Adenosine A2A receptor activation prevents wear particle-induced osteolysis

Abstract

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 A(2A) receptors (A(2A)Rs) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation. We tested the hypothesis that an A(2A)R agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear particle-induced bone resorption. C57BL/6 and A(2A)R knockout (A(2A)R KO) mice received ultrahigh-molecular weight polyethylene particles and were treated daily with either saline or the A(2A)R 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 A(2A)R KO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone resorption markers receptor activator of nuclear factor κB (RANK), RANK ligand, cathepsin K, CD163, and osteopontin were reduced after CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin-1β (IL-1β) and tumor necrosis factor-α 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 A(2A)R agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery.

Figure 1

Bone pitting in mouse calvaria after exposure to wear particles. Mice were administered UHMWPE particles and either treated with saline or the adenosine A_2AR agonist CGS21680. Control mice (sham) were not exposed to particles (n=6). Mice were injected daily and after 2 weeks animals were sacrificed. (A) Representative μCT images of calvaria from wild-type animals. (B) Morphometric quantitation of bone pitting in the presence of varying doses of CGS21680 (1 nM to 1 μM). Data were calculated as the percentage of the area of bone pitting in the saline-treated, particle-exposed mice. Each point represents the mean±SEM of determinations in 6 individual mice per group. ***P<0.001 compared to control (100% pitting), ANOVA. (C) Representative μCT images of calvaria from A_2AR KO animals. (D) Morphometric quantitation of bone pitting in the presence of CGS21680 (1 μM). Data were calculated as the percentage of the area of bone resorption in the saline-treated, particle-exposed mice. Each point represents the mean±SEM of determinations in 4 individual mice per group. NS compared to control, ANOVA (E) Morphometric quantitation of bone resorption in the presence of varying doses of CGS21680 injected every other day in wild-type mice. Data were calculated as the percentage of the area of bone pitting in the saline-treated mice. Data are means ±SEM (n=5). ***P<0.001 compared to saline, ANOVA.

Figure 2

Effect of A_2AR stimulation on osteoclast formation by human bone marrow cells. (A) Human primary bone marrow monocytes were fixed and stained for TRAP after culture for 7 days in the presence of M-CSF and RANKL, with or without CGS21680 (10 μM to 10 nM). TRAP-positive cells containing three or more nuclei were counted as osteoclasts. (B) Number of TRAP+ osteoclast [% relative to control (RANKL+)] expressed as the means of 4 cultures in duplicate in myeloma bone marrow-derived osteoclasts. (C) Number of TRAP+ osteoclast [% relative to control (RANKL+)] expressed as the means of 4 cultures in duplicate in healthy bone marrow-derived osteoclasts. *P<0.05, ***P<0.001, compared to RANKL, # P<0.05 compared to CGS21680 1μM, ANOVA.

Figure 3

Inflammation in mice calvaria after exposure to wear particles in the absence or presence of CGS21680. Calvaria were stained with hematoxylin & eosin to determine the presence of inflammation in the outer bone surface. (A) Calvaria were exposed to saline only (sham) or to UHMWPE particles, the latter in the absence or presence of the adenosine A_2AR agonist CGS21680 (1 μM). (B) The area of inflammatory infiltrate was quantified from (A) and expressed as a percentage of the area of the saline-treated, particle-exposed mice. Data are means±SEM (n=3 per group). ***P<0.001, ANOVA. (C) Calvaria from A_2AR KO mice after exposure to saline only (sham) or UHMWPE particles without or with CGS21680 (1 μM). (D) The area of inflammatory infiltrate was quantified from (C) and expressed as a percentage of the area of the saline-treated, particle-exposed mice. Data are means±SEM (n=3 per group). (E) Comparison between WT and A_2AR KO in saline-treated, particle-exposed mice was quantified expressed as a percentage of the area of the sham mice. Data are means±SEM (n=3 per treatment group). ***P<0.001, ANOVA.

Figure 4

TRAP staining for osteoclasts in mice calvaria. (A) Representative images of saline only (sham) or UHMWPE particle-exposed calvaria with or without CGS21680 (1μM) treatment. Histology for 10 to 100 nM CGS21680 is shown in fig. S2. Red cells are positive for TRAP staining (arrow). (B) Quantification of the number of osteoclast/hpf (high power field) in (A). Osteoclasts were counted in 5 different images for each of 3 mice. Data are means ±SEM (n=3 per group). ***P<0.001 related to saline, ANOVA. (C) Calvaria from A_2ARKO mice after exposure to saline alone (sham) or wear particles in the absence or presence of CGS21680 (1μM). Red cells are positive for TRAP staining (arrow). (D) Quantification of the number of osteoclast/hpf in (C). Osteoclasts were counted in 5 different images for each of 3 mice. Data are means ±SEM (n=3 per group). ***P<0.001 related to saline (ANOVA).

Figure 5

Immunohistochemistry for markers of osteoclasts, macrophages, inflammation, and bone remodeling in wild-type mice. (A) Calvaria were processed and immunohistologic staining carried out on calvaria from sham or UHMWPE-exposed mice, the latter treated with either saline or CGS21680 (1 μM). Shown are representative sections of calvaria (from n=3 mice) stained for cathepsin K, RANK, RANKL, CD163, TNFα, αSMA, osteopontin, osteocalcin, and osteoprotegerin. (B) Immunohistochemistry quantifications of cells/hfp (high power field) for (A). Data are means ± SEM for 5 different slides per untreated mouse (n=3) and each mouse treated with either particulate and saline (n=3) or particulate and 1 μM CGS21680 (n=15) *P<0.05, **P<0.01, ***P<0.001, compared to untreated, ANOVA. Scale bar indicates 50 μm. All images are taken with the same magnification.

Figure 6

Cytokine, chemokine, and bone metabolism markers in mouse calvaria supernatants. Calvaria from untreated (sham) animals and animals exposed to wear particles in the absence or presence of CGS21680 (1 μM) were cultured in medium for 4 hours before collection of supernatants and quantification of cytokines/chemokines and bone metabolism markers. Data are expressed as mean percentages of sham ± SEM from n = 10 mice per treatment group assayed in triplicate. *P<0.5, **P<0.01, ***P<0.001 versus particle-exposed, saline-treated controls; ANOVA.