Direct or indirect stimulation of adenosine A _2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2

Abstract

Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via A 2A receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein-2 (BMP-2; 200 ng), 1 mM CGS21680 (A2AR agonist, EC 50 = 160 nM), or 1 mM dipyridamole (EC 50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 8 wk after surgery (60 6 2%, 79 6 2%, and 75 6 1% bone regeneration, respectively, vs. 32 6 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 mM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatasepositive osteoblasts and diminished tartrate resistance acid phosphatase-positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP-2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP-2 and represents a novel approach to stimulating bone regeneration.-Mediero, A., Wilder, T., Perez-Aso, M., Cronstein, B. N. Direct or indirect stimulation of adenosine A 2A receptors enhances bone regeneration as well as bone morphogenetic protein-2. FASEB J. 29, 1577-1590 (2015). www.fasebj.org

Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

“…The work presented here showed no effects of adenosine, 2-chloroadenosine, GR79236 or BAY606583 on calvarialderived osteoblasts; this is in broad agreement with previous studies which showed exogenous adenosine had no effect on cultured rat osteoblasts [11,29]. However, our results are at variance with several studies which found that adenosine or adenosine analogues, acting via the A 2A or A 2B receptors, stimulate the differentiation and function of human and rodent bone marrow osteoblasts and promote bone regeneration [24,30,31,40]. Our data also do not concur with the reported inhibitory effects of adenosine analogues, acting via A 1 or A 2A receptors, on the differentiation of rodent osteoblast-like cells [41] or human osteoblasts [40].…”

“…In addition, a synthetic A 2B receptor agonist has been shown to increase bone formation, and bone marrow osteoblasts from A 2B receptor knockout mice display reduced levels of bone formation [30]. Recently, it has also been reported that stimulation of the A 2A receptors can enhance bone regeneration [31].…”

Regulation of bone and cartilage by adenosine signaling

“…In other studies A2AR stimulation has been shown to diminish inflammatory osteolysis and increase osteoblast numbers in inflamed bone [55]. In the context of a bone defect, A2AR helps restore bone homeostasis by increasing osteoblasts while decreasing osteoclast number and activity [77]. Yet, He et al observed no effect on human osteoblast differentiation and mineralization with activation or downregulation of the A2AR [78].…”

“…Mediero et al observed enhanced bone regeneration, and an increase in bone volume and osteoblast expression of markers for bone formation including osteocalcin and osteonectin in mouse calvaria treated with either an A2AR agonist (CGS21680) or dipyridamole which blocks adenosine uptake. In addition, osteoclast markers were decreased in vitro and osteoclast numbers were reduced in vivo [77]. Adenosine receptor agonists or agents that increase local adenosine concentration may be preferred approaches to inducing bone regeneration following orthopedic surgeries compared to the currently used recombinant bone morphogenetic proteins, which are known to inflame nearby tissues, stimulate aberrant bone formation and carry higher complication rates [79].…”

“…•Unclear role, no morphologic changes in A1R knockout mice [54,70] •Enhances bone regeneration [77] •Increases osteoblast differentiation and mineralization [82] • •Increases cell viability and cell number [76] •Increases expression of Run × 2, sialoprotein, osteocalcin [68] •Signals via canonical Wnt pathway [72] •Blockade causes delayed fracture healing [68] …”