP5 are increased in IPF patient samples and mice with bleomycin (BLM)-induced pulmonary fibrosis. In particular, higher levels of ACP5 are present in the sera of IPF patients with a diffusing capacity of the lungs for carbonmonoxide (DLCO) less than 40% of the predicted value. Additionally, Acp5 deficiency protects mice from BLM-induced lung injury and fibrosis coupled with a significant reduction of fibroblast differentiation and proliferation. Mechanistic studies reveal that Acp5 is upregulated by transforming growth factor-β1 (TGF-β1) in a TGF-β receptor 1 (TGFβR1)/Smad family member 3 (Smad3)-dependent manner, after which Acp5 dephosphorylates p-β-catenin at serine 33 and threonine 41, inhibiting the degradation of β-catenin and subsequently enhancing β-catenin signaling in the nucleus, which promotes the differentiation, proliferation and migration of fibroblast. More importantly, the treatment of mice with Acp5 siRNA-loaded liposomes or Acp5 inhibitor reverses established lung fibrosis. In conclusions, Acp5 is involved in the initiation and progression of pulmonary fibrosis and strategies aimed at silencing or suppressing Acp5 could be considered as potential therapeutic approaches against pulmonary fibrosis.

Effects of Sparganii Rhizoma on Osteoclast Formation and Osteoblast Differentiation and on an OVX-Induced Bone Loss Model

Postmenopausal osteoporosis is caused by an imbalance between osteoclasts and osteoblasts and causes severe bone loss. Osteoporotic medicines are classified into bone resorption inhibitors and bone formation promoters according to the mechanism of action. Long-term use of bisphosphonate and selective estrogen receptor modulators (SERMs) can cause severe side effects in postmenopausal osteoporosis patients. Therefore, it is important to find alternative natural products that reduce osteoclast activity and increase osteoblast formation. Sparganii Rhizoma (SR) is the dried tuberous rhizome of Sparganium stoloniferum Buchanan-Hamilton and is called “samreung” in Korea. However, to date, the effect of SR on osteoclast differentiation and the ovariectomized (OVX)-induced bone loss model has not been reported. In vitro, tartrate-resistant acid phosphatase (TRAP) staining, western blots, RT-PCR and other methods were used to examine the effect of SR on osteoclast differentiation and osteoblasts. In vivo, we confirmed the effect of SR in a model of OVX-induced postmenopausal osteoporosis. SR inhibited osteoclast differentiation and decreased the expression of TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells 1 (NFATc1) and c-Fos pathway. In addition, SR stimulates osteoblast differentiation and increased protein expression of the bone morphogenetic protein 2 (BMP-2)/SMAD signaling pathway. Moreover, SR protected against bone loss in OVX-induced rats. Our results appear to advance our knowledge of SR and successfully demonstrate its potential role as a osteoclastogenesis-inhibiting and osteogenesis-promoting herbal medicine for the treatment of postmenopausal osteoporosis.

Caffeic Acid Inhibits RANKL and TNFa-induced Osteoclastogenesis by Targeting TAK1-p44/42 MAPK

BACKGROUND: The potential of the caffeic acid in other important Receptor Activator Nuclear Factor kB Ligand (RANKL)-Tumor Necrosis Factor (TNF)a-induced osteoclastogenic signaling pathways has not been known. Therefore, the current study was conducted to explore as well as to understand the inhibition potential of caffeic acid.METHODS: RAW264.7 cells were cultured, treated with caffeic acid, RANKL and TNFa. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed to detect TRAP+ osteoclast-like polynuclear cells. To detect the activity of p44/42 Mitogen Activated Protein Kinase (MAPK), Akt, and Transforming Growth Factor-β-activated Kinase (TAK)1, the phosphorylated forms of the proteins were investigated with the immunoblotting assay.RESULTS: Pre-treatment of caffeic acid inhibited the RANKL and TNFa-induced differentiation of RAW264.7 cells into TRAP+ osteoclast-like polynuclear cells. RANKL and TNFa induced phosphorylation of p44/42 MAPK at Thr202/Tyr204, phosphorylation of Akt at both Ser473 and Thr308 and phosphorylation of TAK1 at Ser412. Pre-treatment with caffeic acid prior to the RANKL and TNFa induction, inhibited the phosphorylation of MAPK, and TAK1, but not Akt.CONCLUSION: Caffeic acid might regulate the RANKL-TNFa-induced osteoclastogenic pathway in RAW264.7 by targeting TAK1, which later activation of p44/42 MAPK was abolished.KEYWORDS: caffeic acid, osteoclastogenesis, p44/42, Erk1/2, Akt, TAK1, RAW264.7

Bortezomib Rescues Ovariectomy-Induced Bone Loss via SMURF-Mediated Ubiquitination Pathway

A balance between bone formation by osteoblasts and bone resorption by osteoclasts is necessary to maintain bone health and homeostasis. As a cancer of plasma cells, multiple myeloma (MM) is accompanied with rapid bone loss and fragility fracture. Bortezomib has been used as a first-line for treating MM for decades. Recently, the potential protection of bortezomib on osteoporosis (OP) is reported; however, the specific mechanism involving bortezomib-mediated antiosteoporotic effect is undetermined. In the present study, we assessed the effects of in vitro bortezomib treatment on osteogenesis and osteoclastogenesis and the protective effect on bone loss in ovariectomized (OVX) mice. Our results indicated that bortezomib treatment increased osteogenic differentiation of MC3T3-E1 cells as evidenced by increased levels of matrix mineralization and osteoblast-specific markers. In bortezomib-treated bone marrow monocytes (BMMs), osteoclast differentiation was suppressed, substantiated by downregulated tartrate-resistant acid phosphatase- (TRAP-) positive multinucleated cells, areas of actin rings, pit formation, and osteoclast-specific genes. Mechanistically, bortezomib exerted a protective effect against OP through the Smad ubiquitination regulatory factor- (SMURF-) mediated ubiquitination pathway. Furthermore, in vivo intraperitoneal injection of bortezomib attenuated the bone microarchitecture in OVX mice. Accordingly, our findings corroborated that bortezomib might have future applications in the treatment of postmenopausal OP.

Intermittent Hypoxic Therapy Inhibits Allogenic Bone-Graft Resorption by Inhibition of Osteoclastogenesis in a Mouse Model

Systemic Intermittent Hypoxic Therapy (IHT) relies on the adaptive response to hypoxic stress. We investigated allogenic bone-graft resorption in the lumbar spine in 48 mice. The mice were exposed to IHT for 1 week before surgery or 1 week after surgery and compared with controls after 1 and 4 weeks. Complete graft resorption was observed in 33–36% of the animals in the control group, but none in the preoperative IHT group. Increased bone-graft volume was demonstrated by micro-computed tomography in the preoperative IHT group after 1 week (p = 0.03) while a non-significant difference was observed after 4 weeks (p = 0.12). There were no significant differences in the postoperative IHT group. Increased concentration of immune cells was localized in the graft area, and more positive tartrate-resistant acid phosphatase (TRAP) staining was found in controls compared with IHT allogenic bone grafts. Systemic IHT resulted in a significant increase of the major osteoclast inhibitor osteoprotegerin as well as osteogenic and angiogenic regulators Tgfbr3, Fst3l, Wisp1, and Vegfd. Inflammatory cytokines and receptor activator of nuclear factor kappa-B ligand (RANKL) stimulators IL-6, IL-17a, IL-17f, and IL-23r increased after 1 and 4 weeks, and serum RANKL expression remained constant while Ccl3 and Ccl5 decreased. We conclude that the adaptive response to IHT activates numerous pathways leading to inhibition of osteoclastic activity and inhibition of allogenic bone-graft resorption.

