STING inhibition accelerates the bone healing process while enhancing type H vessel formation.

The stimulator of interferon genes (STING), one of the critical factors of innate immunity, is indicated to be closely related to angiogenesis. This study examined STING's role in angiogenesis and the formation of type H vessels, a specific subtype of bone vessels that regulates bone healing. Different concentrations of 2',3'-cGAMP, and H-151 or C-176 were applied to activate or inhibit STING, respectively. Human umbilical vein endothelial cells were used to examine the effect of STING on angiogenesis in vitro; cell viability, cell migration, and quantitative real-time polymerase chain reactions were performed. Also, the metatarsal experiment was applied as ex vivo proof. Bone fracture or defect mice models were used to examine the effect of STING in vivo; the bone healing process was evaluated by radiography weekly and by µCT on the 14th day after surgery. The formation of type H vessels (CD31hi Emcnhi endothelial cells) and osteogenesis (OCN-positive cells) was assessed using the cryosection and paraffin section. STING activation inhibited angiogenesis both in vitro and ex vivo and slowed down the bone healing process in vivo. Histological analysis showed an increased callus formation, fewer type H vessels, and almost no callus mineralization in the STING activation group compared to the control group. By contrast, H-151 (a hsSTING inhibitor) promoted angiogenesis at a low dose. Moreover, inhibition of mmSTING by C-176 enhanced type H vessels' formation, implying osteogenesis promotion in bone healing (higher bone volume density and more OCN-positive cells). Our data suggested that STING inhibition accelerates the bone healing process while enhancing type H vessel formation.

PP121 suppresses RANKL-Induced osteoclast formation in vitro and LPS-Induced bone resorption in vivo.

Bone resorption, caused by osteoclasts (OCs), is important to bone homeostasis. The abnormalities of bone resorption may induce a series of diseases, including osteoarthritis, osteoporosis and aseptic peri-implant loosening. The latest research developed,a novel tyrosine and phosphoinositide kinase dual inhibitor, named PP121, inhibited Src in anaplastic thyroid carcinoma cell. However, the therapeutic function of PP121 on abnormal bone resorption is still uncertain. In the present study, we showed that PP121 could potently suppress osteoclast differentiation, osteoclast-specific gene expression and bone resorption via suppressing Src/MAPK (ERK and p38)/Akt-mediated NFATc1 induction in vitro. \It was found that PP121 could suppress the formation of osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, inhibit bone resorption and downregulate the mRNA level of osteoclast-specific markers, including calcitonin receptor (CTR), tartrate resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase 3 (MMP3), Cellular oncogene fos (C-Fos) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Consistent with in vitro observation, we found that PP121 greatly ameliorated LPS-induced bone resorption. Our results provide promising evidence of the therapeutic potential of PP121 for osteolytic diseases related to excessive osteoclast-mediated bone resorption.

Overcoming chemotherapy resistance using pH-sensitive hollow MnO2 nanoshells that target the hypoxic tumor microenvironment of metastasized oral squamous cell carcinoma.

BACKGROUND: Smart nanoscale drug delivery systems that target acidic tumor microenvironments (TME) could offer controlled release of drugs and modulate the hypoxic TME to enhance cancer therapy. The majority of previously reported MnO2 nanostructures are nanoparticles, nanosheets, or nanocomposites incorporated with other types of nanoparticles, which may not offer the most effective method for drug loading or for the controlled release of therapeutic payloads. Previous studies have designed MnO2 nanoshells that achieve tumor-specific and enhanced combination therapy for localized advanced cancer. However, the therapeutic effect of MnO2 nanoshells on metastatic cancer is still uncertain. RESULT: Here, intelligent "theranostic" platforms were synthesized based on hollow mesoporous MnO2 (H-MnO2) nanoshells that were loaded with chemotherapy agents docetaxel and cisplatin (TP) to form H-MnO2-PEG/TP nanoshells, which were designed to alleviate tumor hypoxia, attenuate angiogenesis, trigger the dissolution of Mn2+, and synergize the efficacy of first-class anticancer chemotherapy. The obtained H-MnO2-PEG/TP nanoshells decomposed in the acidic TME, releasing the loaded drugs (TP) and simultaneously attenuated tumor hypoxia and hypoxia-inducible factor-1α (HIF-1α) expression by inducing endogenous tumor hydrogen peroxide (H2O2) decomposition. In vitro experiments showed that compared with the control group, the proliferation, colony formation and migration ability of CAL27 and SCC7 cells were significantly reduced in H-MnO2-PEG/TP group, while cell apoptosis was enhanced, and the expression of hypoxia-inducible factor-1α(HIF-1α) was down-regulated. In vivo experiments showed that tumor to normal organ uptake ratio (T/N ratio) of mice in H-MnO2-PEG/TP group was significantly higher than that in TP group alone (without the nanoparticle), and tumor growth was partially delayed. In the H-MnO2-PEG/TP treatment group, HE staining showed that most of the tumor cells were severely damaged, and TUNEL assay showed cell apoptosis was up-regulated. He staining of renal and liver sections showed no obvious fibrosis, necrosis or hypertrophy, indicating good biosafety. Fluorescence staining showed that HIF-1α expression was decreased, suggesting that the accumulation of MnO2 in the tumor caused the decomposition of H2O2 into O2 and alleviated the hypoxia of the tumor. CONCLUSION: In conclusion, a remarkable in vivo and in vitro synergistic therapeutic effect is achieved through the combination of TP chemotherapy, which simultaneously triggered a series of antiangiogenic and oxidative antitumor reactions.

Metal-Organic Framework Nanoparticles for Ameliorating Breast Cancer-Associated Osteolysis.

Breast cancer metastases to bone poses a significant challenge for the administration of treatment strategies. The bone microenvironment, metastatic tumor cells, osteoclasts, and tumor-associated macrophages (TAMs) all play crucial and synergistic roles in creating a favorable environment for the proliferation, progression, and survival of the metastatic tumor, which in turn induces osteoclast-mediated bone destruction. In this study, we functionalized immunostimulatory cytosine-phosphate-guanosine (CpG)-loaded metal-organic framework (MOF) nanoparticles with bone targeting capabilities by surface modification with FDA approved antiresorptive bisphosphonate, zoledronic acid (ZOL). The functionalized bone targeting immunostimulatory MOF (BT-isMOF) nanoparticles demonstrates strong binding to calcium phosphate in vitro and exhibits specific targeting and accumulation in bone tissues in vivo. In vitro cellular and biochemical analyses demonstrated that the BT-isMOF nanoparticles could potently inhibit osteoclast formation and concomitantly induce macrophages polarization toward the M1 pro-inflammatory phenotype. Finally, using the intratibial murine model of breast cancer bone metastasis, we showed that the administration of BT-isMOF nanoparticles significantly suppressed osteoclast-mediated bone destruction and enhanced polarization of tumor-resident macrophages to M1 phenotype. Together, our data provides promising evidence for the potential therapeutic application of the BT-isMOF nanoparticles in the treatment of breast cancer bone metastases.

Chitooligosaccharide inhibits RANKL-induced osteoclastogenesis and ligation-induced periodontitis by suppressing MAPK/ c-fos/NFATC1 signaling.

Considering the high rate of osteoclast-related diseases worldwide, research targeting osteoclast formation/function is crucial. In vitro, we demonstrated that chitooligosaccharide (CS) dramatically inhibited osteoclastogenesis as well as osteoclast function dose-dependently. CS suppressed osteoclast-specific genes expression during osteoclastogenesis. Furthermore, we found that CS attenuated receptor activator of nuclear factor kappa B ligand (RANKL)-mediated mitogen-activated protein kinase (MAPK) pathway involving p38, erk1/2, and jnk, leading to the reduced expression of c-fos and nuclear factor of activated T cells c1 (NFATc1) during osteoclast differentiation. In vivo, we found CS protected rats from periodontitis-induced alveolar bone loss by micro-computerized tomography and histological analysis. Overall, CS inhibited RANKL-induced osteoclastogenesis and ligature-induced rat periodontitis model, probably by suppressing the MAPK/c-fos/NFATc1 signaling pathway. Therefore, CS may be a safe and promising treatment for osteoclast-related diseases.

Redox-dependent and independent effects of thioredoxin interacting protein.

Thioredoxin interacting protein (TXNIP) is an important physiological inhibitor of the thioredoxin (TXN) redox system in cells. Regulation of TXNIP expression and/or activity not only plays an important role in redox regulation but also exerts redox-independent physiological effects that exhibit direct pathophysiological consequences including elevated inflammatory response, aberrant glucose metabolism, cellular senescence and apoptosis, cellular immunity, and tumorigenesis. This review provides a brief overview of the current knowledge concerning the redox-dependent and independent roles of TXNIP and its relevance to various disease states. The implications for the therapeutic targeting of TXNIP will also be discussed.

Mechanism of delayed frost growth on superhydrophobic surfaces with jumping condensates: more than interdrop freezing.

Delayed frost growth on superhydrophobic surfaces (SHSs) with jumping condensates has been found by many researchers recently. However, the mechanism of this phenomenon has not been elucidated clearly. In this study, copper SHSs with or without jumping condensates were selected as the substrates for observing condensation icing at a relative humidity (RH) of 60%. The results showed that only SHS with jumping condensates showed delayed condensation icing. Moreover, when such SHSs were placed upward and the surface temperature was held at -10 °C, some discrete frozen drops first appeared on the SHSs. The following icing mainly occurred on these discrete global crystals and then expanded around them until covering the entire surface. Little macroscopic interdrop freezing phenomenon was found. The growth of the frost front is mainly dominated by jumping freezing (the condensed droplets jumped onto the ice crystals and were frozen) or direct vapor-ice deposition. Using microscopy, we found interdrop freezing occurred, in addition to the two mechanisms mentioned above. By placing the SHS downward at -10 °C and intentionally introducing or eliminating tiny dusts, we confirmed that there were no superhydrophobic defects on our SHSs. The discrete frozen drops first appearing on the SHSs were triggered by tiny dusts falling on the surface before or during condensation icing. The key approach in delaying or resisting frost growth on SHSs with jumping condensates is to retard initial ice crystal formation, e.g., eliminating the edge effect and keeping the SHSs clean.

Macrophage-Capturing Self-Assembly Photosensitizer Nanoparticles Induces Immune Microenvironment Re-Programming and Golgi-Responsive Immunogenic Cell Death in Head and Neck Carcinoma.

Head and neck carcinoma treatment is shifted toward the combination of therapy causing immune checkpoint blockade (ICB) and immunogenic cell death. In this study, a CSFRi-chimeric TAMCSFR+-targeting extracellular vesicle (EV@CSFRi) platform is developed and designed an intracellular protoporphyrin conjugated with RVRR peptide sequence for furin-cleavage to perform Golgi-targeting and generating ROS (GT-RG). The graphical abstract illustrates the self-assembly of GT-RG nanoparticles into nanofiber through the hydrophily of RVRR and hydrophobicity of RG, and the red line indicates the site of furin cleavage. As is shown in the Graphical abstract, the Golgi-targeting Protoporphyrin-RVRR platform is composed with CSFRi-chimeric extracellular vesicles and forms the tumor-responsive TAM-reprogramming bilayers (GT-RGEV@CSFRi). The GT-RGEV@CSFRi acted as a multifunctional theranostic platform, which can induce immunogenic cell death and further help modulate TAM, thus suppressing the HNC xenograft model by combination therapy with anti-PD-1.

Micron-resolution Imaging of Cortical Bone under 14 T Ultrahigh Magnetic Field.

Compact, mineralized cortical bone tissues are often concealed on magnetic resonance (MR) images. Recent development of MR instruments and pulse techniques has yielded significant advances in acquiring anatomical and physiological information from cortical bone despite its poor 1 H signals. This work demonstrates the first MR research on cortical bones under an ultrahigh magnetic field of 14 T. The 1 H signals of different mammalian species exhibit multi-exponential decays of three characteristic T2 or T2 * values: 0.1-0.5 ms, 1-4 ms, and 4-8 ms. Systematic sample comparisons attribute these T2 /T2 * value ranges to collagen-bound water, pore water, and lipids, respectively. Ultrashort echo time (UTE) imaging under 14 T yielded spatial resolutions of 20-80 microns, which resolves the 3D anatomy of the Haversian canals. The T2 * relaxation characteristics further allow spatial classifications of collagen, pore water and lipids in human specimens. The study achieves a record of the spatial resolution for MR imaging in bone and shows that ultrahigh-field MR has the unique ability to differentiate the soft and organic compartments in bone tissues.

Inhibition of furin by bone targeting superparamagnetic iron oxide nanoparticles alleviated breast cancer bone metastasis.

Breast cancer bone metastasis poses significant challenge for therapeutic strategies. Inside the metastatic environment, osteoclasts and tumor cells interact synergistically to promote cancer progression. In this study, the proprotein convertase furin is targeted due to its critical roles in both tumor cell invasion and osteoclast function. Importantly, the furin inhibitor is specifically delivered by bone targeting superparamagnetic iron oxide (SPIO) nanoparticles. Our in vitro and in vivo data demonstrate that this system can effectively inhibit both osteoclastic bone resorption and breast cancer invastion, leading to alleviated osteolysis. Therefore, the bone targeting & furin inhibition nanoparticle system is a promising therapeutic and diagnostic strategy for breast cancer bone metastasis.

Nicorandil Inhibits Osteoclast Formation Base on NF-κB and p-38 MAPK Signaling Pathways and Relieves Ovariectomy-Induced Bone Loss.

Osteolytic bone disorders are characterized by an overall reduction in bone mineral density which enhances bone ductility and vulnerability to fractures. This disorder is primarily associated with superabundant osteoclast formation and bone resorption activity. Nicorandil (NIC) is a vasodilatory anti-anginal drug with ATP-dependent potassium (KATP) channel openings. However, NIC is adopted to manage adverse cardiovascular and coronary events. Recent research has demonstrated that NIC also possesses anti-inflammatory peculiarity through the regulation of p38 MAPK and NF-κB signaling pathways. Both MAPK and NF-κB signaling pathways play pivotal roles in RANKL-induced osteoclast formation and bone resorption function. Herein, we hypothesized that NIC may exert potential biological effects against osteoclasts, and revealed that NIC dose-dependently suppressed bone marrow macrophage (BMM) precursors to differentiate into TRAP + multinucleated osteoclasts in vitro. Furthermore, osteoclast resorption assays demonstrated anti-resorptive effects exhibited by NIC. NIC had no impact on osteoblast differentiation or mineralization function. Based on Biochemical analyses, NIC relieved RANKL-induced ERK, NF-κB and p38 MAPK signaling without noticeable effects on JNK MAPK activation. However, the attenuation of NF-κB and p38 MAPK activation was sufficient to hamper the downstream induction of c-Fos and NFATc1 expression. Meanwhile, NIC administration markedly protected mice from ovariectomy (OVX)-induced bone loss through in vivo inhibition of osteoclast formation and bone resorption activity. Collectively, this work demonstrated the potential of NIC in the management of osteolytic bone disorders mediated by osteoclasts.

Solid-State Nuclear Magnetic Resonance Identifies Abnormal Calcium Phosphate Formation in Diseased Bones.

The crystallites of calcium phosphate (CaP) in bones consist of hydroxyl apatite (HA) and amorphous calcium phosphate (ACP). These nanoscale structures of CaP are sculptured by biological bone formation and resorption processes and are one of the crucial factors that determine the overall strength of the constructs. We used one- and two-dimensional 1H-31P solid-state nuclear magnetic resonance (SSNMR) to investigate the nanoscopic structural changes of CaP. Two quantitative measurables are deduced based on the heterogeneous linewidth of 31P signal and the ratio of ACP to HA, which characterize the mineral crystallinity and the relative proportion of ACP, respectively. We analyzed bones from different murine models of osteopetrosis and osteoporosis and from human samples with osteoporosis and osteoarthritis. It shows that the ACP content increases notably in osteopetrotic bones that are characterized by defective osteoclastic resorption, whereas the overall crystallinity increases in osteoporotic bones that are marked by overactive osteoclastic resorption. Similar pathological characteristics are observed for the sclerotic bones of late-stage osteoarthritis, as compared to those of the osteopetrotic bones. These findings suggest that osteoclast-related bone diseases not only alter the bone density macroscopically but also lead to abnormal formation of CaP crystallites. The quantitative measurement by SSNMR provides a unique perspective on the pathology of bone diseases at the nanoscopic level.

Chitosan derived nitrogen-doped carbon dots suppress osteoclastic osteolysis via downregulating ROS.

Osteoclasts are the main cells involved in normal bone remodeling and pathological bone destruction in vivo. Overactivation of osteoclasts can lead to osteolytic diseases, including breast cancer, bone tumors, arthritis, the aseptic loosening of orthopedic implants, and Paget's disease. Excessive reactive oxygen species are the main cause of osteoclast overactivation. We have synthesized chitosan derived nitrogen-doped carbon dots (N-CDs) with a high synthetic yield and the ability to scavenge reactive oxygen species (ROS). N-CDs effectively abrogated RANKL-induced elevation in ROS generation and therefore impaired the activation of NF-κB and MAPK pathways. Osteoclastogenesis and bone resorption was effectively attenuated in vitro. Furthermore, the in vivo administration of N-CDs in mice protected them against lipopolysaccharide (LPS)-induced calvarial bone destruction and breast cancer cell-induced tibial bone loss. Based on the good biocompatibility of N-CDs and the ability to efficiently remove ROS, a nanomaterial treatment scheme was provided for the first time for the clinical treatment of osteolytic diseases.

Identification of Fibroblast Activation Protein as an Osteogenic Suppressor and Anti-osteoporosis Drug Target.

Osteogenic suppressors such as Sclerostin not only regulate skeletal development and regeneration but also serve as anti-osteoporosis drug targets. However, very few druggable suppressors have been identified due to limited understanding of the molecular mechanisms governing osteogenesis. Here, we show that fibroblast activation protein (Fap), a serine protease inhibited by the bone growth factor Osteolectin, is an osteogenic suppressor. Genetic deletion of Fap significantly ameliorates limb trabecular bone loss during aging. Pharmacological inhibition of Fap significantly promotes bone formation and inhibits bone resorption in wild-type mice by differentially regulating canonical Wnt and nuclear factor κB (NF-κB) pathways. Pharmacological inhibition of Fap promotes osteoblast differentiation, inhibits osteoclast differentiation, and significantly attenuates osteoporosis in ovariectomized mice. Epistasis analyses in zebrafish show that Osteolectin functions as an endogenous inhibitor of Fap to promote vertebrae mineralization. Taken together, we identify Fap as an important osteogenic suppressor and a potential drug target to treat osteoporosis.

Decellularized Periosteum-Covered Chitosan Globule Composite for Bone Regeneration in Rabbit Femur Condyle Bone Defects.

Critical-sized bone defects are incapable of self-healing and are commonly seen in clinical practice. The authors explore a new treatment for this, decellularized periosteum is applied to chitosan globules (chitosan-DP globules) as a hybrid material. The efficacy of chitosan-DP globules on rabbit femoral condyle bone defects is assessed with biocompatibility, biomechanics, and osteogenic efficiency measurements, and compared with the results of chitosan globules and empty control. No difference in cytotoxicity is observed among chitosan-DP globules, chitosan globules, and the empty control. Chitosan-DP globules possesse a better surface for cell adhesion than did chitosan globules. Chitosan-DP globules demonstrate superior efficiency for osteogenesis in the defect area compared to chitosan globules as per microcomputed tomography examination and push-out testing, with relatively minor histological differences. Both chitosan globule groups show more satisfactory results than those for the empty control. The results implicate chitosan-DP globules as a promising solution for bone defects.

The amelioration of cartilage degeneration by photo-crosslinked GelHA hydrogel and crizotinib encapsulated chitosan microspheres.

The present study aimed to investigate the synergistic therapeutic effect of decreaseing cartilage angiogenesis via exposure to crizotinib encapsulated by chitosan microspheres and photo-crosslinked hydrogel, with the goal of evaluating crizotinib as a treatment for osteoarthritis. First, we developed and evaluated the characteristics of hydrogels and chitosan microspheres. Next, we measured the effect of crizotinib on the cartilage degeneration induced by interleukin-1ß in chondrocytes. Crizotinib ameliorated the pathological changes induced by interleukin-1ß via its anti-angiogenesis function. In addition, we surgically induced osteoarthritis in mice, which were then injected intra-articularly with crizotinib-loaded biomaterials. Cartilage matrix degradation, expression of vascular endothelial growth factor and extracellular signal-regulated kinases 1/2 were evaluated after surgery. Treatment with the combination of crizotinib-loaded biomaterials retarded the progression of surgically induced osteoarthritis. Crizotinib ameliorated cartilage matrix degradation by promoting anti-angiogenesis and impeding extracellular signal-regulated kinases 1/2 signaling pathway. Our results demonstrate that the combination of photo-crosslinked hydrogel and crizotinib-loaded chitosan microspheres might represent a promising strategy for osteoarthritis treatment.

An injectable nucleus pulposus cell-modified decellularized scaffold: biocompatible material for prevention of disc degeneration.

We developed a nucleus pulposus cell (NPC)-modulated decellularized small intestinal submucosa (SIS) scaffold, and assessed the ability of this material to prevent Intervertebral disc degeneration (IVD) degeneration. Decellularized porcine SIS was squashed into particles and the biological safety and efficiency of these particles were evaluated. Next, SIS particles were seeded with rabbit NPCs, cultured for two months in vitro, decellularized again and suspended for intervertebral injection. We demonstrated that use of the decellularization protocol resulted in the removal of cellular components with maximal retention of extracellular matrix. The xenogeneic decellularized SIS did not display cytotoxicity in vitro and its application prevented NPC degradation. Furthermore, the xenogeneic SIS microparticles were effective in preventing IVD progression in vivo in a rabbit disc degeneration model. In conclusion, our study describes an optimized method for decellularized SIS preparation and demonstrated that the material is safe and effective for treating IVD degeneration.

Biodegradable and biocompatible high elastic chitosan scaffold is cell-friendly both in vitro and in vivo.

Biodegradable and biocompatible macromolecule chitosan has been favored for a variety of clinical applications. We reported herein the fabrication of a novel chitosan scaffold with high elasticity. This scaffold can be easily compressed and thus enable the insertion of such scaffold into surgical lesions during minimal invasive surgeries. In addition, this novel scaffold can restore its shape when released. We evidenced that this high elastic scaffold has better biocompatibility than traditional chitosan scaffold. Therefore, this new chitosan material might lead to the manufacture of a variety of novel biodegradable biomedical materials and devices.

Why condensate drops can spontaneously move away on some superhydrophobic surfaces but not on others.

The coalesce-induced condensate drop motion on some superhydrophobic surfaces (SHSs) has attracted increasing attention because of its wide potential applications. However, microscopic mechanism of spontaneous motion has not been discussed thoroughly. In this study, we fabricated two types of superhydrophobic copper surfaces with sisal-like nanoribbon structures and defoliation-like nanosheet structures by different wet chemical oxidation process and followed by same fluorization treatment. With lotus leaf and butterfly wing as control samples, the spontaneous motion phenomenon of condensate drops on these four kinds of SHSs was investigated by using optical microscope under ambient conditions. The results showed that among all four types of SHSs, only superhydrophobic copper surfaces with sisal-like nanoribbon structures showed obvious spontaneous motion of condensate drops, especially when the relative humidity was higher. The microscopic mechanism of spontaneous motion was discussed in relation to the states of condensate drops on different nanostructures. It shows that the instantaneous Cassie state of condensed droplets prior to coalescence plays a key role in determining whether the coalesced drop departs, whereas only SHS possessing nanostructures with small enough Wenzel roughness parameter r (at least <2.1) and nanogaps forming high enough Laplace pressure favors the formation of the instantaneous Cassie state by completing the Wenzel-Cassie transition.