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OBJECTIVE: Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A(1) receptor blockade or deletion on bone density. METHODS: The bone mineral density (BMD) in adenosine A(1) receptor-knockout (A(1)R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A(1)R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. RESULTS: Micro-CT analysis of bones from A(1)R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A(1)R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A(1)R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A(1)R-knockout mice. CONCLUSION: These results suggest that the adenosine A(1) receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Evidence from in vitro, in vivo, and clinical studies indicates that adenosine mediates, at least in part, the antiinflammatory effects of methotrexate (MTX), although the biochemical events involved have not been fully elucidated. This study was undertaken to investigate whether MTX exerts antiinflammatory effects in mice that lack ecto-5'-nucleotidase (ecto-5'-NT) (CD73) and are unable to convert AMP to adenosine extracellularly, in order to determine whether adenosine is generated intracellularly and transported into the extracellular space or is generated from the extracellular dephosphorylation of AMP to adenosine. METHODS: Male CD73 gene-deficient mice and age-matched wild-type mice received intraperitoneal injections of saline or MTX (1 mg/kg/week) for 5 weeks. Air pouches were induced on the back by subcutaneous injection of air; 6 days later, inflammation was induced by injection of carrageenan. RESULTS: Fewer leukocytes, but higher levels of tumor necrosis factor alpha (TNFalpha), accumulated in the air pouches of vehicle-treated CD73-deficient mice compared with those of wild-type mice. As expected, MTX treatment reduced the number of leukocytes and TNFalpha levels in the exudates and increased exudate adenosine concentrations in wild-type mice. In contrast, MTX did not reduce exudate leukocyte counts or TNFalpha levels or increase exudate adenosine levels in CD73-deficient mice. CONCLUSION: These results demonstrate that the antiinflammatory actions of MTX are mediated, at least in part, by increased release of adenine nucleotides that are hydrolyzed extracellularly to adenosine via an ecto-5'-NT-dependent pathway. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Adenosine regulates inflammation and tissue repair, and adenosine A(2A) receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A(2A) receptors contribute to the pathogenesis of dermal fibrosis. METHODS: Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, (14)C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A(2A) receptor-deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A(2A) receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. RESULTS: Adenosine A(2A) receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A(2A), but not A(1) or A(2B), receptor antagonism. Adenosine A(2A) receptor ligation stimulated ERK phosphorylation, and A(2A) receptor-mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A(2A) receptor-deficient and A(2A) receptor antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis. CONCLUSION: These results demonstrate that adenosine A(2A) receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Low-dose methotrexate (MTX), a mainstay in the treatment of rheumatoid arthritis, is effective in only 60-70% of patients, a finding mirrored by poor antiinflammatory efficacy in some animal models, most notably collagen-induced arthritis. To determine whether genetic factors or the model itself is responsible for the poor response to MTX, we directly compared the responses of 4 inbred mouse strains to MTX in the air-pouch model of acute inflammation. METHODS: The exudate leukocyte count and adenosine concentration were determined in inbred mice treated with MTX (0.75 mg/kg intraperitoneally every week for 4 weeks) or vehicle 4 hours after injection of carrageenan into the air pouch using previously described methods. Quantitative trait locus mapping was performed using an in silico, or computer-based, method to identify loci potentially associated with each phenotype. RESULTS: MTX significantly reduced the exudate leukocyte count in C57BL/6J and BALB/cJ mice, but not DBA/1J (the strain used in the collagen-induced arthritis model) or DBA/2J mice. In a parallel manner, MTX increased adenosine concentration in inflammatory exudates of C57BL/6J and BALB/cJ mice, but not DBA/1J or DBA/2J mice. Antiinflammatory and adenosine responses to MTX in DBA/1J x C57BL/6J F(1) and F(2) offspring were most consistent with single genetic loci being responsible for each phenotype. In silico mapping identified partially overlapping loci containing candidate genes involved in both responses. CONCLUSION: Genetic factors contribute to the antiinflammatory efficacy of MTX, and a single locus involved in MTX-induced adenosine up-regulation is likely responsible for the observed resistance to MTX in DBA/1J mice. [ABSTRACT FROM AUTHOR]

Hair follicle neogenesis (HFN) occurs after large skin excisions in mice, serving as a rare regenerative model in mammalian wound healing. Wound healing typically results in fibrosis in mice and humans. We previously showed that small skin excisions in mice result in scarring devoid of HFN, displaying features of nonregenerative healing, and hedgehog (Hh) activation in the dermis of such wounds can induce HFN. In this study, we sought to verify the role of dermal Wnt/β-catenin signaling in HFN because this pathway is essential for hair follicle development but is also paradoxically well-characterized in fibrosis of adult wounds. By deletion of β-catenin in large wound myofibroblasts, we show that Wnt/β-catenin signaling is required for endogenous mechanisms of HFN. By utilizing a combined mouse model that simultaneously induces deletion of β-catenin and constitutive activation of Smoothened in myofibroblasts, we also found that β-catenin is required for Hh-driven dermal papilla formation. Transcriptome analysis confirms that Wnt/β-catenin and Hh pathways are activated in dermal papilla cells. Our results indicate that Wnt-active fibrotic status may also create a permissive state for the regenerative function of Hh, suggesting that activation of both Wnt and Hh pathways in skin wound fibroblasts must be ensured in future strategies to promote HFN., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: β-tricalcium phosphate (β-TCP) has been successfully utilized as a 3D printed ceramic scaffold in the repair of non-healing bone defects; however, it requires the addition of growth factors to augment its regenerative capacity. Synthetic bone mineral (SBM) is a novel and extrudable carbonate hydroxyapatite with ionic substitutions known to facilitate bone healing. However, its efficacy as a 3D printed scaffold for hard tissue defect repair has not been explored., Objective: To evaluate the biocompatibility and cell viability of human osteoprecursor (hOP) cells seeded on 3D printed SBM scaffolds via in vitro analysis., Methods: SBM and β-TCP scaffolds were fabricated via 3D printing and sintered at various temperatures. Scaffolds were then subject to qualitative cytotoxicity testing and cell proliferation experiments utilizing (hOP) cells., Results: SBM scaffolds sintered at lower temperatures (600 °C and 700 °C) induced greater levels of acute cellular stress. At higher sintering temperatures (1100 °C), SBM scaffolds showed inferior cellular viability relative to β-TCP scaffolds sintered to the same temperature (1100 °C). However, qualitative analysis suggested that β-TCP presented no evidence of morphological change, while SBM 1100 °C showed few instances of acute cellular stress., Conclusion: Results demonstrate SBM may be a promising alternative to β-TCP for potential applications in bone tissue engineering.

OA is a common and debilitating condition that restricts mobility and diminishes the quality of life. Recent work indicates that the generation of adenosine at the cell surface is an important mediator of chondrocyte homeostasis, and topical application of adenosine in a slow-release form (liposomes) can halt the progression of OA and diminish the pain associated with OA. Here, we review the evidence indicating that adenosine, acting at A 2A receptors, plays a critical role in endogenous and exogenous treatment and reversal of OA.

Background: Three-dimensional printed bioceramic scaffolds composed of 100% β-tricalcium phosphate augmented with dipyridamole (3DPBC-DIPY) can regenerate bone across critically sized defects in skeletally mature and immature animal models. Before human application, safe and effective bone formation should be demonstrated in a large translational animal model. This study evaluated the ability of 3DPBC-DIPY scaffolds to restore critically sized calvarial defects in a skeletally immature, growing minipig., Methods: Unilateral calvarial defects (~1.4 cm) were created in 6-week-old Göttingen minipigs ( n = 12). Four defects were filled with a 1000 μm 3DPBC-DIPY scaffold with a cap (a solid barrier on the ectocortical side of the scaffold to prevent soft-tissue infiltration), four defects were filled with a 1000 μm 3DPBC-DIPY scaffold without a cap, and four defects served as negative controls (no scaffold). Animals were euthanized 12 weeks postoperatively. Calvariae were subjected to micro-computed tomography, 3D reconstruction with volumetric analysis, qualitative histologic analysis, and nanoindentation., Results: Scaffold-induced bone growth was statistically greater than in negative controls ( P ≤ 0.001), and the scaffolds with caps produced significantly more bone generation compared with the scaffolds without caps ( P ≤ 0.001). Histologic analysis revealed woven and lamellar bone with haversian canals throughout the regenerated bone. Cranial sutures were observed to be patent, and there was no evidence of ectopic bone formation or excess inflammatory response. Reduced elastic modulus and hardness of scaffold-regenerated bone were found to be statistically equivalent to native bone ( P = 0.148 for reduced elastic modulus of scaffolds with and without caps and P = 0.228 and P = 0.902 for hardness of scaffolds with and without caps, respectively)., Conclusion: 3DPBC-DIPY scaffolds have the capacity to regenerate bone across critically sized calvarial defects in a skeletally immature translational pig model., Clinical Relevance Statement: This study assessed the bone generative capacity of 3D-printed bioceramic scaffolds composed of 100% β-tricalcium phosphate and augmented with dipyridamole placed within critical-sized calvarial defects in a growing porcine model., (Copyright © 2023 by the American Society of Plastic Surgeons.)

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Loss of bone is a common medical problem and, while it can be treated with available therapies, some of these therapies have critical side effects. We have previously demonstrated that CGS21680, a selective A 2A adenosine receptor agonist, prevents bone loss, but its on-target toxicities (hypotension, tachycardia) and frequent dosing requirements make it unusable in the clinic. We therefore generated a novel alendronate-CGS21680 conjugate (MRS7216), to target the agonist to bone where it remains for long periods thereby diminishing the frequency of administration and curtailing side effects. MRS7216 was synthesized from CGS21680 by sequential activation of the carboxylic acid moiety and reacting with an appropriate amino acid (PEG, alendronic acid) under basic conditions. MRS7216 was tested on C57BL/6J (WT) mice with established osteoporosis (OP) and WT or A2A KO mice with wear particle-induced inflammatory osteolysis (OL). Mice were treated weekly with MRS7216 (10mg/kg). Bone formation was studied after in vivo labeling with calcein/Alizarin Red, and μCT and histology analyses were performed. In addition, human primary osteoblasts and osteoclasts were cultured using bone marrow discarded after hip replacement. Receptor binding studies demonstrate that MRS7216 efficiently binds the A2A adenosine receptor. MRS7216-treated OP and OL mice had significant new bone formation and reduced bone loss compared to vehicle or alendronate-treated mice. Histological analysis showed that MRS7216 treatment significantly reduced osteoclast number and increased osteoblast number in murine models. Interestingly, cultured human osteoclast differentiation was inhibited, and osteoblast differentiation was stimulated by the compound indicating that MRS7216 conjugates represent a novel therapeutic approach to treat osteoporosis and osteolysis., (© 2022. The Author(s).)

Caffeine is a regular part of the diet of many adults (coffee, tea, soft drinks, and energy drinks). Multiple molecular effects of caffeine suggest that it may promote bone loss. Given the extensive consumption of caffeine worldwide, any impact of caffeine consumption on bone strength and/or density would have large population health implications. The most well-established pharmacological effect of caffeine is non-specific antagonism of adenosine receptors. Adenosine regulates bone metabolism in a complex manner, with in vitro studies suggesting that direct stimulation of adenosine A 2A and A 2B receptors induces bone formation by activating osteoblasts and suppressing osteoclast differentiation and function. Thus, competitive inhibition of adenosine A 2 receptors by caffeine may inhibit bone formation and promote bone resorption. However, antagonism of adenosine A 1 receptors may have opposing effects. Caffeine has also been suggested to affect bone through derangement of calcium metabolism, alteration of vitamin D responses, and other mechanisms. In clinical and population-based studies, the impact of caffeine consumption on bone metabolism offers a mixed picture, with some but not all studies suggesting a potential link between caffeine intake and reduced bone mineral density or increased fracture risk. Differences in methodology, selected populations, and duration/timing of the studies may account for study outcome discrepancies. The in vitro effects of caffeine on cells involved in bone metabolism suggest that caffeine intake may promote osteoporosis, and some but not all clinical studies support a modest adverse caffeine impact. Herein, we describe the basic biology of caffeine as it pertains to bone, review the clinical literature to date, and consider the implications of the current data on clinical practice and future studies., (© 2021. Crown.)

Our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors (2011) contained a number of emerging developments with respect to this G protein-coupled receptor subfamily, including protein structure, protein oligomerization, protein diversity, and allosteric modulation by small molecules. Since then, a wealth of new data and results has been added, allowing us to explore novel concepts such as target binding kinetics and biased signaling of adenosine receptors, to examine a multitude of receptor structures and novel ligands, to gauge new pharmacology, and to evaluate clinical trials with adenosine receptor ligands. This review should therefore be considered a further update of our previous reports from 2001 and 2011. SIGNIFICANCE STATEMENT: Adenosine receptors (ARs) are of continuing interest for future treatment of chronic and acute disease conditions, including inflammatory diseases, neurodegenerative afflictions, and cancer. The design of AR agonists ("biased" or not) and antagonists is largely structure based now, thanks to the tremendous progress in AR structural biology. The A 2A - and A 2B AR appear to modulate the immune response in tumor biology. Many clinical trials for this indication are ongoing, whereas an A 2A AR antagonist (istradefylline) has been approved as an anti-Parkinson agent., (U.S. Government work not protected by U.S. copyright.)

Skeletal conditions represent a considerable challenge to health systems globally. Barriers to effective therapeutic development include a lack of accurate preclinical tissue and disease models. Most recently, work was attempted to present a novel whole organ approach to modeling human bone and cartilage tissues. These self-assembling skeletal organoids mimic the cellular milieu and extracellular organization present in native tissues. Bone organoids demonstrated osteogenesis and micro vessel formation, and cartilage organoids showed evidence of cartilage development and maturation. Skeletal organoids derived from both bone and cartilage tissues yielded spontaneous polarization of their cartilaginous and bone components. Using these hybrid skeletal organoids, we successfully generated "mini joint" cultures, which we used to model inflammatory disease and test Adenosine (A 2A ) receptor agonists as a therapeutic agent. The work and respective results indicated that skeletal organoids can be an effective biological model for tissue development and disease as well as to test therapeutic agents., (© 2021 Wiley Periodicals LLC.)

Background: β-Tricalcium phosphate (β-TCP) is one of the most common synthetic bone grafting materials utilized in craniofacial reconstruction; however, it is limited by a slow degradation rate. The aim of this study was to leverage 3-dimensional (3D) printing in an effort to accelerate the degradation kinetics of β-TCP., Methods: Twenty-two 1-month-old New Zealand white rabbits underwent creation of calvarial and alveolar defects, repaired with 3D-printed β-TCP scaffolds coated with 1000 μM of osteogenic agent dipyridamole. Rabbits were euthanized after 2, 6, and 18 months after surgical intervention. Bone regeneration, scaffold degradation, and bone mechanical properties were quantified., Results: Histological analysis confirmed the generation of vascularized and organized bone. Microcomputed tomography analysis from 2 to 18 months demonstrated decreased scaffold volume within calvarial (23.6% ± 2.5%, 5.1% ± 2.2%; P < 0.001) and alveolar (21.5% ± 2.2%, 0.2% ± 1.9%; P < 0.001) defects, with degradation rates of 54.6%/year and 90.5%/year, respectively. Scaffold-inducted bone generation within the defect was volumetrically similar to native bone in the calvarium (55.7% ± 6.9% vs 46.7% ± 6.8%; P = 0.064) and alveolus (31.4% ± 7.1% vs 33.8% ± 3.7%; P = 0.337). Mechanical properties between regenerated and native bone were similar., Conclusions: Our study demonstrates an improved degradation profile and replacement of absorbed β-TCP with vascularized, organized bone through 3D printing and addition of an osteogenic agent. This novel additive manufacturing and tissue engineering protocol has implications to the future of craniofacial skeletal reconstruction as a safe and efficacious bone tissue engineering method., Competing Interests: Conflicts of interest and sources of funding: P.G.C. and B.N.C. have filed a patent about the use of tissue repair devices and scaffolds (No. US20150150681A1). B.N.C. holds patents numbers 5,932,558, 6,020,321, 6,555,545, and 7,795,427. This work was supported by the National Institutes of Health (R33HD090664), NYU Langone Health Preclinical Imaging Laboratory, a shared resource partially supported by the NIH/SIG (1S10OD018337-01), the Laura and Isaac Perlmutter Cancer Center Support Grant NIH/NCI (5P30CA016087) and the NIBIB Biomedical Technology Resource Center Grant NIH (P41 EB017183)., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Methotrexate (MTX) is widely used for the treatment of rheumatoid arthritis due to its well-known anti-inflammatory role in immune cells but its impact on brown and beige adipose tissue biology has not yet been investigated. Here, we present the novel evidence that MTX treatment increases the gene expression of thermogenic genes in brown and beige adipose tissues in a fat cell autonomous manner. Furthermore, we show that treatment of mice with MTX is associated with cold resistance, improved glucose homeostasis, decreased inflammation, and reduced hepatosteatosis in high-fat diet states. Overall, our data provide novel evidence of a role of MTX on thermogenic tissues not previously appreciated., Competing Interests: The authors declare that they have no conflict of interest with the contents of this article., (© 2021 The Authors. FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

