Faculty Bibliography

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..

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.

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.

Background/Purpose: 3D-printed bioactive ceramic (3DPBC) scaffolds composed of beta-tricalcium phosphate (b-TCP) and coated in the osteogenic agent dipyridamole have been previously shown to heal critically sized calvarial defects in an adult animal model. This bone tissue engineering construct has yet to be applied in a pediatric craniofacial model and there has been evidence that other osteogenic agents such as BMP-2 can prematurely fuse growing sutures. The purpose of this study is to apply the described bone tissue engineering construct in a pediatric growing animal model and 1) quantify osteogenic potential in a growing calvarium; 2) maximize the scaffold design and dipyridamole (DIPY) concentration for the growing calvarium; and 3) characterize the effects of this bone tissue engineering construct on the growing suture. Methods/Description: Bilateral calvarial defects (10 mm) were created in 5-week-old New Zealand White rabbits (n = 14) 2 mm posterior and lateral to the coronal suture and sagittal sutures, respectively. 3DPBC scaffolds were constructed in quadrant form composed by varying pore dimensions (220, 330, and 500 mum). Each scaffold was collagen coated and soaked in varying concentrations of DIPY (100, 1000, and 10 000 muM). Controls comprised empty defects and collagen-coated scaffolds. Scaffolds were then placed into the calvarial defects to fill the bone space. Animals were euthanized 8 weeks postoperatively. Calvaria were analyzed using micro-computed tomography and 3D reconstruction.Mixed model analyses were conducted considering pore size and dosage effects on bone growth (a = 0.05).
Result(s): Scaffold group healing presented bone formation throughout the scaffold structure (defect marginal and central regions) while bone healing in empty sites was restricted to the defect margins, confirming its critical size dimension at 8 weeks in vivo. No significant difference in bone formation was detected when experimental groups were collapsed over pore size (P > .40). When pore size was collapsed over DIPY concentration, higher mean values were observed for the DIPYimmersed groups, and significance was shown between the 1000-muM and collagen groups (P < .05). Pore size and DIPY interaction was more pronounced for the 330-mum pore size where both the 100-and 1000-mum dosages presented significantly higher bone formation compared to collagen (P < .05). Across all concentrations of DIPY, including 10 000 mM (10 times greater than the experimental concentration, yielding the highest bone formation), sutures remained patent.
Conclusion(s):We present an effective bone tissue engineering scaffold design and dipyridamole concentration that significantly improve bone growth in a pediatric growing calvarial model and preserves cranial suture patency