Faculty Bibliography

Background/Purpose: Previous in vivo and in vitro animal studies demonstrate that the adenosine A2A receptor (A2AR) agonist dipyridamole (DIPY) stimulates robust osteogenic differentiation and proliferation without adverse effects on craniofacial suture development. However, no studies to date have been performed on human tissue. This study compares the effects of DIPY, BMP-2, and standard osteogenic media on osteogenic differentiation by human mesenchymal stem cells to lay the foundation for translating this bone tissue engineering approach to pediatric craniofacial reconstruction. Methods/Description: Pediatric mesenchymal stem cells were isolated from surplus bone taken from consented patients undergoing craniofacial surgery. Cells were cultured at early passage for 3 weeks in 1 of 7 experimental conditions: control media; osteogenic media (control + 100 muM beta-glycerophosphate, 0.1 muM dexamethasone and 100 mg/ mL L-ascorbic acid); osteogenic media + 200 ng/mL BMP-2; osteogenic media + 10, 100, 1000, or 10 000 muM DIPY. All experiments were performed in biological triplicates. Samples were analyzed using Alkaline phosphatase (ALP) assay at 6 hours, 24 hours, 48 hours, and 7 days as a marker of early osteogenic differentiation. At the end of the 3-week differentiation period, cells underwent immunocytochemistry to verify phalloidin, osteocalcin, and collagen I expression. Alizarin red staining was used to detect mineralization. Statistical analysis used 1-way ANOVA with Tukeys post hoc correction and multiple t test comparison of means.
Result(s): In all osteogenic conditions, relative peak ALP activity occurred at 48 hours. One thousand micrometer DIPY showed significantly increased peak ALP activity compared to BMP-2 (3.6 +/- 0.1 fold increase vs 3.1 +/- 0.1; P = .006). There was no significant difference between 1000 muM DIPY and osteogenic media (4.1 +/- 0.1; P = .36). At 3 weeks, immunocytochemistry revealed differentiation in all osteogenic conditions compared to control. One thousand micrometer DIPY cells showed greater evidence of mature osteogenic differentiation including cuboidal cell morphology and deposition of collagen I in an extracellular fibrillar network pattern compared to both control osteogenic media and BMP-2. Alizarin red quantification demonstrated significantly increased extracellular matrix mineralization at 100 muM(2.4+/-0.4; P = .002), 1000 muM (4.3+/-0.6; P = .001), and 10 000 muM (5.1 +/- 0.2; P < .0001) DIPY compared to nonosteogenic control medium (1.0 +/- 0.1). Matrix mineralization was not significantly different between BMP-2 (2.4 +/- 0.2) and 1000 muM DIPY (P = .08). ImageJ analysis revealed increased proportion of osteocalcin expressing cells (40.0% +/- 2.8%) in stem cells treated with 1000 muM of dipyridamole compared to control (1.0% +/- 0.6%), osteogenic (5.8% +/- 1.0%), and BMP-2 (16.9% +/- 2.2%; P < .0001).
Conclusion(s): Dipyridamole promotes early osteogenic differentiation and maturation of human bone-derived mesenchymal stem cells. These data suggest that dipyridamole may be an effective tissue engineering strategy for pediatric craniofacial reconstruction