Adenosine signaling plays a critical role in the maintenance of articular cartilage and may serve as a novel therapeutic for osteoarthritis (OA), a highly prevalent and morbid disease without effective therapeutics in the current market. Mice lacking adenosine A2A receptors (A2AR) develop spontaneous OA by 16 weeks of age, a finding relevant to human OA since loss of adenosine signaling due to diminished adenosine production (NT5E deficiency) also leads to development of OA in mice and humans. To better understand the mechanism by which A2AR and adenosine generation protect from OA development, we examined differential gene expression in neonatal chondrocytes from WT and A2AR null mice. Analysis of differentially expressed genes was analyzed by KEGG pathway analysis, and oPOSSUM and the flatiron database were used to identify transcription factor binding enrichment, and tissue-specific network analyses and patterns were compared to gene expression patterns in chondrocytes from patients with OA. There was a differential expression of 2211 genes (padj<0.05). Pathway enrichment analysis revealed that pro-inflammatory changes, increased metalloprotease, reduced matrix organization, and homeostasis are upregulated in A2AR null chondrocytes. Moreover, stress responses, including autophagy and HIF-1 signaling, seem to be important drivers of OA and bear marked resemblance to the human OA transcriptome. Although A2AR null mice are born with grossly intact articular cartilage, we identify here the molecular foundations for early-onset OA in these mice, further establishing their role as models for human disease and the potential use of adenosine as a treatment for human disease., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Adenosine A 2A receptor mediates the promotion of wound healing and revascularization of injured tissue, in healthy and animals with impaired wound healing, through a mechanism depending upon tissue plasminogen activator (tPA), a component of the fibrinolytic system. In order to evaluate the contribution of plasmin generation in the proangiogenic effect of adenosine A 2A receptor activation, we determined the expression and secretion of t-PA, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and annexin A2 by human dermal microvascular endothelial cells stimulated by the selective agonist CGS-21680. The plasmin generation was assayed through an enzymatic assay and the proangiogenic effect was studied using an endothelial tube formation assay in Matrigel. Adenosine A 2A receptor activation in endothelial cells diminished the release of PAI-1 and promoted the production of annexin A2, which acts as a cell membrane co-receptor for plasminogen and its activator tPA. Annexin A2 mediated the increased cell membrane-associated plasmin generation in adenosine A 2A receptor agonist treated human dermal microvascular endothelial cells and is required for tube formation in an in vitro model of angiogenesis. These results suggest a novel mechanism by which adenosine A 2A receptor activation promotes angiogenesis: increased endothelial expression of annexin A2, which, in turn, promotes fibrinolysis by binding tPA and plasminogen to the cell surface., Competing Interests: BC holds or has filed applications for patents on the use of adenosine A2A receptor agonists to promote wound healing and use of A2A receptor antagonists to inhibit fibrosis; use of adenosine A1 receptor antagonists to treat osteoporosis and other diseases of bone; the use of adenosine A1 and A2B Receptor antagonists to treat fatty liver; and the use of adenosine A2A receptor agonists to prevent prosthesis loosening. He is consultant (within the past two years) for King Pharmaceuticals (licensee of patents on wound healing and fibrosis above), CanFite Biopharmaceuticals, Savient Pharmaceuticals, Bristol-Myers Squibb, Roche Pharmaceuticals, Cellzome, Tap (Takeda) Pharmaceuticals, Prometheus Laboratories, Regeneron (Westat, DSMB), Sepracor, Amgen, Endocyte, Protalex, Allos, Inc. Combinatorx, Kyowa Hakka, received honoraria from Tap (Takeda) Pharmaceuticals and holds stock in CanFite Biopharmaceuticals received for membership in Scientific Advisory Board. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valls, Soldado, Arasa, Perez-Aso, Williams, Cronstein, Noguera, Terencio and Montesinos.)

Autophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced OA cartilage damage in a murine model of obesity-induced OA. The same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established post-traumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat OA.

In OA chondrocytes, there is diminished mitochondrial production of ATP and diminished extracellular adenosine resulting in diminished adenosine A2A receptor (A2AR) stimulation and altered chondrocyte homeostasis which contributes to the pathogenesis of OA. We tested the hypothesis that A2AR stimulation maintains or enhances mitochondrial function in chondrocytes. The effect of A2AR signaling on mitochondrial health and function was determined in primary murine chondrocytes, a human chondrocytic cell line (T/C-28a2), primary human chondrocytes, and a murine model of OA by transmission electron microscopy analysis, mitochondrial stress testing, confocal live imaging for mitochondrial inner membrane polarity, and immunohistochemistry. In primary murine chondrocytes from A2AR -/- null mice, which develop spontaneous OA by 16 weeks, there is mitochondrial swelling, dysfunction, and reduced mitochondrial content with increased reactive oxygen species (ROS) burden and diminished mitophagy, as compared to chondrocytes from WT animals. IL-1-stimulated T/C-28a2 cells treated with an A2AR agonist had reduced ROS burden with increased mitochondrial dynamic stability and function, findings which were recapitulated in primary human chondrocytes. In an obesity-induced OA mouse model, there was a marked increase in mitochondrial oxidized material which was markedly improved after intraarticular injections of liposomal A2AR agonist. These results are consistent with the hypothesis that A2AR ligation is mitoprotective in OA., (© 2020 Federation of American Societies for Experimental Biology.)

Competing Interests: Competing interests: None declared.

Despite the introduction of numerous biologic agents for the treatment of rheumatoid arthritis (RA) and other forms of inflammatory arthritis, low-dose methotrexate therapy remains the gold standard in RA therapy. Methotrexate is generally the first-line drug for the treatment of RA, psoriatic arthritis and other forms of inflammatory arthritis, and it enhances the effect of most biologic agents in RA. Understanding the mechanism of action of methotrexate could be instructive in the appropriate use of the drug and in the design of new regimens for the treatment of RA. Although methotrexate is one of the first examples of intelligent drug design, multiple mechanisms potentially contribute to the anti-inflammatory actions of methotrexate, including the inhibition of purine and pyrimidine synthesis, transmethylation reactions, translocation of nuclear factor-κB (NF-κB) to the nucleus, signalling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and nitric oxide production, as well as the promotion of adenosine release and expression of certain long non-coding RNAs.

Background: Three-dimensionally-printed bioceramic scaffolds composed of β-tricalcium phosphate delivering the osteogenic agent dipyridamole can heal critically sized calvarial defects in skeletally mature translational models. However, this construct has yet to be applied to growing craniofacial models. In this study, the authors implanted three-dimensionally-printed bioceramic/dipyridamole scaffolds in a growing calvaria animal model and evaluated bone growth as a function of geometric scaffold design and dipyridamole concentration. Potential adverse effects on the growing suture were also evaluated., Methods: Bilateral calvarial defects (10 mm) were created in 5-week-old (approximately 1.1 kg) New Zealand White rabbits (n = 16 analyzed). Three-dimensionally-printed bioceramic scaffolds were constructed in quadrant form composed of varying pore dimensions (220, 330, and 500 μm). Each scaffold was coated with collagen and soaked in varying concentrations of dipyridamole (100, 1000, and 10,000 μM). Controls consisted of empty defects. Animals were killed 8 weeks postoperatively. Calvariae were analyzed using micro-computed tomography, three-dimensional reconstruction, and nondecalcified histologic sectioning., Results: Scaffold-induced bone growth was statistically greater than bone growth in empty defects (p = 0.02). Large scaffold pores, 500 μm, coated in 1000 μM dipyridamole yielded the most bone growth and lowest degree of scaffold presence within the defect. Histology showed vascularized woven and lamellar bone along with initial formation of vascular canals within the scaffold lattice. Micro-computed tomographic and histologic analysis revealed patent calvarial sutures without evidence of ectopic bone formation across all dipyridamole concentrations., Conclusion: The authors present an effective pediatric bone tissue-engineering scaffold design and dipyridamole concentration that is effective in augmentation of calvarial bone generation while preserving cranial suture patency.

The joint is a complex anatomical structure consisting of different tissues, each with a particular feature, playing together to give mobility and stability at the body. All the joints have a similar composition including cartilage for reducing the friction of the movement and protecting the underlying bone, a synovial membrane that produces synovial fluid to lubricate the joint, ligaments to limit joint movement, and tendons for the interaction with muscles. Direct or indirect damage of one or more of the tissues forming the joint is the foundation of different pathological conditions. Many molecular mechanisms are involved in maintaining the joint homeostasis as well as in triggering disease development. The molecular pathway activated by the purinergic system is one of them.The purinergic signaling defines a group of receptors and intermembrane channels activated by adenosine, adenosine diphosphate, adenosine 5'-triphosphate, uridine triphosphate, and uridine diphosphate. It has been largely described as a modulator of many physiological and pathological conditions including rheumatic diseases. Here we will give an overview of the purinergic system in the joint describing its expression and function in the synovium, cartilage, ligament, tendon, and bone with a therapeutic perspective., (Copyright © 2020 Corciulo and Cronstein.)

Objectives: To detail the greatest areas of unmet scientific and clinical needs in rheumatology., Methods: The 21st annual international Advances in Targeted Therapies meeting brought together more than 100 leading basic scientists and clinical researchers in rheumatology, immunology, epidemiology, molecular biology and other specialties. During the meeting, breakout sessions were convened, consisting of 5 disease-specific groups with 20-30 experts assigned to each group based on expertise. Specific groups included: rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, systemic lupus erythematosus and other systemic autoimmune rheumatic diseases. In each group, experts were asked to identify unmet clinical and translational research needs in general and then to prioritise and detail the most important specific needs within each disease area., Results: Overarching themes across all disease states included the need to innovate clinical trial design with emphasis on studying patients with refractory disease, the development of trials that take into account disease endotypes and patients with overlapping inflammatory diseases, the need to better understand the prevalence and incidence of inflammatory diseases in developing regions of the world and ultimately to develop therapies that can cure inflammatory autoimmune diseases., Conclusions: Unmet needs for new therapies and trial designs, particularly for those with treatment refractory disease, remain a top priority in rheumatology., Competing Interests: Competing interests: KLW reports personal fees from Pfizer, grants and personal fees from BMS, personal fees from AbbVie, personal fees from UCB, personal fees from Lilly, personal fees from Galapagos, personal fees from GSK, personal fees from Roche outside the submitted work. JB reports personal fees from AbbVie outside the submitted work. MD reports grants and personal fees from Pfizer, grants and personal fees from Abbvie, grants and personal fees from Lilly, grants and personal fees from Novartis, grants and personal fees from UCB, grants and personal fees from Merck, grants and personal fees from Roche, during the conduct of the study. JTR reports grants from Pfizer, personal fees from Abbvie, personal fees from Gilead, personal fees from Santen, personal fees from Roche, personal fees from Novartis, personal fees from UCB, personal fees from Corvus, personal fees from Horizon, personal fees from Celldex, personal fees from Eyevensys, personal fees from UpToDate, personal fees from Janssen outside the submitted work. XM reports personal fees from BMS, personal fees from GILEAD, personal fees from PFIZER, personal fees from SAMSUNG, personal fees from UCB outside the submitted work. BNC has multiple patents, none of which are relevant. In addition he has multiple grants from NIH. JSS reports grants and personal fees from AbbVie, personal fees from Amgen, personal fees from AstraZeneca, personal fees from Astro, personal fees from Bristol-Myers Squibb, personal fees from Celgene, personal fees from Celltrion, personal fees from ILTOO, grants and personal fees from Janssen, grants and personal fees from Lilly, personal fees from MSD, grants and personal fees from Novartis-Sandoz, personal fees from Novo-Nordisk, grants and personal fees from Roche, personal fees from Samsung Bioepis, personal fees from Sanofi, personal fees from UCB, grants and personal fees from Pfizer outside the submitted work. DF reports grant/research support from Actelion, grant/research support from Amgen, grant/research support from BMS, grant/research support from Corbus, grant/research support from Galapagos GSK, grant/research support from NIH, grant/research support from Novartis, grant/research support from Pfizer, grant/research support from Sanofi, grant/research support from Roche/Genentech, personal fees from Actelion, personal fees from Amgen, personal fees from BMS, personal fees from Corbus, personal fees from Galapagos, personal fees from Novartis, personal fees from Pfizer outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

This study investigates a comprehensive model of bone regeneration capacity of dypiridamole-loaded 3D-printed bioceramic (DIPY-3DPBC) scaffolds composed of 100% beta-tricalcium phosphate (β -TCP) in an immature rabbit model through the time of facial maturity. The efficacy of this construct was compared to autologous bone graft, the clinical standard of care in pediatric craniofacial reconstruction, with attention paid to volume of regenerated bone by 3D reconstruction, histologic and mechanical properties of regenerated bone, and long-term safety regarding potential craniofacial growth restriction. Additionally, long-term degradation of scaffold constructs was evaluated. At 24 weeks in vivo, DIPY-3DPBC scaffolds demonstrated volumetrically significant osteogenic regeneration of calvarial and alveolar defects comparable to autogenous bone graft with favorable biodegradation of the bioactive ceramic component in vivo. Characterization of regenerated bone reveals osteogenesis of organized, vascularized bone with histologic and mechanical characteristics comparable to native bone. Radiographic and histologic analyses were consistent with patent craniofacial sutures. Lastly, through application of 3D morphometric facial surface analysis, our results support that DIPY-3DPBC scaffolds do not cause premature closure of sutures and preserve normal craniofacial growth. Based on this novel evaluation model, this DIPY-3DPBC scaffold strategy is a promising candidate as a safe, efficacious pediatric bone tissue engineering strategy.

There are over two million long bone defects treated in the United States annually, of which ~5% will not heal without significant surgical intervention. While autogenous grafting is the standard of care in simple defects, a customized scaffold for large defects in unlimited quantities is not available. Recently, a three-dimensionally (3D)-printed bioactive ceramic (3DPBC) scaffold has been successfully utilized in the of repair critical-sized (CSD) long bone defects in vivo. In this study, 3DPBC scaffolds were augmented with dipyridamole (DIPY), an adenosine A2A receptor (A 2A R) indirect agonist, because of its known effect to enhance bone formation. CSD full thickness segmental defects (~11 mm × full thickness) defects were created in the radial diaphysis in New Zealand white rabbits (n = 24). A customized 3DPBC scaffold composed of β-tricalcium phosphate was placed into the defect site. Groups included scaffolds that were collagen-coated (COLL), or immersed in 10, 100, or 1,000 μM DIPY solution. Animals were euthanized 8 weeks post-operatively and the radii/ulna-scaffold complex retrieved en bloc, for micro-CT, histological, and mechanical analysis. Bone growth was assessed exclusively within scaffold pores and evaluated by microCT and advanced reconstruction software. Biomechanical properties were evaluated utilizing nanoindentation to assess the newly regenerated bone for elastic modulus (E) and hardness (H). MicroCT reconstructions illustrated bone in-growth throughout the scaffold, with an increase in bone volume dependent on the DIPY dosage. The histological evaluation did not indicate any adverse immune response while revealing progressive remodeling of bone. These customized biologic 3DPBC scaffolds have the potential of repairing and regenerating bone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2499-2507, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background: Alveolar clefts are traditionally treated with secondary bone grafting, but this is associated with morbidity and graft resorption. Although recombinant human bone morphogenetic protein-2 (rhBMP-2) is under investigation for alveolar cleft repair, safety concerns remain. Dipyridamole is an adenosine receptor indirect agonist with known osteogenic potential. This study compared dipyridamole to rhBMP-2 at alveolar cleft defects delivered using bioceramic scaffolds., Methods: Skeletally immature New Zealand White rabbits underwent unilateral, 3.5 × 3.5-mm alveolar resection adjacent to the growing suture. Five served as negative controls. The remaining defects were reconstructed with three-dimensionally printed bioceramic scaffolds coated with 1000 μm of dipyridamole (n = 6), 10,000 μm of dipyridamole (n = 7), or 0.2 mg/ml of rhBMP-2 (n = 5). At 8 weeks, new bone was quantified. Nondecalcified histologic evaluation was performed, and new bone was evaluated mechanically. Statistical analysis was performed using a generalized linear mixed model and the Wilcoxon rank sum test., Results: Negative controls did not heal, whereas new bone formation bridged all three-dimensionally printed bioceramic treatment groups. The 1000-μm dipyridamole scaffolds regenerated 28.03 ± 7.38 percent, 10,000-μm dipyridamole scaffolds regenerated 36.18 ± 6.83 percent (1000 μm versus 10,000 μm dipyridamole; p = 0.104), and rhBMP-2-coated scaffolds regenerated 37.17 ± 16.69 percent bone (p = 0.124 versus 1000 μm dipyridamole, and p = 0.938 versus 10,000 μm dipyridamole). On histology/electron microscopy, no changes in suture biology were evident for dipyridamole, whereas rhBMP-2 demonstrated early signs of suture fusion. Healing was highly cellular and vascularized across all groups. No statistical differences in mechanical properties were observed between either dipyridamole or rhBMP-2 compared with native bone., Conclusion: Dipyridamole generates new bone without osteolysis and early suture fusion associated with rhBMP-2 in skeletally immature bone defects.

To develop a comprehensive listing of the greatest unmet scientific and clinical needs in rheumatology. The 20th annual international Targeted Therapies meeting brought more than 100 leading basic scientists and clinical researchers in rheumatology, immunology, epidemiology, molecular biology and other specialties. During the meeting, breakout sessions were convened, consisting of five disease-specific groups with 20-30 experts assigned to each group based on expertise. Specific groups included rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, systemic lupus erythematosus, connective tissue diseases and a basic science immunology group spanning all of these clinical domains. In each group, experts were asked to consider recent accomplishments within their clinical domain in the last year and update the unmet needs in three categorical areas: basic/translational science, clinical science and therapeutic development, and clinical care. While progress was noted among some of previously identified needs, both new needs were identified and themes from prior meetings were re-iterated: the need for better understanding the heterogeneity within each disease, and for identifying preclinical states of disease allowing treatment and prevention of disease in those at risk, and the elusive ability to cure disease. Within the clinical care realm, improved comorbidity management and patient-centred care continue to be unmet needs, and the need for new and affordable therapeutics was highlighted. Unmet needs for new and accessible targeted therapies, disease prevention and ultimately cure remain a priority in rheumatology., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Adenosine receptor activation has been investigated as a potential therapeutic approach to heal bone. Bone has enhanced regenerative potential when influenced by either direct or indirect adenosine receptor agonism. As investigators continue to elucidate how adenosine influences bone cell homeostasis at the cellular and molecular levels, a small but growing body of literature has reported successful in vivo applications of adenosine delivery. This review summarizes the role adenosine receptor ligation plays in osteoblast and osteoclast biology and remodeling/regeneration. It also reports on all the modalities described in the literature at this point for delivery of adenosine through in vivo models for bone healing and regeneration., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A 2A receptors (A2ARs) and Wingless/Integrated-β-catenin pathways. Cytosolic β-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular β-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) β-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular β-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 μM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 β-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular β-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, β-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A 2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

Short biologic half-lives limit the therapeutic utility of many small molecules. One approach to extending the half-life of pharmacologically active small molecules is conjugation to less degradable nanoparticles; here we report the synthesis and activity of six targeted polymeric (PEG-b-PLA) nanoparticles for use as adenosine receptor agonists. Using click chemistry, PLA-b-PEG400-N 3 and PLA-b-PEG2000 block copolymers were bound to adenosine at the 3',4'-OH, 5'-OH, and 6-NH 2 positions with an acetylene group. Activity of the conjugates as adenosine receptor ligands was tested by their capacity to stimulate cAMP increases in RAW264.7 murine macrophage cells. Only adenosine-conjugated nanoparticles (A-3',4'-OH-TPN2), in which PEG2000 was bound to adenosine on the 3',4' hydroxyl groups, stimulated cAMP increases and these increases were blocked by selective antagonists of both adenosine A2A and A2B receptors, consistent with ligation of these receptors. Adenosine nanoparticles were tested in vivo in a rat model of post-traumatic osteoarthritis; intra-articular injection of adenosine nanoparticles prevented the development of osteoarthritis in this model. These studies suggest that attachment of adenosine to biodegradable nanoparticles provides a novel approach to achieving prolonged therapeutic effects.

Background: Autologous bone grafts remain a standard of care for the reconstruction of large bony defects, but limitations persist. The authors explored the bone regenerative capacity of customized, three-dimensionally printed bioactive ceramic scaffolds with dipyridamole, an adenosine A2A receptor indirect agonist known to enhance bone formation., Methods: Critical-size bony defects (10-mm height, 10-mm length, full-thickness) were created at the mandibular rami of rabbits (n = 15). Defects were replaced by a custom-to-defect, three-dimensionally printed bioactive ceramic scaffold composed of β-tricalcium phosphate. Scaffolds were uncoated (control), collagen-coated, or immersed in 100 μM dipyridamole. At 8 weeks, animals were euthanized and the rami retrieved. Bone growth was assessed exclusively within scaffold pores, and evaluated by micro-computed tomography/advanced reconstruction software. Micro-computed tomographic quantification was calculated. Nondecalcified histology was performed. A general linear mixed model was performed to compare group means and 95 percent confidence intervals., Results: Qualitative analysis did not show an inflammatory response. The control and collagen groups (12.3 ± 8.3 percent and 6.9 ± 8.3 percent bone occupancy of free space, respectively) had less bone growth, whereas the most bone growth was in the dipyridamole group (26.9 ± 10.7 percent); the difference was statistically significant (dipyridamole versus control, p < 0.03; dipyridamole versus collagen, p < 0.01 ). There was significantly more residual scaffold material for the collagen group relative to the dipyridamole group (p < 0.015), whereas the control group presented intermediate values (nonsignificant relative to both collagen and dipyridamole). Highly cellular and vascularized intramembranous-like bone healing was observed in all groups., Conclusion: Dipyridamole significantly increased the three-dimensionally printed bioactive ceramic scaffold's ability to regenerate bone in a thin bone defect environment.

Mini-sabbaticals are formal short-term training and educational experiences away from an investigator's home research unit. These may include rotations with other research units and externships at government research or regulatory agencies, industry and non-profit programs, and training and/or intensive educational programs. The National Institutes of Health have been encouraging training institutions to consider offering mini-sabbaticals, but given the newness of the concept, limited data are available to guide the implementation of mini-sabbatical programs. In this paper, we review the history of sabbaticals and mini-sabbaticals, report the results of surveys we performed to ascertain the use of mini-sabbaticals at Clinical and Translational Science Award hubs, and consider best practice recommendations for institutions seeking to establish formal mini-sabbatical programs.