Inhibitory Effects of Astaxanthin on CML-HSA-Induced Inflammatory and RANKL-Induced Osteoclastogenic Gene Expression in RAW 264.7 Cells

Objective: Elevated levels of serum Nε-carboxymethyllysine (CML), a well-known advanced glycation end-product (AGE), were observed in patients with inflammation or osteoporosis. Astaxanthin was reported to possess anti-inflammatory and antioxidant effects. In the present study, we investigated the effects of commercially available dietary supplement AstaReal ACTR (ASR) capsule content as astaxanthin on CML-HSA-induced inflammatory and receptor activator of nuclear factor-kappa-Β ligand (RANKL)-induced osteoclastogenic gene expression. Methods: RAW 264.7 murine macrophage cells were stimulated with CML-HSA to trigger inflammatory gene expression and treated with either a vehicle control or varied concentrations of astaxanthin. Inflammatory gene expression was measured using an enzyme-linked immunosorbent assay (ELISA) or qPCR. We triggered osteoclastogenesis using RANKL, and osteoclastogenic gene expression was measured through tartrate-resistant acid phosphatase (TRAP) activity, staining, immunofluorescence, and qPCR analyses. Results: CML-HSA showed a stimulatory effect on inflammatory gene expression, and astaxanthin reduced the expression by at least two-fold. The levels of autoinflammatory gene expression were reduced by astaxanthin. The RANKL-induced osteoclastogenesis was significantly inhibited by astaxanthin, with reductions in the activation of nuclear factor-κB (NF-κB), the expression of NFATc1 (nuclear factor of activated T cells 1), multinucleated cell formation, and the expression of mature osteoclast marker genes. Conclusion: Astaxanthin has potential as a remedy for CML-HSA-induced inflammation and RANKL-induced excessive bone loss.

Utility of Thermal Cross-Linking in Stabilizing Hydrogels with Beta-Tricalcium Phosphate and/or Epigallocatechin Gallate for Use in Bone Regeneration Therapy

β-tricalcium phosphate (β-TCP) granules are commonly used materials in dentistry or orthopedic surgery. However, further improvements are required to raise the operability and bone-forming ability of β-TCP granules in a clinical setting. Recently, we developed epigallocatechin gallate (EGCG)-modified gelatin sponges as a novel biomaterial for bone regeneration. However, there is no study on using the above material for preparing hydrogel incorporating β-TCP granules. Here, we demonstrate that vacuum heating treatment induced thermal cross-linking in gelatin sponges modified with EGCG and incorporating β-TCP granules (vhEc-GS-β) so that the hydrogels prepared from vhEc-GS-β showed high stability, β-TCP granule retention, operability, and cytocompatibility. Additionally, microcomputed tomography morphometry revealed that the hydrogels from vhEc-GS-β had significantly higher bone-forming ability than β-TCP alone. Tartrate-resistant acid phosphatase staining demonstrated that the number of osteoclasts increased at three weeks in defects treated with the hydrogels from vhEc-GS-β compared with that around β-TCP alone. The overall results indicate that thermal cross-linking treatment for the preparation of sponges (precursor of hydrogels) can be a promising process to enhance the bone-forming ability. This insight should provide a basis for the development of novel materials with good operativity and bone-forming ability for bone regenerative medicine.

Effects of Alendronate and Dexamethasone on Osteoclast Gene Expression and Bone Resorption in Mouse Marrow Cultures

Osteoclasts are cells whose main function is the resorption of bone matrix. However, several factors, including medications, can interfere with the resorption process. Alendronate (ALN), a nitrogen-containing type of bisphosphonate, and dexamethasone (DEX), a glucocorticoid, are drugs that may affect the resorption activity. The aim of this study is to investigate the effects of ALN, and/or DEX on osteoclast gene expression and resorption activity in primary mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D3, a model for the bone microenvironment. Cultures were treated only with ALN (10−5 M), DEX (10−6 M), and with a combination of both agents. Viability assays performed at days 5, 7, and 9 showed the highest number of viable cells at day 7. All the following assays were then performed at day 7 of cell culture: tartrate resistant acid phosphatase (TRAP) histochemistry, receptor activator of nuclear factor kappa B ligand (RANKL) immunofluorescence, osteoprotegerin (OPG), and RANKL gene expression by qPCR and resorption analysis by scanning electron microscopy. Treatment with ALN, DEX, and the combination of both did not promote significant changes in the number of TRAP+ cells, although larger giant cells were detected in groups treated with DEX. DEX treatment increased the gene expression of RANKL and reduced OPG. The treatment with ALN reduced the depth of the resorption pits, but their inhibitory effect was less effective when administered with DEX: