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Osteoarthritis (OA) is the most common joint disease worldwide and a leading cause of disability. The Wnt/β-catenin cascade is essential in articular cartilage development and homeostasis. It has proved that both overexpression and loss of β-catenin lead to cartilage degeneration and OA symptoms. However, the mechanism of Wnt/β-catenin balance in healthy cartilage remains unclear. In the present work, we confirmed that the Wnt/β-catenin activation and PTHrP suppression in cartilage during the post-traumatic OA process. Then, we demonstrated that Wnt/β-catenin upregulated PTHrP expression through binding to its promoter (P2), and induce mRNA (AT6) transcript expression, while PTHrP repressed Wnt/β-catenin activity, and formed a Wnt/β-catenin-PTHrP negative feedback loop in the very primary chondrocytes to maintain cartilage homeostasis. However, this negative feedback loop vanished in dedifferentiated chondrocytes, hypertrophic chondrocytes, and IL-1β treated very primary chondrocytes. We further found that miR-106b-5p was increased in these “aberrant” chondrocytes and directly targeted PTHrP mRNA to abolish the feedback loop. PKC-ζ was activated by PTHrP through phosphorylation at Thr410/403, and subsequently induced β-catenin phosphorylation and ubiquitination. Finally, we disclosed that exogenous PTHrP attenuated OA progression exogenous PTHrP attenuated OA progression. Together, these findings reveal that PTHrP is a vital mediator to keep Wnt/β-catenin activity homeostasis in healthy cartilage through a negative feedback loop, and PTHrP might be a therapeutic target for OA and cartilage regeneration.

Introduction Magnesium (Mg) has a prophylactic potential against the onset of hyperlipidemia. Similar to statin, Mg is recommended as lipid-lowering medication for hypercholesterolemia and concomitantly exhibits an association with increased bone mass. The combination of statin with Mg ions (Mg2+) may be able to alleviate the high-fat diet (HFD)-induced bone loss and reduce the side-effects of statin. This study aimed to explore the feasibility of combined Mg2+ with simvastatin (SIM) for treating HFD-induced bone loss in mice and the involving mechanisms. Materials and methods C57BL/6 male mice were fed with a HFD or a normal-fat diet (NFD). Mice were intraperitoneally injected SIM and/or orally received water with additional Mg2+ until sacrificed. Enzyme-linked immunosorbent assay was performed to measure cytokines and cholesterol in serum and liver lysates. Bone mineral density (BMD) and microarchitecture were assessed by micro-computed tomography (μCT) in different groups. The adipogenesis in palmitate pre-treated HepG2 cells was performed under various treatments. Results μCT analysis showed that the trabecular bone mass was significantly lower in the HFD-fed group than that in NFD-fed group since week 8. The cortical thickness in HFD-fed group had a significant decrease at week 24, as compared with NFD-fed group. The combination of Mg2+ and SIM significantly attenuated the trabecular bone loss in HFD-fed mice via arresting the osteoclast formation and bone resorption. Besides, such combination also reduced the hepatocytic synthesis of cholesterol and inhibited matrix metallopeptidase 13 (Mmp13) mRNA expression in pre-osteoclasts. Conclusions The combination of Mg2+ and SIM shows a synergistic effect on attenuating the HFD-induced bone loss. Our current formulation may be a cost-effective alternative treatment to be indicated for obesity-related bone loss., Graphical abstract Illustration of the underlying mechanism of combined therapy of Mg2+ and simvastatin that leads to a synergistic effect on attenuating the high-fat diet-induced bone loss via impairing osteoclast maturation and bone resorption in bone marrow. Meanwhile, the combination can directly limit the hepatocytic synthesis of cholesterol.Image 1, Highlights • High-fat diet-fed mouse has a susceptibility to lower trabecular bone mass as compared with that of normal-fat diet-fed mouse. • The combination of Mg2+ and simvastatin attenuates the trabecular bone loss in high-fat diet-fed mice. • The combination of Mg2+ and simvastatin reduces the hepatocytic synthesis of cholesterol.

The neuronal engagement of the peripheral nerve system plays a crucial role in regulating fracture healing, but how to modulate the neuronal activity to enhance fracture healing remains unexploited. Here it is shown that electrical stimulation (ES) directly promotes the biosynthesis and release of calcitonin gene‐related peptide (CGRP) by activating Ca2+/CaMKII/CREB signaling pathway and action potential, respectively. To accelerate rat femoral osteoporotic fracture healing which presents with decline of CGRP, soft electrodes are engineered and they are implanted at L3 and L4 dorsal root ganglions (DRGs). ES delivered at DRGs for the first two weeks after fracture increases CGRP expression in both DRGs and fracture callus. It is also identified that CGRP is indispensable for type‐H vessel formation, a biological event coupling angiogenesis and osteogenesis, contributing to ES‐enhanced osteoporotic fracture healing. This proof‐of‐concept study shows for the first time that ES at lumbar DRGs can effectively promote femoral fracture healing, offering an innovative strategy using bioelectronic device to enhance bone regeneration., Electrical stimulation (ES) at dorsal root ganglions (DRGs) triggers calcium influx and activates the Ca2+/CaMKII/CREB signaling cascade, resulting in the transcription of calcitonin gene‐related peptide (CGRP). After synthesized in DRGs, CGRP is transported to peripheral nerve terminals and released following depolarization by ES. The released CGRP and subsequently increased type‐H vessel formation promote osteoporotic fracture healing.

In this study, perforated cannulated magnesium (Mg) hip stents were fabricated via modified Mg injection molding and conventional machining, respectively. Additionally, the stent canal was filled with paraffin to simulate injection of biomaterials. The microstructure, mechanical performance, corrosion behavior, and biocompatibility were comparably studied. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) showed higher affinity of interstitial element such as oxygen and carbon as consequences of routine molding process. After immersion in SBF, machining stents showed reduced degradation rate and increased deposition of calcium phosphate compared to molding stents. Corrosion resistance was improved via paraffin-filling. Consistently, the hemolysis and in vitro osteoblast cell culture models showed favourable biocompatibility in machining stents compared to molding ones, which was improved by paraffin-filling treatment as well. These results implied that the feasibility of the prepared machining stents as the potential in vivo orthopaedic application where slower degradation is required, which could be enhanced by designing canal-filling injection of biomaterials as well.

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Here, we demonstrated that magnesium (Mg) nail implantation into the marrow cavity degraded gradually accompanied with about 4-fold increase of new bone formation and over 5-fold of new vessel formation as compared with DO alone group in the 5 mm femoral segmental defect rat model at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. CGRP concentration-dependently promoted endothelial cell migration and tube formation. Meanwhile, CGRP promoted the phosphorylation of focal adhesion kinase (FAK) at Y397 site and elevated the expression of vascular endothelial growth factor A (VEGFA). Moreover, inhibitor/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. We revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications.

Displaced fractures of patella often require open reduction surgery and internal fixation to restore the extensor continuity and articular congruity. Fracture fixation with biodegradable magnesium (Mg) pins enhanced fracture healing. We hypothesized that fixation with Mg pins and their degradation over time would enhance healing of patellar fracture radiologically, mechanically, and histologically. Transverse patellar fracture surgery was performed on thirty-two 18-weeks old female New Zealand White Rabbits. The fracture was fixed with a pin made of stainless steel or pure Mg, and a figure-of-eight stainless steel band wire. Samples were harvested at week 8 or 12, and assessed with microCT, tensile testing, microindentation, and histology. Microarchitectural analysis showed that Mg group showed 12% higher in the ratio of bone volume to tissue volume at week 8, and 38.4% higher of bone volume at week 12. Tensile testing showed that the failure load and stiffness of Mg group were 66.9% and 104% higher than the control group at week 8, respectively. At week 12, Mg group was 60.8% higher in ultimate strength than the control group. Microindentation showed that, compared to the Control group, Mg group showed 49.9% higher Vickers hardness and 31% higher elastic modulus at week 8 and 12, respectively. At week 12, the new bone of Mg group remodelled to laminar bone, but those of the control group remained woven bone-like. Fixation of transverse patellar fracture with Mg pins and its degradation enhanced new bone formation and mechanical properties of the repaired patella compared to the Control group., Graphical abstract Image 1, Highlights • Kirschner wires (K-wire) with tension band wire is widely used fixation implants for repairing of displaced patellar fractures. • Fixation of patellar fracture with Mg pins enhanced new bone formation and mechanical properties of the repaired patella. • With a stainless steel tension band wire, Mg pins may be an alternative to K-wire for fixation of patellar fractures.

Distraction osteogenesis (DO) is a well-established surgical technique for treating bone defect and limb lengthening. The major drawback of DO is the long treatment period as the external fixator has to be kept in place until consolidation is completed. Calcitonin gene-related peptide (CGRP) has been reported to promote angiogenesis by affecting endothelial progenitor cells (EPCs) in limb ischemia and wound healing. Thus, the goal of this study was to evaluate the angiogenic effect of exogenous CGRP on bone regeneration in a rat DO model. Exogenous CGRP was directly injected into the bone defect after each cycle of distraction in vivo. Microcomputed tomography, biomechanical test, and histological analysis were performed to assess the new bone formation. Angiography and immunofluorescence were performed to assess the formation of blood vessels. CD31+CD144+ EPCs in the bone defect were quantified with flow cytometry. In in vitro study, bone marrow stem cells (BMSCs) were used to investigate the effect of CGRP on EPCs production during endothelial differentiation. Our results showed that CGRP significantly promoted bone regeneration and vessel formation after consolidation. CGRP significantly increased the fraction of CD31+CD144+EPCs and the capillary density in the bone defect at the end of distraction phase. CGRP increased EPC population in the endothelial differentiation of BMSCs in vitro by activating PI3K/AKT signaling pathway. Furthermore, differentiated EPCs rapidly assembled into tube-like structures and promoted osteogenic differentiation of BMSCs. In conclusion, CGRP increased EPC population and promoted blood vessel formation and bone regeneration at the defect region in a DO model. Impact statement Distraction osteogenesis (DO) is a well-established surgical technique for limb lengthening and bone defect. The disadvantage of this technique is that external fixator is needed to be kept in place for about 12 months. This may result in increased risk of infection, financial burden, and negative psychological impacts. In this study, we have injected calcitonin gene-related peptide (CGRP) into the defect region after distraction and found that CGRP enhanced vessel formation and bone regeneration in a rat DO model. This suggests that a controlled delivery system for CGRP could be developed and applied clinically for accelerating bone regeneration in patients with DO.

One of the effective strategies for prevention of steroid-associated osteonecrosis (SAON) is to inhibit bone resorption and fat formation and promote bone formation at osteonecrotic sensitive skeletal sites. We identified a novel phytomolecule that showed positive effects on osteogenesis, anti-bone resorption and anti-adipogenesis in vitro and also developed a bone-targeting delivery system (BTDS) for in vivo experimental study. The study investigated if our innovative synthesized BTDS carrying this phytomolecule would be able to effectively prevent SAON in a rat model. SAON was induced by combined injections of lipopolysaccharide and methylprednisolone. SAON rats were divided into four groups, one SAON untreated control group and three SAON treatment groups with different types of delivery systems (Asp8-liposome-icaritin, liposome-icaritin and Asp8-liposome) for two weeks. SAON lesions were identified and osteoclasts activity, osteogenesis and adipogenesis at these sites were evaluated by immunohistochemistry. Ex vitro study was also designed to evaluate the osteogenic and adipogenic potential of the isolated bone marrow stromal cells (BMSCs) via real-time PCR and histochemical staining. Our results showed that as a bone surface-specific BTDS, Asp8-liposome-icaritin effectively prevented steroids-treated rats from SAON with significantly decreased osteocytes apoptosis, down-regulated osteoclatsogenesis and up-regulated osteogenesis. However, both liposome-icaritin and Asp8-liposome treatment did not show significant efficacy for SAON prevention. In summary, this proof-concept-study showed for the first time that the innovative Asp8-liposome-icaritin BTDS was effective for prevention of SAON in terms of bone resorption prevention, adipogenesis suppression, and bone-formation enhancement.

Two patients have suffered from chronic tracheobronchitis for twenty years and were treated by traditional therapy. However, chronic tracheobronchitis has never been completely cured. They develop lasting drastic morning coughing because of throat abnormal sensation caused by sticky phlegm. The chronic tracheobronchitis was obviously relieved, in most conditions was totally disappeared, since the third month they regularly took isosorbide dinitrate (ISDN) for angina pectoris. This unexpected effect of ISDN is remarkable, and given the smooth muscle relaxation mechanism, this may provide a potential therapeutic idea that takes advantage of similar drugs both for angina pectoris and chronic tracheobronchitis.

Interleukin 17A (IL-17A) is critical in the pathogenesis of autoimmune diseases through driving inflammatory cascades. However, the role of IL-17 in osteoarthritis (OA) is not well understood. Tumor necrosis factor–receptor–associated factor 3 (TRAF3) is a receptor proximal negative regulator of IL-17 signaling. It remains unclear whether TRAF3 exerts regulatory effects on cartilage degradation and contributes to the pathogenesis of OA. In this study, we found that TRAF3 notably suppressed IL-17–induced NF-κB and mitogen-activated protein kinase activation and, subsequently, the production of matrix-degrading enzymes. TRAF3 depletion enhanced IL-17 signaling, along with increased matrix-degrading enzyme production. In vivo, cartilage destruction caused by surgery-induced OA was alleviated markedly both in 1l17a–deficient mice and in TRAF3 transgenic mice. In contrast, silencing TRAF3 through adenoviruses worsened cartilage degradation in experimental OA. Moreover, the destructive effect of IL-17 on cartilage was abolished in TRAF3 transgenic mice in an IL-17 intra-articular injection animal model. Similarly, genetic deletion of IL-17 blocked TRAF3 knockdown—mediated promotion of cartilage destruction, suggesting that the protective effect of TRAF3 on cartilage is mediated by its suppression of IL-17 signaling. Collectively, our results suggest that TRAF3 negatively regulates IL-17–mediated cartilage degradation and pathogenesis of OA, and may serve as a potential new therapy target for OA.

This study examines two strategies for improving the analysis of an hourly update three-dimensional variational data assimilation (3DVAR) system and the subsequent quantitative precipitation forecast (QPF). The first strategy is to assimilate synoptic and radar observations in different steps. This strategy aims to extract both large-scale and convective-scale information from observations typically representing different scales. The second strategy is to add a divergence constraint to the momentum variables in the 3DVAR system. This technique aims at improving the dynamic balance and suppressing noise introduced during the assimilation process. A detailed analysis on how the new techniques impact convective-scale QPF was conducted using a severe storm case over Colorado and Kansas during 8 and 9 August 2008. First, it is demonstrated that, without the new strategies, the QPF initialized with an hourly update analysis performs worse than its 3-hourly counterpart. The implementation of the two-step assimilation and divergence constraint in the hourly update system results in improved QPF throughout most of the 12-h forecast period. The diagnoses of the analysis fields show that the two-step assimilation is able to preserve key convective-scale as well as large-scale structures that are consistent with the development of the real weather system. The divergence constraint is effective in improving the balance between the momentum control variables in the analysis, which leads to less spurious convection and improved QPF scores. The improvements of the new techniques were further verified by eight convective cases in 2014 and shown to be statistically significant.

ObjectivesWnt16 is implicated in bone fracture and bone mass accrual both in animals and humans. However, its functional roles and molecular mechanism in chondrocyte differentiation and osteoarthritis (OA) pathophysiology remain largely undefined. In this study, we analysed its mechanistic association and functional relationship in OA progression in chondrocyte lineage.MethodsThe role of Wnt16 during skeletal development was examined by Col2a1-Wnt16 transgenic mice and Wnt16fl/fl;Col2a1-Cre (Wnt16-cKO) mice. OA progression was assessed by micro-CT analysis and Osteoarthritis Research Society International score after anterior cruciate ligament transection (ACLT) surgery with Wnt16 manipulation by adenovirus intra-articular injection. The molecular mechanism was investigated in vitro using 3D chondrocyte pellet culture and biochemical analyses. Histological analysis was performed in mouse joints and human cartilage specimens.ResultsWnt16 overexpression in chondrocytes in mice significantly inhibited chondrocyte hypertrophy during skeletal development. Wnt16 deficiency exaggerated OA progression, whereas intra-articular injection of Ad-Wnt16 markedly attenuated ACLT-induced OA. Cellular and molecular analyses showed that, instead of β-catenin and calcium pathways, Wnt16 activated the planar cell polarity (PCP) and JNK pathway by interacting mainly with AP2b1, and to a lesser extend Ror2 and CD146, and subsequently induced PTHrP expression through phosphor-Raptor mTORC1 pathway.ConclusionsOur findings indicate that Wnt16 activates PCP/JNK and crosstalks with mTORC1-PTHrP pathway to inhibit chondrocyte hypertrophy. Our preclinical study suggests that Wnt16 may be a potential therapeutic target for OA treatment.

The momentum variables of streamfunction and velocity potential are used as control variables in a number of operational variational data assimilation systems. However, in this study it is shown that, for limited-area high-resolution data assimilation, the momentum control variables ψ and χ (ψχ) pose potential difficulties in background error modeling and, hence, may result in degraded analysis and forecast when compared with the direct use of x and y components of wind (UV). In this study, the characteristics of the modeled background error statistics, derived from an ensemble generated from Weather Research and Forecasting (WRF) Model real-time forecasts of two summer months, are first compared between the two control variable options. Assimilation and forecast experiments are then conducted with both options for seven convective events in a domain that encompasses the Rocky Mountain Front Range using the three-dimensional variational data assimilation (3DVar) system of the WRF Model. The impacts of the two control variable options are compared in terms of their skills in short-term qualitative precipitation forecasts. Further analysis is performed for one case to examine the impacts when radar observations are included in the 3DVar assimilation. The main findings are as follows: 1) the background error modeling used in WRF 3DVar with the control variables ψχ increases the length scale and decreases the variance for u and υ, which causes negative impact on the analysis of the velocity field and on precipitation prediction; 2) the UV-based 3DVar allows closer fits to radar wind observations; and 3) the use of UV control variables improves the 0–12-h precipitation prediction.

Osteoarthritis (OA) is a highly prevalent and debilitating joint disorder characterized by the degeneration of articular cartilage. However, no effective medical therapy has been found yet for such condition. In this study, we directly confirmed the existence of articular cartilage stem cells (ACSCs) in vivo and in situ for the first time both in normal and OA articular cartilage, and explored their chondrogenesis in Interleukin-1β (IL-1β) induced inflammation environment and disclose whether the inhibition of NF-κB signaling can induce ACSCs activation thus improve the progression of experimental OA. We found an interesting phenomenon that ACSCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for self-repair. During the in vitro mechanism study, we discovered IL-1β can efficiently activate the NF-κB pathway and potently impair the responsiveness of ACSCs, whereas the NF-κB pathway inhibitor rescued the ACSCs chondrogenesis. The final in vivo experiments further confirmed ACSCs' activation were maintained by NF-κB pathway inhibitor, which induced cartilage regeneration, and protected articular cartilage from injury in an OA animal model. Our results provided in vivo evidence of the presence of ACSCs, and disclosed their action in the early OA stage and gradual quiet as OA process, presented a potential mechanism for both cartilage intrinsic repair and its final degradation, and demonstrated the feasibility of inducing endogenous adult tissue-specific mesenchymal stem cells for articular cartilage repair and OA therapy. Stem Cells 2015;33:3125–3137

Study Design. Intermittent Cyclic Mechanical Tension (ICMT) was applied to end plate chondrocytes by using an FX-4000T Flexercell Tension Plus unit (Flexcell International Corporation, Hillsborough, NC). Changes of end plate chondrocytes were observed after ICMT stimulation. Objective. To investigate the relationship between mechanical stimulation and calcification of end plate chondrocytes. Summary of Background Data. Previous study showed that end plate calcification was related to mechanical stress, but there was no clear evidence to indicate whether or not mechanical stimulation could induce calcification of end plate chondrocytes in vitro. Methods. Rat end plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) was applied for 25 days, 4 hours a day and continued to culture for 5 days. End plate chondrocytes were incubated for 12 hours in the presence or absence of 10 ng/mL of transforming growth factor-beta 1 (TGF-beta 1) (prepared from a stock solution at 10 mu g/mL in 2 mM citric acid containing 2 mg/mL bovine serum albumin) in MEM/F-12 containing a final concentration of 1% FCS. End plate chondrocytes calcification was stained by alizarin red S (AR-S). End plate chondrocytes viability was examined by LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA). Related gene expression was examined by reverse transcription-polymerase chain reaction and Western blot. Results. LIVE/DEAD assay verified that the nonloading (NC) group and the ICMT group end plate chondrocytes remained adherent, with no change in viability after the application of ICMT. Alizarin red staining showed that ICMT induced the calcification of end plate chondrocytes. Real-time reverse transcription-polymerase chain reaction showed that mRNA expression of endogenous TGF-beta 1 decreased and mRNA expression of type I, type X, osteocalcin and osteopontin increased after ICMT. The ankh gene expression of both mRNA and protein levels decreased in the ICMT stimulation. The ankh gene expression of both mRNA and protein levels increased in TGF-beta 1 stimulation. Compared with NC group, the alkaline phosphatase activities significantly increased in ICMT group. Conclusion. Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-beta 1.

Cytoxicity is an essential concern for polyethyleneimine 25 kDa (PEI 25 kDa), a widely reported, highly effective transfection agent used in gene delivery. In our recent experiments, Small molecular weight cross-linked poly(ethylene imine) by biscarbamate linkage (PEI-Bu) (Mn: 3278, Mw: 4289) can reduce target cell apoptosis induced by polycationic transfection, and has almost the same DNA condensation capability as PEI 25 kDa. PEI-Bu showed significantly higher activity and lower cytotoxicity than PEI 25 kDa in transfecting the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) gene to rat synoviocytes, an optimal target for arthritis treatment. The expression of IL-1Ra in synoviocytes then suppresses the expression of metalloproteases 13 (MMP13) gene, which is responsible for cartilage destruction regulated by IL-1β in arthritis. In conclusion, PEI-Bu is a promising tool for delivering IL-1Ra gene to synoviocytes for arthritis therapy.