Canonical pathways for validating steroid-associated osteonecrosis in mice.

The present study aimed to establish and evaluate a preclinical model of steroid-associated osteonecrosis (SAON) in mice. Sixteen 24-week-old male C57BL/6 mice were used to establish SAON by two intraperitoneal injections of lipopolysaccharide (LPS), followed by three subcutaneous injections of methylprednisolone (MPS). Each injection was conducted on working day, with an interval of 24 h. Six cycles of injections were conducted. Additional twelve mice (age- and gender-matched) were used as normal controls. At 2 and 6 weeks after completing induction, bilateral femora and bilateral tibiae were collected for histological examination, micro-CT scanning, and bulk RNA sequencing. All mice were alive until sacrificed at the indicated time points. The typical SAON lesion was identified by histological evaluation at week 2 and week 6 with increased lacunae and TUNEL+ osteocytes. Micro-CT showed significant bone degeneration at week 6 in SAON model. Histology and histomorphometry showed significantly lower Runx2+ area, mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR/BS), type H vessels, Ki67+ (proliferating) cells, and higher marrow fat fraction, osteoclast number and TNFα+ areas in SAON group. Bulk RNA-seq revealed changed canonical signaling pathways regulating cell cycle, angiogenesis, osteogenesis, and osteoclastogenesis in the SAON group. The present study successfully established SAON in mice with a combination treatment of LPS and MPS, which could be considered a reliable and reproducible animal model to study the pathophysiology and molecular mechanism of early-stage SAON and to develop potential therapeutic approaches for the prevention and treatment of SAON.

Excess glucocorticoids inhibit murine bone turnover via modulating the immunometabolism of the skeletal microenvironment.

Elevated bone resorption and diminished bone formation have been recognized as the primary features of glucocorticoid-associated skeletal disorders. However, the direct effects of excess glucocorticoids on bone turnover remain unclear. Here, we explored the outcomes of exogenous glucocorticoid treatment on bone loss and delayed fracture healing in mice and found that reduced bone turnover was a dominant feature, resulting in a net loss of bone mass. The primary effect of glucocorticoids on osteogenic differentiation was not inhibitory; instead, they cooperated with macrophages to facilitate osteogenesis. Impaired local nutrient status - notably, obstructed fatty acid transportation - was a key factor contributing to glucocorticoid-induced impairment of bone turnover in vivo. Furthermore, fatty acid oxidation in macrophages fueled the ability of glucocorticoid-liganded receptors to enter the nucleus and then promoted the expression of BMP2, a key cytokine that facilitates osteogenesis. Metabolic reprogramming by localized fatty acid delivery partly rescued glucocorticoid-induced pathology by restoring a healthier immune-metabolic milieu. These data provide insights into the multifactorial metabolic mechanisms by which glucocorticoids generate skeletal disorders, thus suggesting possible therapeutic avenues.

Time-Sequential and Multi-Functional 3D Printed MgO2 /PLGA Scaffold Developed as a Novel Biodegradable and Bioactive Bone Substitute for Challenging Postsurgical Osteosarcoma Treatment.

Osteosarcoma (OS) is the most commonly occurring primary bone malignant tumor. The clinical postsurgical OS treatment faces big challenges for the staged therapeutic requirements of early anti-tumor, anti-bacterial, and long-lasting osteogenesis. Herein, multi-functional bioactive scaffolds with time-sequential functions of preventing tumor recurrence, inhibiting bacterial infection, and promoting bone defect repair are designed as a novel strategy. Nanocomposite scaffold magnesium peroxide (MgO2 )/poly (lactide-co-glycolide) is prepared by low-temperature 3D printing for controllable releasing magnesium ions (Mg2+ ) and reactive oxygen species in a time-sequential manner. The scaffold with 20 wt% MgO2 (20MP) is verified with desired mechanical properties, as well as exhibits staged release behavior of bioactive elements with hydrogen peroxide (H2 O2 ) release for the first 3 weeks, and long-lasting Mg2+ release for 12 weeks. The released H2 O2 initiates chemodynamic therapy to induce apoptosis and ferroptosis in tumor cells, along with activating the anticancer immune microenvironment by M1 polarization of macrophages. The released Mg2+ subsequently enhances bone repair by activating the Wnt3a/GSK-3ß/ß-catenin signaling pathway to promote osteogenic differentiation of bone marrow mesenchymal stem cells and create osteopromotive immune microenvironment by M2 polarization of macrophages. In conclusion, the multi-functional 20MP scaffold demonstrates time-sequential therapeutic properties as an innovative strategy for OS-associated bone defect treatment.

Engineered biochemical cues of regenerative biomaterials to enhance endogenous stem/progenitor cells (ESPCs)-mediated articular cartilage repair.

As a highly specialized shock-absorbing connective tissue, articular cartilage (AC) has very limited self-repair capacity after traumatic injuries, posing a heavy socioeconomic burden. Common clinical therapies for small- to medium-size focal AC defects are well-developed endogenous repair and cell-based strategies, including microfracture, mosaicplasty, autologous chondrocyte implantation (ACI), and matrix-induced ACI (MACI). However, these treatments frequently result in mechanically inferior fibrocartilage, low cost-effectiveness, donor site morbidity, and short-term durability. It prompts an urgent need for innovative approaches to pattern a pro-regenerative microenvironment and yield hyaline-like cartilage with similar biomechanical and biochemical properties as healthy native AC. Acellular regenerative biomaterials can create a favorable local environment for AC repair without causing relevant regulatory and scientific concerns from cell-based treatments. A deeper understanding of the mechanism of endogenous cartilage healing is furthering the (bio)design and application of these scaffolds. Currently, the utilization of regenerative biomaterials to magnify the repairing effect of joint-resident endogenous stem/progenitor cells (ESPCs) presents an evolving improvement for cartilage repair. This review starts by briefly summarizing the current understanding of endogenous AC repair and the vital roles of ESPCs and chemoattractants for cartilage regeneration. Then several intrinsic hurdles for regenerative biomaterials-based AC repair are discussed. The recent advances in novel (bio)design and application regarding regenerative biomaterials with favorable biochemical cues to provide an instructive extracellular microenvironment and to guide the ESPCs (e.g. adhesion, migration, proliferation, differentiation, matrix production, and remodeling) for cartilage repair are summarized. Finally, this review outlines the future directions of engineering the next-generation regenerative biomaterials toward ultimate clinical translation.

Blockage of Osteopontin-Integrin ß3 Signaling in Infrapatellar Fat Pad Attenuates Osteoarthritis in Mice.

The knowledge of osteoarthritis (OA) has nowadays been extended from a focalized cartilage disorder to a multifactorial disease. Although recent investigations have reported that infrapatellar fat pad (IPFP) can trigger inflammation in the knee joint, the mechanisms behind the role of IPFP on knee OA progression remain to be defined. Here, dysregulated osteopontin (OPN) and integrin ß3 signaling are found in the OA specimens of both human and mice. It is further demonstrated that IPFP-derived OPN participates in OA progression, including activated matrix metallopeptidase 9 in chondrocyte hypertrophy and integrin ß3 in IPFP fibrosis. Motivated by these findings, an injectable nanogel is fabricated to provide sustained release of siRNA Cd61 (RGD- Nanogel/siRNA Cd61) that targets integrins. The RGD- Nanogel possesses excellent biocompatibility and desired targeting abilities both in vitro and in vivo. Local injection of RGD- Nanogel/siRNA Cd61 robustly alleviates the cartilage degeneration, suppresses the advancement of tidemark, and reduces the subchondral trabecular bone mass in OA mice. Taken together, this study provides an avenue for developing RGD- Nanogel/siRNA Cd61 therapy to mitigate OA progression via blocking OPN-integrin ß3 signaling in IPFP.

Injectable bioactive glass/sodium alginate hydrogel with immunomodulatory and angiogenic properties for enhanced tendon healing.

Tendon healing is a complex process involving inflammation, proliferation, and remodeling, eventually achieving a state of hypocellularity and hypovascularity. Currently, few treatments can satisfactorily restore the structure and function of native tendon. Bioactive glass (BG) has been shown to possess immunomodulatory and angiogenic properties. In this study, we investigated whether an injectable hydrogel fabricated of BG and sodium alginate (SA) could be applied to enhance tenogenesis following suture repair of injured tendon. We demonstrated that BG/SA hydrogel significantly accelerated tenogenesis without inducing heterotopic ossification based on histological analysis. The therapeutic effect could attribute to increased angiogenesis and M1 to M2 phenotypic switch of macrophages within 7 days post-surgery. Morphological characterization demonstrated that BG/SA hydrogel partially reverted the pathological changes of Achilles tendon, including increased length and cross-sectional area (CSA). Finally, biomechanical test showed that BG/SA hydrogel significantly improved ultimate load, failure stress, and tensile modulus of the repaired tendon. In conclusion, administration of an injectable BG/SA hydrogel can be a novel and promising therapeutic approach to augment Achilles tendon healing in conjunction with surgical intervention.

Bioactive glass-elicited stem cell-derived extracellular vesicles regulate M2 macrophage polarization and angiogenesis to improve tendon regeneration and functional recovery.

Effective countermeasures for tendon injury remains unsatisfactory. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs)-based therapy via regulation of Mφ-mediated angiogenesis has emerged as a promising strategy for tissue regeneration. Still, approaches to tailor the functions of EVs to treat tendon injuries have been limited. We reported a novel strategy by applying MSC-EVs boosted with bioactive glasses (BG). BG-elicited EVs (EVB) showed up-regulation of medicinal miRNAs, including miR-199b-3p and miR-125a-5p, which play a pivotal role in M2 Mφ-mediated angiogenesis. EVB accelerated angiogenesis via the reprogrammed anti-inflammatory M2 Mφs compared with naïve MSC-EVs (EVN). In rodent Achilles tendon rupture model, EVB local administration activated anti-inflammatory responses via M2 polarization and led to a spatial correlation between M2 Mφs and newly formed blood vessels. Our results showed that EVB outperformed EVN in promoting tenogenesis and in reducing detrimental morphological changes without causing heterotopic ossification. Biomechanical test revealed that EVB significantly improved ultimate load, stiffness, and tensile modulus of the repaired tendon, along with a positive correlation between M2/M1 ratio and biomechanical properties. On the basis of the boosted nature to reprogram regenerative microenvironment, EVB holds considerable potential to be developed as a next-generation therapeutic modality for enhancing functional regeneration to achieve satisfying tendon regeneration.

Controlled Release of Bone Morphogenetic Protein-2 Augments the Coupling of Angiogenesis and Osteogenesis for Accelerating Mandibular Defect Repair.

Reconstruction of a mandibular defect is challenging, with high expectations for both functional and esthetic results. Bone morphogenetic protein-2 (BMP-2) is an essential growth factor in osteogenesis, but the efficacy of the BMP-2-based strategy on the bone regeneration of mandibular defects has not been well-investigated. In addition, the underlying mechanisms of BMP-2 that drives the bone formation in mandibular defects remain to be clarified. Here, we utilized BMP-2-loaded hydrogel to augment bone formation in a critical-size mandibular defect model in rats. We found that implantation of BMP-2-loaded hydrogel significantly promoted intramembranous ossification within the defect. The region with new bone triggered by BMP-2 harbored abundant CD31+ endomucin+ type H vessels and associated osterix (Osx)+ osteoprogenitor cells. Intriguingly, the new bone comprised large numbers of skeletal stem cells (SSCs) (CD51+ CD200+) and their multi-potent descendants (CD51+ CD105+), which were mainly distributed adjacent to the invaded blood vessels, after implantation of the BMP-2-loaded hydrogel. Meanwhile, BMP-2 further elevated the fraction of CD51+ CD105+ SSC descendants. Overall, the evidence indicates that BMP-2 may recapitulate a close interaction between functional vessels and SSCs. We conclude that BMP-2 augmented coupling of angiogenesis and osteogenesis in a novel and indispensable way to improve bone regeneration in mandibular defects, and warrants clinical investigation and application.

Biosynthesized Bandages Carrying Magnesium Oxide Nanoparticles Induce Cortical Bone Formation by Modulating Endogenous Periosteal Cells.

Bone grafting is frequently conducted to treat bone defects caused by trauma and tumor removal, yet with significant medical and socioeconomic burdens. Space-occupying bone substitutes remain challenging in the control of osteointegration, and meanwhile activation of endogenous periosteal cells by using non-space-occupying implants to promote new bone formation becomes another therapeutic strategy. Here, we fabricated a magnesium-based artificial bandage with optimal micropatterns for activating periosteum-associated biomineralization. Collagen was self-assembled on the surface of magnesium oxide nanoparticles embedded electrospun fibrous membranes as a hierarchical bandage structure to facilitate the integration with periosteum in situ. After the implantation on the surface of cortical bone in vivo, magnesium ions were released to generate a pro-osteogenic immune microenvironment by activating the endogenous periosteal macrophages into M2 phenotype and, meanwhile, promote blood vessel formation and neurite outgrowth. In a cortical bone defect model, magnesium-based artificial bandage guided the surrounding newly formed bone tissue to cover the defected area. Taken together, our study suggests that the strategy of stimulating bone formation can be achieved with magnesium delivery to periosteum in situ and the proposed periosteal bandages act as a bioactive media for accelerating bone healing.

Magnesium-Encapsulated Injectable Hydrogel and 3D-Engineered Polycaprolactone Conduit Facilitate Peripheral Nerve Regeneration.

Peripheral nerve injury is a challenging orthopedic condition that can be treated by autograft transplantation, a gold standard treatment in the current clinical setting. Nevertheless, limited availability of autografts and potential morbidities in donors hampers its widespread application. Bioactive scaffold-based tissue engineering is a promising strategy to promote nerve regeneration. Additionally, magnesium (Mg) ions enhance nerve regeneration; however, an effectively controlled delivery vehicle is necessary to optimize their in vivo therapeutic effects. Herein, a bisphosphonate-based injectable hydrogel exhibiting sustained Mg2+ delivery for peripheral nerve regeneration is developed. It is observed that Mg2+ promoted neurite outgrowth in a concentration-dependent manner by activating the PI3K/Akt signaling pathway and Sema5b. Moreover, implantation of polycaprolactone (PCL) conduits filled with Mg2+ -releasing hydrogel in 10 mm nerve defects in rats significantly enhanced axon regeneration and remyelination at 12 weeks post-operation compared to the controls (blank conduits or conduits filled with Mg2+ -absent hydrogel). Functional recovery analysis reveals enhanced reinnervation in the animals treated with the Mg2+ -releasing hydrogel compared to that in the control groups. In summary, the Mg2+ -releasing hydrogel combined with the 3D-engineered PCL conduit promotes peripheral nerve regeneration and functional recovery. Thus, a new strategy to facilitate the repair of challenging peripheral nerve injuries is proposed.

Macrophages in epididymal adipose tissue secrete osteopontin to regulate bone homeostasis.

Epididymal white adipose tissue (eWAT) secretes an array of cytokines to regulate the metabolism of organs and tissues in high-fat diet (HFD)-induced obesity, but its effects on bone metabolism are not well understood. Here, we report that macrophages in eWAT are a main source of osteopontin, which selectively circulates to the bone marrow and promotes the degradation of the bone matrix by activating osteoclasts, as well as modulating bone marrow-derived macrophages (BMDMs) to engulf the lipid droplets released from adipocytes in the bone marrow of mice. However, the lactate accumulation induced by osteopontin regulation blocks both lipolysis and osteoclastogenesis in BMDMs by limiting the energy regeneration by ATP6V0d2 in lysosomes. Both surgical removal of eWAT and local injection of either clodronate liposomes (for depleting macrophages) or osteopontin-neutralizing antibody show comparable amelioration of HFD-induced bone loss in mice. These results provide an avenue for developing therapeutic strategies to mitigate obesity-related bone disorders.

Implantable Electrical Stimulation at Dorsal Root Ganglions Accelerates Osteoporotic Fracture Healing via Calcitonin Gene-Related Peptide.

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.

Comprehensive Analysis of Key Genes, Signaling Pathways and miRNAs in Human Knee Osteoarthritis: Based on Bioinformatics.

Background: Osteoarthritis (OA) is one of the main causes of disability in the elderly population, accompanied by a series of underlying pathologic changes, such as cartilage degradation, synovitis, subchondral bone sclerosis, and meniscus injury. The present study aimed to identify key genes, signaling pathways, and miRNAs in knee OA associated with the entire joint components, and to explain the potential mechanisms using computational analysis. Methods: The differentially expressed genes (DEGs) in cartilage, synovium, subchondral bone, and meniscus were identified using the Gene Expression Omnibus 2R (GEO2R) analysis based on dataset from GSE43923, GSE12021, GSE98918, and GSE51588, respectively and visualized in Volcano Plot. Venn diagram analyses were performed to identify the overlapping DEGs (overlapping DEGs) that expressed in at least two types of tissues mentioned above. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, protein-protein interaction (PPI) analysis, and module analysis were conducted. Furthermore, qRT-PCR was performed to validate above results using our clinical specimens. Results: As a result, a total of 236 overlapping DEGs were identified, of which 160 were upregulated and 76 were downregulated. Through enrichment analysis and constructing the PPI network and miRNA-mRNA network, knee OA-related key genes, such as HEY1, AHR, VEGFA, MYC, and CXCL12 were identified. Clinical validation by qRT-PCR experiments further supported above computational results. In addition, knee OA-related key miRNAs such as miR-101, miR-181a, miR-29, miR-9, and miR-221, and pathways such as Wnt signaling, HIF-1 signaling, PI3K-Akt signaling, and axon guidance pathways were also identified. Among above identified knee OA-related key genes, pathways and miRNAs, genes such as AHR, HEY1, MYC, GAP43, and PTN, pathways like axon guidance, and miRNAs such as miR-17, miR-21, miR-155, miR-185, and miR-1 are lack of research and worthy for future investigation. Conclusion: The present informatic study for the first time provides insight to the potential therapeutic targets of knee OA by comprehensively analyzing the overlapping genes differentially expressed in multiple joint components and their relevant signaling pathways and interactive miRNAs.

Biodegradable magnesium combined with distraction osteogenesis synergistically stimulates bone tissue regeneration via CGRP-FAK-VEGF signaling axis.

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.

Biodegradable magnesium pins enhanced the healing of transverse patellar fracture in rabbits.

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.

Synergistic effects of magnesium ions and simvastatin on attenuation of high-fat diet-induced bone loss.

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.

Calcitonin Gene-Related Peptide Enhances Distraction Osteogenesis by Increasing Angiogenesis.

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.

Tumor Necrosis Factor Receptor Associated Factor 3 Modulates Cartilage Degradation through Suppression of Interleukin 17 Signaling.

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.

Wnt16 attenuates osteoarthritis progression through a PCP/JNK-mTORC1-PTHrP cascade.

OBJECTIVES: Wnt16 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. METHODS: The 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. RESULTS: Wnt16 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 ß-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. CONCLUSIONS: Our 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.

Reciprocal inhibition of YAP/TAZ and NF-κB regulates osteoarthritic cartilage degradation.

Osteoarthritis is one of the leading causes of pain and disability in the aged population due to articular cartilage damage. This warrants investigation of signaling mechanisms that could protect cartilage from degeneration and degradation. Here we show in a murine model of experimental osteoarthritis that YAP activation by transgenic overexpression or by deletion of its upstream inhibitory kinases Mst1/2 preserves articular cartilage integrity, whereas deletion of YAP in chondrocytes promotes cartilage disruption. Our work shows that YAP is both necessary and sufficient for the maintenance of cartilage homeostasis in osteoarthritis. Mechanistically, inflammatory cytokines, such as TNFα or IL-1ß, trigger YAP/TAZ degradation through TAK1-mediated phosphorylation. Furthermore, YAP directly interacts with TAK1 and attenuates NF-κB signaling by inhibiting substrate accessibility of TAK1. Our study establishes a reciprocal antagonism between Hippo-YAP/TAZ and NF-κB signaling in regulating the induction of matrix-degrading enzyme expression and cartilage degradation during osteoarthritis pathogenesis.