Purpose: In a previous study we demonstrated that adenosine plays an important role in maintaining homeostasis in the joint. Adenosine has a short half-life (1-4 seconds in whole blood) limiting its therapeutic potential. Previously we showed that intrarticular injection of adenosine in a liposomal formulation prevents and reverses osteoarthritis (OA) progression in a post-traumatic OA (PTOA) animal model. Here we adopt a different strategy in prolonging the therapeutic half-life of adenosine by conjugating adenosine to the biodegradable carrier poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles. Method(s): Six targeted polymeric (PEG-b-PLA) nanoparticles were synthetized by binding PLA-b-PEG400-N3 and PLA-b-PEG2000 block copolymers to adenosine at the 3',4'-OH, 5'-OH, and 6-NH2 positions with an acetylene group. The efficacy of the new molecules to stimulate cAMP production was tested in RAW264.7 murine macrophage cells. PTOA was induced in Sprague Dawley rats (n=3 for each group) following non-surgical rupture of anterior cruciate ligament (ACL). Rats were treated with intra-articular injections of 100 ul of a suspension containing PLA-PEG2000-3,4-OH-adenosine (Nano-Ade; 1 mg/kg), or PLA-PEG2000 nanoparticles (Nano) or with saline for 6 weeks. The animals received the first injection 7 days after ACL rupture and injections were performed every 10 days. Knee swelling in the rats was measured before every injection. At the end of the experiment rats were sacrificed and both legs were collected for immunohistochemistry and microcomputed tomography (muCT) analysis. The capacity of the various nanoparticle preparations to bind to and activate adenosine receptors was studied as the capacity to stimulate cAMP accumulation and inhibit IL-1-stimulated IL-6, MMP13, col10a1 and NFkB translocation to the nucleus in primary murine chondrocytes. Result(s): Only adenosine-conjugated nanoparticles in which PEG2000 was bound to adenosine on the 3',4' hydroxyl groups stimulated cAMP increase (163% vs control; p<0.05) and this increase were blocked by selective antagonists of both adenosine A2A and A2B receptors. Intra-articular injection of the Nano-Ade in PTOA rats diminished swelling in affected knees (p<0.001 vs Nano). We observed that Nano-Ade diminished the IL-1beta-stimulated expression of IL-6 mRNA expression (from 214+/-107 to 79+/-89 vs Control group), an effect that was partially reversed by A2AR (ZM241385 and SCH58261, 1muM each) and A2BR (PSB1115) antagonists. In contrast, the Nano-Ade diminished IL-1beta-stimulated MMP13 and Collagen-10 mRNA and protein expression via stimulation of A2BR since the effect was reversed by the selective A2B antagonist but not by the A2AR-selective antagonist. Nano-Ade also diminished IL-1beta stimulated activation of NF-kB, a central intracellular signal for inflammation (67% decrease vs IL-1beta treated cells; p<0.001). In rats treated with Nano-Ade there was markedly reduced fibrillation of the cartilage surface (H&E staining) and less proteoglycan loss (Safranin-O staining) resulting in a significantly decreased OARSI score (Nano=2.99+/-1.20, Nano-Ade=0.88+/-0.69; p<0.03 vs Nano). Analysis of muCT scanned images showed an increase in cartilage volume in Nano-Ade treated rats (143% vs Nano; p=0.023). We also found that Nano-Ade reduced RNA expression for MMP-13 (83% reduction compare to IL-1beta stimulated cells; p<0.001) and Collagen-10 (81% reduction compare to IL-1beta stimulated cells; p<0.01) in primary murine chondrocytes stimulated with Il-1. Conclusion(s): Taken together these results demonstrate that the adenosine-functionalized particles bind to and activate adenosine A2A and A2B receptors on murine cells. Activation of adenosine receptors by PLA-PEG2000-3,4-OH-adenosine prevents OA progression and reduce pro-inflammatory mediators in murine chondrocytes.

Background: The most common cause of total joint replace-ment revision procedures is loosening of the implant due to bone erosion around the prosthesis. In most patients, wear particle induced osteolysis is responsible for prosthesis failure. We have previously identified CGS21680 (CGS), an A2A receptor agonist, as a possible treatment. Thus, we generated and tested a novel alendronate-CGS conjugate (MRS7216) that localizes to bone. We hypothesized that MRS7216 administration could prevent inflammatory osteolysis and bone erosion by enhancing A2A signaling, while preventing CGS associated side effects.
Method(s): Osteolysis was induced in 6-8 week-old C57BL/6J mice (5 mice per treatment group). Mice received a single dose or weekly 10 mg/kg intraperitoneal dose of MRS7216 conjugate, beginning at the time of surgery. Other treatment groups were given equivalent weekly doses of either alendronate-PEG6 (AlenP) or saline. After 2 weeks, mice were sacrificed. MicroCT and immunohistochemistry analyses were performed. The studies were approved by the NYU Institutional Animal Care and Use Committee.
Result(s): Studies revealed that mice treated with a weekly dose of MRS7216 had significant (40%) reduction in bone damage and (81%) reduction in osteoclasts compared to saline treated mice. AlenP did not prevent bone erosion (p<0.05) and had significant (55%) decrease in osteoclasts compared to saline group. Staining in MRS7216-treated group, showed an increase in osteoblasts compared to saline and AlenP (p<0.06).
Conclusion(s): MRS7216 conjugate suppresses wear particle-induced osteolysis, inhibits osteoclastogenesis and increases osteoblast number. MRS7216 could be a novel treatment in prevention of osteolysis and potentially promote bone regeneration. Supported by the Medical Student Training in Aging Research Program, National Institutes of Health, National Institute of Aging, Bethesda, Maryland to New York University School of Medicine

Background/Purpose: Our tissue engineering laboratory has previously demonstrated that dipyridamole-coated, 3D printed bioceramic (3DPBC) scaffolds comprised of B-tricalcium phosphate generate significantly more bone compared to negative controls in short-term growing animal model studies. No detrimental effects to the cranial suture were observed in any experimental animals. The longterm osteogenic efficacy and safety of our 3DPBC scaffold for tissue engineering in growing calvaria was assessed by describing bone regeneration compared to autogenous bone graft, scaffold degradation kinetics, and the effects of the construct on cranial growth over time. Methods/Description: Twenty-two 1-month-old (immature) New Zealand white rabbits underwent unilateral 11-mm craniotomy within 2 mm of the coronal and sagittal sutures. Rabbits' calvarial defects were repaired by 1 of 2 interventions: 3DPBC scaffolds coated with 1000 mM dipyridamole (n = 14) or autogenous calvarial bone graft (n = 8). Six rabbits from the 3DPBC scaffold group were sacrificed at 8 weeks. The remaining rabbits (n = 8 each group) were observed until craniofacial growth was completed (6 months) and then euthanized. Bone regeneration, scaffold degradation, and cranial suture patency were analyzed in Amira software using reconstructed microcomputed tomography (muCT) images. Cranial growth was assessed by comparing bilateral cephalometric measurements based on muCT images. Bone growth and suture patency were qualitatively evaluated through histologic analysis.
Result(s): After 6 months of healing, animals with defects repaired with 3DPBC scaffolds regenerated an average of 53.9% +/- 3.6% (mean +/- SEM) bone, compared to 53.5% +/- 3.6% in defects repaired with bone graft (P = .95). Unoperated calvarial bone porosity was 49.4%+/-2.0%. Scaffolds showed significant degradation at 6 months (15.1% +/-0.7%) compared to 8 weeks (23.2% +/- 0.9%; P<=.001). Comparative measurements of operated and unoperated sides showed no significant differences in asymmetry between scaffold and bone graft animals (P > .24). Analysis of histologic sections revealed well-vascularized, organized bone formation within scaffold interstices with no evidence of ectopic bone formation, excess inflammatory cells, or suture fusion.
Conclusion(s): Dipyridamole-coated 3D-printed bioceramic scaffolds bone regeneration is comparable to autogenous bone graft without showing signs of adverse events such as premature cranial suture fusion, or detrimental effects to facial growth. The scaffold demonstrates favorable absorption kinetics, highlighting the potential for this technology in pediatric bone tissue engineering

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.

Background: Mutations in plakophilin-2 (PKP2) are the most common cause of familial Arrhythmogenic Right Ventricular Cardiomyopathy, a disease characterized by ventricular arrhythmias, sudden death and progressive fibrofatty cardiomyopathy. The relation between loss of PKP2 expression and structural cardiomyopathy remains under study, though paracrine activation of pro-fibrotic intracellular signaling cascades is a likely event. Previous studies have indicated that ATP release into the intracellular space, and activation of adenosine receptors, can regulate fibrosis in various tissues. However, the role of this mechanism in the heart, and in the specific case of a PKP2-initiated cardiomyopathy, remains unexplored. The aim of this study was to investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy.
Methods and Results: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. A cardiac-specific, tamoxifenactivated PKP2 knock-out murine model (PKP2-cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Transcriptomic data of PKP2-cKO mice revealed overexpression of genes involved in adenosine-receptor cascades. Treatment with Istradefylline (an adenosine 2A receptorblocker) tempered the progression of fibrosis and mechanical failure observed in PKP2-cKO mice (see Fig. B,C). In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects.
Conclusion(s): Paracrine adenosine 2A receptor activation contributes to the progression of fibrosis and impaired cardiac function in animals deficient in PKP2. Given the limitations of the animal model, translation to the case of patients with PKP2 deficiency needs to be done with caution. (Figure Presented)

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.

Osteopenia and fragility fractures have been associated with HIV infection. Tenofovir, a common antiviral in HIV treatment, also leads to increases in bone catabolism markers and decreased bone mineral density (BMD) in children and young adults. In murine models and human cell lines, tenofovir inhibits ATP release and decreases extracellular adenosine levels. Adenosine, and adenosine A2A receptor, inhibits osteoclast formation, and increasing local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptaken, stimulates new bone formation as well as rhBMP-2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and determined whether dipyridamole may be a useful treatment to counteract the deleterous bone effects of tenofovir. M-CSF/RANKL-induced-primary murine osteoclast was studied as the number of TRAP-positive-cells after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were study by RT-PCR, and MAPK/NFkB expression by WesternBlot. Male C57Bl/6 mice were treated as follow: saline 0.9% (control), tenofovir 75mg/Kg/day, dipyridamole 25mg/Kg/day, combination tenofovir/dipyridamole (n = 10, 4weeks). Calcein/AlizarinRed-labelling of newly formed bone was used, and long bones were prepared for microCT/histology. Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC₅₀  = 44.5nM) that was reversed by dipyridamole (IC₅₀  = 0.3µM). Tenofovir increased Cathepsin K and NFATc1 mRNA levels and dipyridamole reversed the effect. Dipyridamole reversed the effect of tenofovir on pERK1/2, pp38 and NFkB nuclear translocation. Mice treated with tenofovir lost nearly 10% of body weight (p < 0.001). MicroCT revealed decrease BMD and altered trabecular bone in tenofovir-treated mice, reversed by dipyridamole. TRAP-staining showed increased osteoclasts in tenofovir-treated mice (p < 0.005) an effect reversed by dipyridamole. Similar results were obtained for Cathepsin K and CD68. RANKL-positive-cells were increased in tenofovir-treated mice whereas OPG-positive-cells decreased, and both effects were reversed by dipyridamole. These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment.