Faculty Bibliography

Background/Purpose : Implant loosening due to loss of bone is the most common cause of total joint replacement revision surgeries. One of the main cause of osteolysis is the shed of wear particles from the implant as it causes an increased local inflammation and osteoclast number and activity. It is known that an A 2A adenosine receptor selective agonist (CGS21680, CGS) prevents bone loss in wear particle-induced osteolysis model in mice. But frequent administration requirements and its potential to cause side effects make it a less than optimal treatment. 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. Methods : 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 PEG 6 linker was incorporated to alendronic acid by direct coupling. Osteolysis in 6-8-week-old C57BL/6J (WT) or A 2A KO mice was induced by surgical implantation of 3mg of ultrahigh-molecular-weight-polyethylene 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-PEG 6 (AlenP) or saline respectively. An additional control group underwent sham surgery. New bone formation was studied by Calcein/Alizarin Red-labeling. 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. Results : Receptor binding studies demonstrate that the Ki for CGS, 7216 conjugates and the control AlenP molecules were 21.5 nM, 69.2 nM and >10,000 nM respectively, indicating that MRS7216 efficiently binds the A2A adenosine receptor. MicroCT studies showed that WT mice treated with weekly doses of MRS7216 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. Similarly in A 2A KO mice MRS7216 treatment did not prevent bone damage. 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 WT mice compared to the saline treated group. The osteoclast depletion was more dramatic in MRS7216 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 per HPF compared to saline (55%, p=0.01) and to AlenP group (45%, p=0.03). Furthermore Double bone labeling with calcein/alizarin red showed a significant increase on femurs bone formation of MRS7216 treated group compared to saline and to Alendronate group (p=0.0092 and p=0.0345). Conclusion : Alendronate-CGS conjugates represent a novel and specific therapeutic approach to inhibit osteolysis and stimulate new bone formation to prevent prosthetic failure in patients with prosthetic joints or other bone pathologies

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/CONCLUSIONS: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.

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.

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-N₃ and PLA-b-PEG2000 block copolymers were bound to adenosine at the 3',4'-OH, 5'-OH, and 6-NH₂ 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.

Methotrexate has been used in treatment of rheumatoid arthritis (RA) since the 1980s and to this day is often the first line medication for RA treatment. In this review, we examine multiple hypotheses to explain the mechanism of methotrexate efficacy in RA. These include folate antagonism, adenosine signaling, generation of reactive oxygen species (ROS), decrease in adhesion molecules, alteration of cytokine profiles, and polyamine inhibition amongst some others. Currently, adenosine signaling is probably the most widely accepted explanation for the methotrexate mechanism in RA given that methotrexate increases adenosine levels and on engagement of adenosine with its extracellular receptors an intracellular cascade is activated promoting an overall anti-inflammatory state. In addition to these hypotheses, we examine the mechanism of methotrexate in RA from the perspective of its adverse effects and consider some of the newer genetic markers of methotrexate efficacy and toxicity in RA. Lastly, we briefly discuss the mechanism of additive methotrexate in the setting of TNF-α inhibitor treatment of RA. Ultimately, finding a clear explanation for the pathway and mechanism leading to methotrexate efficacy in RA, there may be a way to formulate more potent therapies with fewer side effects.

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/Purpose: Alveolar cleft surgery is the most common bone reconstruction performed in patients with a cleft. Osteogenic agents such as BMP-2 have been used to restore the bony cleft without the morbidity of bone graft, but concerns remain regarding premature fusion of sutures, exuberant bone formation, and malignant degeneration. We have recently demonstrated that dipyridamole-coated, 3D printed bio-ceramic (3DPBC) scaffolds generate comparable bone amounts to BMP2 and significantly greater bone compared to negative controls in short-term growing animal model studies. No detrimental effects to growth sutures were noted in any animals. This study investigates the long-term osteogenic properties, degradation kinetics, and effects on facial growth of these tissue engineering constructs in growing animal models. Methods/Description: Twenty-two 1-month-old (immature) New Zealand white rabbits underwent creation of unilateral 3.5 x 3.5 mm alveolar defects. Each alveolar defect was repaired with either 3DPBC scaffolds coated with 1000 muM dipyridamole (n = 14) or with autogenous bone graft from the radius (n = 8). Six rabbits from the 3DPBC scaffold group were sacrificed at 8 weeks. The remaining rabbits (n = 8 each group) were euthanized following completion of craniofacial growth (6 months). Bone regeneration, scaffold degradation, and maxillary suture patency were calculated using CT images reconstructed and analyzed in Amira software. Facial symmetry was evaluated using dense-surface 3D modeling and validated with bilateral cephalometric measurements of maxillary projection. Bone growth and suture patency were qualitatively evaluated through histologic analysis.
Result(s): After 6 months, animals with defects repaired with 3DPBC scaffolds regenerated an average of 52.9% +/- 3.3% bone (mean +/- SEM), compared to 40.7%+/-4.0% in defects repaired with bone graft (P = .02). This is compared to unoperated alveolus occupied by 39.3% +/- 1.6% bone. Scaffolds showed significant degradation at 6 months (6.7% +/- 1.6%) compared to at 8 weeks (27.1% +/- 1.9%; P >= .001). Morphometric analysis using dense surface modeling showed similar symmetry indices of 55.0 +/- 3.3 for scaffold animals and 61.7% +/- 1.6% for bone graft animals (P = .10). Comparative measurements of operated and unoperated sides showed no significant differences in asymmetry between scaffold and bone graft animals (P = .86). Histologic analysis of scaffold samples revealed vascularized, organized bone within scaffold interstices without evidence of ectopic bone, excess inflammatory cells, or suture fusion.
Conclusion(s): In a growing animal model, dipyridamole-coated 3DPBC scaffolds can regenerate bone comparable to autogenous bone graft by radiographic and histologic analysis. Over 6 months, scaffolds show significant, favorable degradation and do not result in premature suture fusion or disruption of facial growth compared to bone graft. These results support long-term safety and efficacy of this tissue engineering strategy in the repair of alveolar cleft defects