Faculty Bibliography

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.

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.

Le méthotrexate est utilisé dans le traitement de la polyarthrite rhumatoïde (PR) depuis les années 1980 et est souvent à ce jour le médicament de première intention pour le traitement de la PR. Dans cette revue, nous examinons plusieurs hypothèses pour expliquer le mécanisme à l'origine de l'efficacité du méthotrexate dans la PR. Celles-ci comprennent l'antagonisme du folate, la signalisation par l'adénosine, la génération d'espèces réactives de l'oxygène (ROS), la diminution des molécules d'adhérence, la modification des profils cytokiniques et l'inhibition des polyamines, entre autres. Actuellement, la signalisation par l'adénosine est probablement l'explication la plus largement acceptée du mécanisme du méthotrexate dans la PR, car le méthotrexate augmente les taux d'adénosine et suite à l'engagement de l'adénosine avec ses récepteurs extracellulaires, une cascade intracellulaire est activée et favorise un état antiinflammatoire global. Outre ces hypothèses, nous examinons le mécanisme du méthotrexate dans la PR sous l'angle de ses effets indésirables et considérons certains des nouveaux marqueurs génétiques de l'efficacité et de la toxicité du méthotrexate dans la PR. Enfin, nous discutons brièvement du mécanisme du méthotrexate en association avec un traitement de la PR par un inhibiteur du TNF-. En fin de compte, en trouvant une explication claire de la voie et du mécanisme conduisant à l'efficacité du méthotrexate dans la PR, il pourrait exister un moyen de formuler des thérapies plus puissantes avec moins d'effets secondaires.

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/CONCLUSIONS: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.

OBJECTIVES/OBJECTIVE: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.

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 2 million long bone defects treated in the USA annually, of which ~5% will not heal without significant surgical intervention. While autogenous grafting is 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 long bone defects in vivo. In this study, 3DPBC scaffolds were augmented with Dipyridamole, an adenosine A2A receptor (A_2A R) indirect agonist, because of its known effect to enhance bone formation. Critical-sized full thickness segmental defects (~11mm x 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 10μM, 100μM, or 1000μM Dipyridamole 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 Dipyridamole dosage. 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. This article is protected by copyright. All rights reserved.

Background/Purpose : Tenofovir is an anti-retroviral agent commonly used to treat human immunodeficiency virus (HIV)-infected patients as part of the drug regimen known as highly active anti-retroviral therapy (HAART). As many as 15% of patients taking tenofovir develop osteopenia resulting in pathological fractures. Recent studies in our lab indicate that tenofovir-induced osteopenia is due to reduction of extracellular adenosine resulting from tenofovir-mediated blockade of Pannexin-1, a transporter for ATP which is hydrolysed in the extracellular space to adenosine, and the effect of tenofovir on bone can be reversed by dipyridamole, an agent that blocks adenosine re-uptake. To further confirm this effect we studied the effect tenofovir on bone in Pannexin-1 knockout mice (PANX1KO) mice. Methods : PANX1KO animals were treated daily with 75mg/Kg of tenofovir, 25 mg/kg tenofovir or both during 4 weeks, after that bone mineral density (BMD) was measured. Additionally, primary osteoclasts were differentiated in the presence of different concentrations of Tenofovir and dipyridamole. The differentiation stage and extracellular ATP levels were studied. Results : Consistent with previous experiments, tenofovir treatment of extracellular ATP in cultures of wild type (WT) bone marrow-derived primary osteoclasts as well as an increase in osteoclast differentiation. However, the addition of dipyridamole inhibited osteoclast differentiation (p=0.0068). The dipyridamole-induced inhibition was reversed with Tenofovir in a dose dependent manner (0.0055). In contrast, osteoclasts from PANX1KO mice did not decrease ATP release when treated with Tenofovir (p= 0.3292). Additionally, PANX1KO mice osteoclast differentiation was also inhibited by dipyridamole (p=0.0005), which was not reversed by dipyridamole treatment (p= 0.9756). In WT mice, Tenofovir treatment reduced bone mineral density by 10% (p< 0.05, n=10) and this effect was reversed in the animals who received Dipyridamole in addition to Tenofovir. In contrast, tenofovir did not affect bone mineral density in PANX1KO mice (p >0.99, n=4). Conclusion : Tenofovir, a commonly used drug, induces osteopenia in many patients. The results presented here support the hypothesis that tenofovir reduces bone density by inhibiting ATP release with subsequent adenosinemediated inhibition of osteoclast differentiation. These effects can be reversed by treatment with dipyridamole