Combination of cobalt, chromium and titanium nanoparticles increases cytotoxicity in vitro and pro-inflammatory cytokines in vivo.

Background: The mixture of different metallic nanoparticles released from intended and unintended wearing of orthopaedic implants such as the Co/Cr cup and head, Co/Cr sleeves or tapers and their interface with Ti stems in the case of hip prostheses are a leading cause of adverse inflammatory responses and cytotoxicity to the host. Methods: This study assessed the in vitro cytotoxic effects of three metallic nanoparticles (Co, Cr and Ti) separately and in combination on macrophages. The in vivo effects were also evaluated after peri-tibial soft tissue injection in mice. Results: The results demonstrated that Co, Cr, and Ti nanoparticles and their combination were phagocytosed by macrophages both in vitro and in vivo. High doses of nanoparticles from each individual metal caused a variable rate of cell death in vitro. However, the mixture of Co/Cr/Ti nanoparticles was more toxic than the Co, Cr or Ti metals alone at low doses. Intracellular distribution of Co, Cr, and Ti in the combined group was heterogeneous and associated with distinct morphological features. The results from in vivo experiments showed a significant increase in the mRNA levels of interleukin (IL)-1ß, IL-6, IL-8 and tumour necrosis factor (TNF)-α in peri-tibial soft tissue following the administration of Co alone as well as the combination of nanoparticles. Conclusion: This study demonstrated that the combination of Co/Cr/Ti nanoparticles was more cytotoxic than any of the individual metals in vitro and induced higher expression of genes encoding pro-inflammatory cytokines than single metals in vivo. The in vivo model utilised in this study might provide a useful tool for rapid assessment of the effects of unintended release of metal nanoparticles from implants in pre-/post-marketing studies. Translational potential of this article: This study highlights the importance of preclinical assessments of potential nanoparticles produced by wear and tear of metal implants using macrophages and animal models, in particular their combinational toxicity in addition to the assessments of the bulk metallic materials.

3D printed magnesium-doped ß-TCP gyroid scaffold with osteogenesis, angiogenesis, immunomodulation properties and bone regeneration capability in vivo.

Bioceramics have been used in orthopedic surgery for several years. Magnesium (Mg) is an essential element in bone tissue and plays an important role in bone metabolism. Mg-doped bioceramics has attracted the attention of researchers recently. However, the optimal doping amount of Mg in ß-TCP and the immunomodulatory property of Mg-doped ß-TCP (Mg-TCP) have not been determined yet. In this study, ß-TCP scaffolds doped with different contents of magnesium oxide (0 wt%, 1 wt%, 3 wt%, and 5 wt%) with gyroid structure were printed by digital light processing (DLP) method, and the physicochemical and biological functions were then investigated. Mg-doping improved the physicochemical properties of the ß-TCP scaffolds. In vitro experiments confirmed that the doping of Mg in ß-TCP scaffolds promoted the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenic differentiation of endothelial progenitor cells (EPCs), where the 5Mg-TCP has the optimal properties when using the "one cell type" method. It was also found that all Mg-TCP facilitated the polarization of RAW264.7 cells to the M2 phenotype, especially the 3Mg-TCP. However, 3Mg-TCP displayed the optimal osteogenic and angiogenic potential when using a "multiple cell type" method, which referred to culturing the BMSCs or EPCs in the macrophage-conditioned medium. Finally, the in vivo experiments were conducted and the results confirmed that the 3Mg-TCP scaffolds possessed the satisfying bone defect repair capability both after 6 and 12 weeks of implantation. This study suggests that 3Mg-TCP scaffolds provide the optimal biological performance and thus have the potential for clinical translation.

Three-dimensional printing of gyroid-structured composite bioceramic scaffolds with tuneable degradability.

Customisation of bioactivity and degradability of porous bioceramic scaffolds is a formidable challenge in the field of regenerative medicine. In this study, we developed gyroid-structured ternary composite scaffolds (biphasic calcium phosphate (BCP) and 45S5 bioglass® (BG)) using digital light processing 3D printing technology based on material and structural design. Additionally, the mechanical strength, bioactivity, degradability, and biocompatibility of the composite ceramic scaffolds were evaluated. The results revealed that BG reacted with BCP to generate major active crystalline phases of CaSiO3 and Na3Ca6(PO4)5. These active crystalline phases accelerated the exchange rate of Si4+, Ca2+, and PO43- with HCO3- in simulated body fluids and resulted in the rapid formation of carbonated hydroxyapatite (CHA), analogous to the formation of natural bone tissue. Interestingly, the precipitated CHA showed petal- and needle-like morphologies, which provided a large surface area to promote cell adhesion and proliferation. Furthermore, an increase in the BG content improved the degradability of ternary composite scaffolds after soaking in Tris-HCl solution. The tuneable degradability increased by three times at 30 wt% BG and sharply increased by 6.8 times at 40 wt% BG. This study provides a promising strategy to design scaffolds with improved bioactivity and tuneable degradability to assist a diverse population suffering from orthopedic conditions.

Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Promotes Inflammation and Accelerates Osteoarthritis by Activating ß-Catenin.

Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation, subchondral bone remodeling and osteophyte formation. Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) has not been fully investigated in the pathogenesis of OA. In this study, we found that SHP2 expression was significantly increased after interleukin-1ß (IL-1ß) treatment in primary mouse chondrocytes. Inhibition of SHP2 using siRNA reduced MMP3, MMP13 levels, but increased AGGRECAN, COL2A1, SOX9 expression in vitro. On the contrary, overexpression of SHP2 exerted the opposite results and promoted cartilage degradation. Mechanistically, SHP2 activated Wnt/ß-catenin signaling possibly through directly binding to ß-catenin. SHP2 also induced inflammation through activating Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways. Our in vivo studies showed that SHP2 knockdown effectively delayed cartilage destruction and reduced osteophyte formation in the mouse model of OA induced by destabilization of the medial meniscus (DMM). Altogether, our study identifies that SHP2 is a novel and potential therapeutic target of OA.

Inhibiting Monoacylglycerol Lipase Suppresses RANKL-Induced Osteoclastogenesis and Alleviates Ovariectomy-Induced Bone Loss.

Osteoporosis is a common chronic metabolic bone disease characterized by reduced trabecular bone and increased bone fragility. Monoacylglycerol lipase (MAGL) is a lipolytic enzyme to catalyze the hydrolysis of monoglycerides and specifically degrades the 2-arachidonoyl glycerol (2-AG). Previous studies have identified that 2-AG is the mainly source for arachidonic acid and the most abundant endogenous agonist of cannabinoid receptors. Considering the close relationship between inflammatory mediators/cannabinoid receptors and bone metabolism, we speculated that MAGL may play a role in the osteoclast differentiation. In the present study, we found that MAGL protein expression increased during osteoclast differentiation. MAGL knockdown by adenovirus-mediated shRNA in bone marrow-derived macrophages demonstrated the suppressive effects of MAGL on osteoclast formation and bone resorption. In addition, pharmacological inhibition of MAGL by JZL184 suppressed osteoclast differentiation, bone resorption, and osteoclast-specific gene expression. Activation of the Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways was inhibited by JZL184 and deletion of MAGL. Our in vivo study indicated that JZL184 ameliorated bone loss in an ovariectomized mouse model. Furthermore, overexpressing H1 calponin partially alleviated the inhibition caused by JZL184 or MAGL deletion on osteoclastogenesis. Therefore, we conclude that targeting MAGL may be a novel therapeutic strategy for osteoporosis.

NR1D1 modulates synovial inflammation and bone destruction in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, pannus formation, and cartilage and bone destruction. Nuclear receptor subfamily 1 group D member 1 (NR1D1) functions as a transcriptional repressor and plays a vital role in inflammatory reactions. However, whether NR1D1 is involved in synovial inflammation and joint destruction during the pathogenesis of RA is unknown. In this study, we found that NR1D1 expression was increased in synovial tissues from patients with RA and decreased in RA Fibroblast-like synoviocytes (FLSs) stimulated with IL-1ß in vitro. We showed that NR1D1 activation decreased the expression of proinflammatory cytokines and matrix metalloproteinases (MMPs), while NR1D1 silencing exerted the opposite effect. Furthermore, NR1D1 activation reduced reactive oxygen species (ROS) generation and increased the production of nuclear transcription factor E2-related factor 2 (Nrf2)-associated enzymes. Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways were blocked by the NR1D1 agonist SR9009 but activated by NR1D1 silencing. NR1D1 activation also inhibited M1 macrophage polarization and suppressed osteoclastogenesis and osteoclast-related genes expression. Treatment with NR1D1 agonist SR9009 in collagen-induced arthritis (CIA) mouse significantly suppressed the hyperplasia of synovial, infiltration of inflammatory cell and destruction of cartilage and bone. Our findings demonstrate an important role for NR1D1 in RA and suggest its therapeutic potential.

Pristimerin Suppresses RANKL-Induced Osteoclastogenesis and Ameliorates Ovariectomy-Induced Bone Loss.

Osteoporosis is characterized by bone loss and destruction of trabecular architecture, which greatly increases the burden on the healthcare system. Excessive activation of osteoclasts is an important cause of osteoporosis, and suppression of osteoclastogenesis is helpful for the treatment of osteoporosis. Pristimerin, a natural compound, possesses numerous pharmacological effects via inactivating the NF-κB and MAPK pathways, which are closely related to osteoclastogenesis process. However, the relationship between Pristimerin and osteoclastogenesis requires further investigation. In this research, we examined the effect of Pristimerin on osteoclastogenesis and investigated the related mechanisms. Our results showed Pristimerin inhibited RANKL-induced osteoclast differentiation and osteoclastic bone resorption in vitro, with decreased expression of osteoclastogenesis-related markers including c-Fos, NFATc1, TRAP, Cathepsin K, and MMP-9 at both mRNA and protein levels. Furthermore, Pristimerin suppressed NF-κB and MAPK signaling pathways, reduced reactive oxygen species (ROS) production and activated the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling during osteoclastogenesis. Our in vivo experiments showed that Pristimerin remarkably ameliorated ovariectomy-induced bone loss, reduced serum levels of TNF-α, IL-1ß, IL-6, and RANKL, and increased serum level of osteoprotegerin (OPG). Therefore, our research indicated that Pristimerin is a potential chemical for the treatment of osteoporosis.

Vinpocetine inhibits RANKL-induced osteoclastogenesis and attenuates ovariectomy-induced bone loss.

Osteoporosis is a result of impaired bone formation and/or excessive bone resorption. Osteoclasts are the only cells in the body that have a bone resorption function. Inhibiting osteoclast activity and differentiation is a way to treat osteoporosis. The current pharmacological treatment for osteoporosis has many shortcomings, and more effective treatments are needed. Vinpocetine (Vinp), a derivative of the alkaloid vincamine, has been used to treat cerebrovascular disorders and cognitive impairment for a long time. Vinp inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB)-dependent inflammatory responses and oxidative damage in which osteoclasts are often involved. However, the effects of Vinp on the regulation of osteoclast activity remain unknown. In this study, we found that Vinp significantly inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast and F-actin formation and decreased osteoclastic bone resorption in vitro. Vinp also suppressed the expression of osteoclast-specific genes, including NFATc1, c-Fos, tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), and cathepsin K (CTSK) at both the mRNA and protein levels. Vinp reduced activation of NF-κB, MAPK, and AKT signaling during osteoclastogenesis and prevented the production of reactive oxygen species with increased nuclear factor erythroid 2-related factor 2, heme oxygenase 1, and NAD(P)H:quinone acceptor oxidoreductase 1 expression. Animal experiments consistently demonstrated that Vinp treatment significantly attenuated ovariectomy-induced bone loss with a decrease in the osteoclast number and decreases in serum levels of RANKL, TRAP, interleukin-1ß, and tumor necrosis factor-alpha, as well as increased serum levels of osteoprotegerin. Taken together, our findings reveal that Vinp may be a potential pharmacological choice for preventing and treating osteoporosis.

Hesperetin suppresses RANKL-induced osteoclastogenesis and ameliorates lipopolysaccharide-induced bone loss.

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis.

Chemical concentration measurement in blood serum and urine samples using liquid-core optical fiber Raman spectroscopy.

We report measurements of chemical concentrations in clinical blood serum and urine samples using liquid-core optical fiber (LCOF) Raman spectroscopy to increase the collected signal strength. Both Raman and absorption spectra were acquired in the near-infrared region using the LCOF geometry. Spectra of 71 blood serum and 61 urine samples were regressed via partial least squares against reference analyzer values. Significant correlation was found between predicted and reference concentrations for 13 chemicals. Using absorption data to normalize the LCOF enhancement made the results more accurate. The experimental geometry is well suited for high-volume and automated chemical analysis of clear biofluids.

Correction method for absorption-dependent signal enhancement by a liquid-core optical fiber.

The enhancement of a dissolved chemical's Raman scattering by a liquid-core optical fiber (LCOF) geometry is absorption dependent. This dependence leads to a disruption of the usual linear correlation between chemical concentration and Raman peak area. To recover the linearity, we augmented a standard LCOF Raman spectroscopy system with spectrophotometric capabilities, permitting sequential measurements of Raman and absorption spectra within the LCOF. Measurements of samples with identical Raman-scatterer concentrations but different absorption coefficients are described. Using the absorption values, we reduced variations in the measured Raman intensities from 60% to less than 1%. This correction method should be important for LCOF-based Raman spectroscopy of sample sets with variable absorption coefficients, such as urine and blood serum from multiple patients.

Quantitative concentration measurements of creatinine dissolved in water and urine using Raman spectroscopy and a liquid core optical fiber.

We describe the use of a Teflon-AF liquid core optical fiber (LCOF) geometry to enhance the collection of Raman scattering from the biochemical creatinine, dissolved in water and in urine. At short integration times, where shot noise is most troublesome, the enhanced signal leads to greater accuracy in estimating the creatinine concentration from the spectrum. At longer integration times, instabilities in the LCOF geometry manifest themselves, and the predictions are the same as or worse than those from standard cuvette-based spectral measurements. Photobleaching of fluorescence from urine is more extensive and more stable in the LCOF as well. Starting from the measured enhancement of a major creatinine Raman band, we calculate the expected ratio of prediction errors obtained using the two geometries, and it agrees closely with the observed ratio. These results indicate that Raman spectroscopy with these Teflon-AF LCOFs is stable enough for quantitative concentration predictions, accurate to a few percent of the concentration range spanned.

Quantitative analysis of Raman signal enhancement from aqueous samples in liquid core optical fibers.

Raman scattering from aqueous liquids can be collected with high efficiency by enclosing the liquid within a suitable waveguide, as several groups have reported. Here, we present a quantitative model that predicts the relative strength of signals collected from (a) a tubular waveguide and (b) a flat-walled cuvette. Experimental measurements of Raman scattering from aqueous ethanol are made using two geometries, a Teflon-AF waveguide and a standard quartz cuvette. The model correctly predicts the enhancements in several ethanol Raman bands provided by the waveguide geometry. This model should be useful in aligning and characterizing liquid core waveguides, whose manufacture is still undergoing refinements. In particular, the model shows that absorption and scattering losses affect the enhancement factor in different ways.