Faculty Bibliography

Background/Purpose: The most common cause of total joint replacement revision surgeries is loosening of the implant due to loss of bone around the prosthesis.Wear particles shed from the prosthesis plays a critical role by increasing local inflammation and osteoclast number and activity, ultimately causing osteolysis.We have previously reported that an A2A adenosine receptor selective agonist (CGS21680, CGS) prevents osteolysis in wear particle-induced osteolysis model in mice. Frequent administration requirements and potential toxicity make it a less than optimal treatment for inflammatory osteolysis.We therefore generated and tested a novel alendronate-CGS conjugate (MRS7216) that specifically localizes to bone targeting the agonist to the site of tissue injury and thereby diminishing the frequency of administration and curtailing systemic side effects.
Method(s): The conjugate was synthesized from CGS by sequential activation of the carboxylic acid moiety and reacting with the appropriate amino acid under basic conditions. A PEG6 linker was incorporated to alendronic acid by direct coupling. Osteolysis in 6-8-week-old C57BL/6J mice was induced by surgical implantation of 3mg of ultrahigh-molecular-weightpolyethylene particles over the calvaria. Mice received a weekly 10mg/kg intraperitoneal dose of MRS7216 conjugate, starting at the time of surgery. Other groups of mice were treated with equivalent weekly doses of alendronate-PEG6 (AlenP) or saline respectively. An additional control group underwent sham surgery. After 2 weeks, animals were sacrificed and microCT and histology analyses were performed. The studies were approved by the Institutional Animal Care and Use Committee of NYU School of Medicine.
Result(s): Receptor binding studies demonstrate that the Ki for CGS, 7216 conjugates and the control AlenP molecules were 21.5 nM, 69.2 nMand >10,000 nM respectively, indicating thatMRS7216 efficiently binds the A2A adenosine receptor. MicroCT studies showed that mice treated with weekly doses of 7216 had a significant reduction in bone damage of 40% (p=0.04) compared to saline treated mice. In contrast, AlenP molecules did not prevent bone erosion. Histological analysis of TRAP stained samples showed a significant decrease of osteoclast number/high-power field (HPF) of 55% (p=0.03) in AlenP treated mice compared to the saline treated group. The osteoclast depletion was more dramatic inMRS7216 treated group with an 81% reduction of osteoclasts number/HPF (p= 0.002). Additionally alkaline phosphatase staining in MRS7216 treated group, showed a significant increase in osteoblast number/HPF compared to saline (55%, p=0.01) and to AlenP group (45%, p=0.03).
Conclusion(s): Alendronate-CGS conjugates represent a novel therapeutic approach to prevent osteolysis and prosthetic failure in patients with prosthetic joints

STATEMENT OF PURPOSE/OBJECTIVE: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 (SFF), 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. METHODS:) and hardness (H) using nanoindentation. RESULTS:) data for the newly regenerated bone presented statistically homogenous values analogous to native bone at the three-time points, while hardness (H) values were equivalent to the native radial bone at 24 weeks. The negative control samples showed limited healing at 8 weeks. CONCLUSIONS: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.

Background/Purpose: Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 10% of the US population. With age and injury, chondrocytes have diminished mitochondrial content and mitochondrial production of ATP contributing to OA pathogenesis. We have previously reported that chondrocytes release ATP, which is converted extracellularly to adenosine and maintains chondrocyte homeostasis via endogenous stimulation of the A2AR. Injured/ inflamed chondrocytes have lower ATP levels and release less ATP resulting in diminished extracellular adenosine and A2AR stimulation. Mice and humans lacking the capacity to convert extracellular ATP to adenosine (ecto-5'nucleotidase deficient) develop spontaneous OA as do mice lacking A2AR (A2ARKO). We therefore studied the effect of A2AR stimulation on mitochondrial health and function in chondrocytes from WTand A2ARKO mice and in a human chondrocytic cell line.
Method(s): A human chondrocyte cell line, T/C28-a2, or neonatal chondrocytes isolated from WTand A2ARKO mice (C57Bl6 background) were grown in culture, treated with IL-1beta (5ng/ml) or medium (4h, 37oC) and during the last hour of incubation, with either medium or the selective A2AR agonist (CGS21680, 1mM). Mitochondrial function was then analyzed by Seahorse Mito Stress Kits using the Seahorse apparatus. Mitochondrial health was assessed by analyzing mean pixel intensity (MPI) of tetramethylrhodamine (TMRM) live cell staining which correlates with reduced collapsibility of mitochondrial membrane potential. Mitochondrial content and ROS burden were assessed in live cell confocal imaging (MitoTracker; MitoSox) and by immunohistochemistry (anti-ATPase Ab; anti-8hydroxyguanosine Ab, 8OHg) in paraffinembedded tissue from WTand A2ARKO mice.
Result(s): The mitochondrial membrane potential and mitochondrial content were reduced in A2ARKO chondrocytes compared to WT. In WT chondrocytes mitochondrial content increased after IL-1beta treatment and A2AR stimulation increased mitochondrial membrane potential as well. Histologic staining of knee cartilage for 8OHg, a marker of ROSinduced oxidation in mitochondria, of age-matched WTand A2ARKO mice revealed increased ROS burden in OA (A2AR KO) cartilage. In T/C28-a2 cells, neither IL-1beta nor CGS21680 affected basal O2 consumption rates (OCR), maximal respiratory rate or ATP production but IL-1beta-treated T/C28-a2 cells stimulated by CGS21680 increased all three measures of mitochondrial function (p<0.03, one-way ANOVA). Membrane potential (measured by TMRM MPI) decreased in T/ C28-a2 cells after IL-1beta or CGS treatment alone, but IL-1beta + CGS21680 treatment significantly increased MPI, indicating enhanced mitochondrial health. Mitochondrial content is not significantly modulated by IL-1beta, CGS or IL-1beta+CGS in vitro, but IL-1beta treatment significantly increased ROS burden (p<0.0001) and IL-1beta+CGS did not affect ROS-burden in T/C28-a2 cells.
Conclusion(s): Mitochondrial function and biomass decline with age and after injury and diminished mitochondrial function contributes to the development of OA. A2AR stimulation enhances the function of mitochondria in inflamed chondrocytes and contributes to the maintenance of healthy chondrocytes and cartilage

