Osteoarthritis (OA) is characterized by cartilage destruction and chondrocytes have a central role in this process. With age and inflammation chondrocytes have reduced capacity to synthesize and maintain ATP, a molecule important for cartilage homeostasis. Here we show that concentrations of ATP and adenosine, its metabolite, fall after treatment of mouse chondrocytes and rat tibia explants with IL-1β, an inflammatory mediator thought to participate in OA pathogenesis. Mice lacking A2A adenosine receptor (A2AR) or ecto-5′nucleotidase (an enzyme that converts extracellular AMP to adenosine) develop spontaneous OA and chondrocytes lacking A2AR develop an ‘OA phenotype' with increased expression of Mmp13 and Col10a1. Adenosine replacement by intra-articular injection of liposomal suspensions containing adenosine prevents development of OA in rats. These results support the hypothesis that maintaining extracellular adenosine levels is an important homeostatic mechanism, loss of which contributes to the development of OA; targeting adenosine A2A receptors might treat or prevent OA.

Adenosine is generated in increased concentrations at sites of injury/hypoxia and mediates a variety of physiological and pharmacological effects via G protein-coupled receptors (A(1), A(2A), A(2B), and A(3)). Because all adenosine receptors are expressed on osteoclasts, we determined the role of A(2A) receptor in the regulation of osteoclast differentiation. Differentiation and bone resorption were studied as the macrophage colony-stimulating factor-1-receptor activator of NF-κB ligand formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells from primary murine bone marrow-derived precursors. A(2A) receptor and osteoclast marker expression levels were studied by RT-PCR. Cytokine secretion was assayed by enzyme-linked immunosorbent assay. In vivo examination of A(2A) knockout (KO)/control bones was determined by TRAP staining, micro-computed tomography, and electron microscopy. The A(2A) receptor agonist, CGS21680, inhibited osteoclast differentiation and function (half maximal inhibitory concentration, 50 nmol/L), increased the percentage of immature osteoclast precursors, and decreased IL-1β and tumor necrosis factor-α secretion, an effect that was reversed by the A(2A) antagonist, ZM241385. Cathepsin K and osteopontin mRNA expression increased in control and ZM241385-pretreated osteoclasts, and this was blocked by CGS21680. Micro-computed tomography of A(2A)KO mouse femurs showed a significantly decreased bone volume/trabecular bone volume ratio, decreased trabecular number, and increased trabecular space. A(2A)KO femurs showed an increased TRAP-positive osteoclast. Electron microscopy in A(2A)KO femurs showed marked osteoclast membrane folding and increased bone resorption. Thus, adenosine, acting via the A(2A) receptor, inhibits macrophage colony-stimulating factor-1-receptor activator of NF-κB ligand-stimulated osteoclast differentiation and may regulate bone turnover under conditions in which adenosine levels are elevated.

Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 μm/s and a partial pressure of O2 (pO2) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO2, but also decreased the speed of locomotion in the deep paracortex. Although CCR7−/− naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Gαi-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO2, tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.

Bone defects resulting from trauma or infection need timely and effective treatments to restore damaged bone. Using specialized three-dimensional (3-D) printing technology we have created custom 3-D scaffolds of hydroxyapatite (HA)/Beta-Tri-Calcium Phosphate (β-TCP) to promote bone repair. To further enhance bone regeneration we have coated the scaffolds with dipyridamole, an agent that increases local adenosine levels by blocking cellular uptake of adenosine. 15% HA:85% β-TCP scaffolds were designed using Robocad software, fabricated using a 3-D Robocasting system, and sintered at 1100°C for 4h. Scaffolds were coated with BMP-2 (200ng/ml), Dypiridamole 100µM or saline and implanted in C57B6 and adenosine A2A receptor knockout (A2AKO) mice with 3mm cranial critical bone defects for 2-8 weeks. Dipyridamole release from scaffold was assayed spectrophotometrically. MicroCT and histological analysis were performed. micro-computed tomography (microCT) showed significant bone formation and remodeling in HA/β-TCP- dipyridamole and HA/β-TCP -BMP-2 scaffolds when compared to scaffolds immersed in vehicle at 2, 4 and 8 weeks (n=5 per group; p≤ 0.05, p≤ 0.05 and p≤ 0.01, respectively). Histological analysis showed increased bone formation and a trend toward increased remodeling in HA/β-TCP- dipyridamole and HA/β-TCP-BMP-2 scaffolds. coating scaffolds with dipyridamole did not enhance bone regeneration in A2AKO mice. In conclusion, scaffolds printed with HA/β-TCP promote bone regeneration in critical bone defects and coating these scaffolds with agents that stimulate A2A receptors and growth factors can further enhance bone regeneration. These coated scaffolds may be very useful for treating critical bone defects due to trauma, infection or other causes.

Prosthesis loosening, associated with wear-particle–induced inflammation and osteoclast-mediated bone destruction, is a common cause for joint implant failure, leading to revision surgery. Adenosine A2A receptors (A2AR) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation. We tested the hypothesis that an A2AR agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear-particle–induced bone resorption. C57Bl/6 and A2A knockout (A2ARKO) mice received ultrahigh-molecular weight polyethylene particles (UHMWPE) and were treated daily with either saline or the A2AR agonist CGS21680. After 2 weeks, micro-computed tomography of calvaria demonstrated that CGS21680 reduced particle-induced bone pitting and porosity in a dose-dependent manner, increasing cortical bone and bone volume compared to control mice. Histological examination demonstrated diminished inflammation after treatment with CGS21680. In A2AKO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone-resorption markers receptor activator of nuclear factor-kB (RANK), RANK ligand (RANKL), cathepsin K, CD163, and osteopontin were reduced following CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin 1β (IL-1β) and TNFα was significantly decreased, whereas IL-10 was markedly increased in bone by CGS21680. These results in mice suggest that site-specific delivery of an adenosine A2AR agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery.