Background/Purpose Such orthopedic procedures as spinal fusion and repair of bone defects due to trauma, infection or metastatic disease, require formation of new bone. Adenosine, acting via stimulation of the A_2A receptor (A2AR), inhibits osteoclast differentiation. Here we determined whether direct A2AR stimulation or enhancing adenosine concentrations via blockade of purine transport into cells via ent1 with dipyridamole regulates bone formation in a murine calvarial model.

Methods 6-8-wk male C57Bl/6 mice (WT) or A_2AKO were anesthetized; a 3mm trephine defect was formed and covered with a collagen scaffold soaked in saline or CGS21680 (A2AR agonist) or dipyridamole (1mM each) alone or in the presence of ZM241385 1mM (A2AR antagonist). Animals received appropriate treatment daily until sacrifice. Bone Morphogenetic Protein 2 (BMP-2) 200ng was used as a bone formation control. At 0, 2, 4, 6 and 8 weeks calvarias were harvested and prepared for microCT and histology. XenoLight Rediject Bone Probe 680 was injected intravenously at different time points and used to probe bone formation (fluorescence).

Results 8 weeks after surgery microCT examination of WT mouse calvaria demonstrated that both CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 (60±2%, 79±2% and 75±1% bone regeneration, respectively, vs. 32±2% in control, p<0.001, n=5 mice per condition). Both CGS21680 and dipyridamole effects were abrogated by ZM241385 (20±3% and 26±4% bone regeneration, respectively, vs. 32±2% in control, p=ns, n=5 mice per condition). Neither CGS21680 nor dipyridamole enhanced bone formation in A_2AKO mice. In CGS21680- and dipyridamole-treated WT mice there was increased immunostaining for bone formation markers in the bony defects (Alkaline Phosphatase positive cells/hpf increased from 15±1 for control to 21±1 for CGS21680 and 24±1 for dipyridamole, p<0.001). TRAP staining revealed fewer osteoclasts in CGS21680- and dipyridamole-treated defects (17±1 and 16±1 osteoclast/hpf respectively vs. 24±1 Osteoclast/hpf for control, p<0.001) 8 weeks after defect formation, an effect blocked by ZM241385 or absent in A2AR KO mice. In vivo imaging with XenoLight Rediject Bone Probe 680 (a marker of bone formation) reveals a markedly increased fluorescent signal in treated animals, equivalent to BMP-2, when compared to control as soon as one week after bone defect formation and which lasts for at least 7 weeks.

Conclusion Stimulation of A2AR increases the rate of new bone formation at sites of surgical bone defects as well as BMP2, a finding which suggests that targeting A2AR may be an effective way to increase bone formation following orthopedic procedures.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-receptors-stimulate-bone-regeneration/