Injuries to the extremities often require resection of necrotic hard tissue. For large-bone defects, autogenous bone grafting is ideal but, similar to all grafting procedures, is subject to limitations. Synthetic biomaterial-driven engineered healing offers an alternative approach. This work focuses on three-dimensional (3D) printing technology of solid-free form fabrication, more specifically robocasting/direct write. The research hypothesizes that a bioactive calcium-phosphate scaffold may successfully regenerate extensive bony defects in vivo and that newly regenerated bone will demonstrate mechanical properties similar to native bone as healing time elapses. Robocasting technology was used in designing and printing customizable scaffolds, composed of 100% beta tri-calcium phosphate (β-TCP), which were used to repair critical sized long-bone defects. Following full thickness segmental defects (~11 mm × full thickness) in the radial diaphysis in New Zealand white rabbits, a custom 3D-printed, 100% β-TCP, scaffold was implanted or left empty (negative control) and allowed to heal over 8, 12, and 24 weeks. Scaffolds and bone, en bloc, were subjected to micro-CT and histological analysis for quantification of bone, scaffold and soft tissue expressed as a function of volume percentage. Additionally, biomechanical testing at two different regions, (a) bone in the scaffold and (b) in native radial bone (control), was conducted to assess the newly regenerated bone for reduced elastic modulus (E r ) and hardness (H) using nanoindentation. Histological analysis showed no signs of any adverse immune response while revealing progressive remodelling of bone within the scaffold along with gradual decrease in 3D-scaffold volume over time. Micro-CT images indicated directional bone ingrowth, with an increase in bone formation over time. Reduced elastic modulus (E r ) data for the newly regenerated bone presented statistically homogenous values analogous to native bone at the three time points, whereas hardness (H) values were equivalent to the native radial bone only at 24 weeks. The negative control samples showed limited healing at 8 weeks. Custom engineered β-TCP scaffolds are biocompatible, resorbable, and can directionally regenerate and remodel bone in a segmental long-bone defect in a rabbit model. Custom designs and fabrication of β-TCP scaffolds for use in other bone defect models warrant further investigation., (© 2018 John Wiley & Sons, Ltd.)

Craniomaxillofacial congenital anomalies comprise approximately one third of all congenital birth defects and include deformities such as alveolar clefts, craniosynostosis, and microtia. Current surgical treatments commonly require the use of autogenous graft material which are difficult to shape, limited in supply, associated with donor site morbidity and cannot grow with a maturing skeleton. Our group has demonstrated that 3D printed bio-ceramic scaffolds can generate vascularized bone within large, critical-sized defects (defects too large to heal spontaneously) of the craniomaxillofacial skeleton. Furthermore, these scaffolds are also able to function as a delivery vehicle for a new osteogenic agent with a well-established safety profile. The same 3D printers and imaging software platforms have been leveraged by our team to create sterilizable patient-specific intraoperative models for craniofacial reconstruction. For microtia repair, the current standard of care surgical guide is a two-dimensional drawing taken from the contralateral ear. Our laboratory has used 3D printers and open source software platforms to design personalized microtia surgical models. In this review, we report on the advancements in tissue engineering principles, digital imaging software platforms and 3D printing that have culminated in the application of this technology to repair large bone defects in skeletally immature transitional models and provide in-house manufactured, sterilizable patient-specific models for craniofacial reconstruction., (© 2018 Wiley Periodicals, Inc.)

Adenosine receptor activation has been explored as a modulator of the inflammatory process that propagates osteoarthritis. It has been reported that cartilage has enhanced regenerative potential when influenced by adenosine receptor activation. As adenosine's role in maintaining chondrocyte homeostasis at the cellular and molecular levels is explored, successful in vivo applications of adenosine delivery for cartilage repair continue to be reported. This review summarizes the role adenosine receptor ligation plays in chondrocyte homeostasis and regeneration of articular cartilage damaged in osteoarthritis. It also reports on all the modalities reported for delivery of adenosine through in vivo applications.

The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A 2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase + /Alizarin Red + cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. β-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear β-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A 2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

Objective: To introduce a novel preclinical animal model of psoriatic arthritis (PsA) in R26Stat3C stopfl/fl CD4Cre mice, and to investigate the role of Th17 cytokines in the disease pathogenesis., Methods: We characterized a novel murine model of Th17-driven cutaneous and synovio-entheseal disease directed by T cell-specific expression of a hyperactive Stat3 allele. By crossing R26Stat3C stopfl/fl CD4Cre mice onto an interleukin-22 (IL-22)-knockout background or treating the mice with a neutralizing antibody against IL-17, we interrogated how these Th17 cytokines could contribute to the pathogenesis of PsA., Results: R26Stat3C stopfl/fl CD4Cre mice developed acanthosis, hyperkeratosis, and parakeratosis of the skin, as well as enthesitis/tendinitis and periarticular bone erosion in different joints, accompanied by osteopenia. T cell-specific expression of a hyperactive Stat3C allele was found to drive the augmented Th17 response in these animals. Careful characterization of the mouse bone marrow revealed an increase in osteoclast progenitor (OCP) and RANKL-producing cells, which contributed to the osteopenia phenotype observed in the mutant animals. Abrogation of the Th17 cytokines IL-17 or IL-22 improved both the skin and bone phenotype in R26Stat3C stopfl/fl CD4Cre mice, revealing a central role of Th17 cells in the regulation of OCP and RANKL expression on stromal cells., Conclusion: Perturbation of the IL-23/Th17 axis instigates Th17-mediated inflammation in R26Stat3C stopfl/fl CD4Cre mice, leading to cutaneous and synovio-entheseal inflammation and bone pathologic features highly reminiscent of human PsA. Both IL-17A and IL-22 produced by Th17 cells appear to play critical roles in promoting the cutaneous and musculoskeletal inflammation that characterizes PsA., (© 2018, American College of Rheumatology.)

The purine nucleoside adenosine is a present in most body fluids where it regulates a wide variety of physiologic and pharmacologic processes. Adenosine mediates its effects through activating 4 G protein-coupled receptors expressed on the cell membrane: A1, A2A, A2B, and A3. The adenosine receptors are widely distributed in the body, and tissues with high expression include immune tissues, cartilage, bone, heart, and brain. Here we review the source and metabolism of adenosine and the role of adenosine in regulating immunity and cartilage biology., (Copyright © 2018 Elsevier Inc. All rights reserved.)