Background/Purpose: Human Immunodeficiency Virus (HIV) infection devastates the immune system but also affects tissues and organs such as kidney, liver, central nervous system, heart and bone. Bone alterations have been observed in HIV disease for nearly two decades, in particular a higher risk of low bone mineral density (BMD) and fragility fractures. Treatment of patients with Tenofovir alone or in combination (as part of HAART), leads to further changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylated; tenofovir-phosphate inhibits HIV-reverse transcriptase by mimicking AMP. We have recently found that Tenofovir inhibits Pannexin-1/Connexin-43-mediated ATP release from cells and decreases extracellular adenosine levels and fibrosis in murine models. As adenosine and ATP are key regulators of bone homeostasis, we determined whether Tenofovir directly affects bone by an adenosine- or ATP-dependent mechanism.
Method(s): M-CSF/RANKL-induced osteoclast (OC) and stimulated osteoblast (OB) differentiation were studied in primary murine bone marrow culture as the number of TRAP-positive or Alizarin Red-positive cells, respectively, after challenge with Tenofovir (1nM-100muM) alone or in combination with Dipyridamole (1nM-100muM), an agent that increases extracellular adenosine by blocking cellular adenosine uptake. Pannexin-1 and Connexin-43 expression were permanently knocked down in RAW264.7cells by lentiviral infection with appropriate shRNA or scrambled shRNA and these cells were induced to differentiate into OC by RANKL. OC/OB differentiation markers were study by RT-PCR, and intracellular pathways by Western Blot.
Result(s): Tenofovir produced a dose-dependent increase in OC differentiation (EC50=44.5nM) that was reversed by Dipyridamole (IC50=0.3muM). Tenofovir increases Cathepsin K and NFATc1 mRNA levels during OC differentiation, and the effect was reversed by Dipyridamole. When both Pannexin-1 and Connexin-43 were absent, Tenofovir did not increase OC number. Dipyridamole reversed the effect of Tenofovir on pERK1/2, pp38 and NFkB nuclear translocation. Tenofovir inhibits OB differentiation in a dose-dependent manner (IC50=0.4muM) and treatment with Dipyridamole reversed this effect (EC50=10nM). Tenofovir increases RANKL mRNA expression and decreases OPG mRNA expression during OB differentiation; these effects are reversed by Dipyridamole. We have also found alterations in beta catenin signaling pathway due to Tenofovir treatment.
Conclusion(s): Tenofovir enhances osteoclast differentiation and inhibits osteoblast differentiation by an adenosine-dependent mechanism, a finding that suggests that treatment with agents that increase local adenosine concentrations, like Dipyridamole, might prevent bone loss due to Tenofovir treatment

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.

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 A2A 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 A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

OBJECTIVE:CD4Cre mice, and investigate the role of Th17 cytokines in the disease pathogenesis. METHODS:CD4Cre mice onto an IL-22 knockout background or treating them with a neutralizing antibody against IL-17, we interrogated how these Th17 cytokines contribute to disease pathogenesis. RESULTS:CD4Cre mice, revealing a central role of Th17 cells in the regulation of OCP numbers and RANKL expression on stromal cells. CONCLUSION/CONCLUSIONS:CD4Cre mice, leading to cutaneous and synovio-entheseal inflammation, and bone pathology highly reminiscent of psoriatic arthritis. Both IL-17A and IL-22 produced by Th17 cells play critical roles in promoting the cutaneous and musculoskeletal inflammation that characterizes psoriatic arthritis..

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.

Drug discovery and development (DDD) is an interdisciplinary enterprise that spans the translational continuum. Despite DDD's importance, formal training within medical and biomedical schools is lacking. In this tutorial, we outline the current educational landscape in DDD and the growing educational need in this area. Lastly, we describe the Health Innovations and Therapeutics concentration as an example of how to design and implement an educational program in DDD.