Rescue of Impaired Fracture Healing in COX-2−/− Mice via Activation of Prostaglandin E2 Receptor Subtype 4

Abstract

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracturehealing model, COX-2 ؊/؊ mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2 ؊/؊ mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2 ؊/؊ callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2 ؊/؊ mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 longterm limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2 ؊/؊ fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2 ؊/؊ mice. Fracture healing is a complex process orchestrated by precise presentation of growth factors and cytokines that control activation, proliferation, and differentiation of the local mesenchymal stem/progenitor cells. Fracture healing begins with hematoma formation and an inflammatory response. The activated stem/progenitor cells proliferate and further differentiate into osteoblasts and chondrocytes. Endochondral bone formation takes place toward the most central avascular region of the callus. Chondrogenesis initiates directly adjacent to the surface of the cortical bone and is surrounded by less-differentiated mesenchymal progenitor cells. The subsequent expansion of the callus involves the conversion of the lingering mesenchymal progenitor cells into chondrocytes and further proliferation and differentiation of chondrocytes into a calcified cartilage template that permits vascular invasion and bone formation. Areas of intramembranous bone formation flank the area of endochondral ossification, particularly along the bone surface furthest from the central fracture site where the blood supply is typically better preserved. The coordinated endochondral and intramembranous bone formation pathways eventually result in a bridging mineralized callus that re-establishes the integrity of the skeletal element.

Growth Factors and Signalling Molecules for Cartilage Tissue Engineering – from Embryology to Innovative Release Strategies for Guided Tissue Engineering

“…Regarding bone formation and bone resorption the EP4 receptor has been shown to be essential in terms of PGE 2 action in bone (Yoshida et al, 2002). In this respect the critical involvement of EP4 receptor in fracture healing was demonstrated very recently (Yoshida et al, 2002, Xie et al, 2009. Furthermore, EP2 and EP4 receptors were shown to be required for PGE 2 -dependent chondrocyte differentiation (Miyamato et al, 2003).…”

Periosteum

“…It is therefore a critical regulator in inflammation. By in situ hybridization, Cox-2 was found in chondroprogenitors and mesenchymal cells along the periosteal surface, and its expression was correlated with the early induction of chondrogenesis and early expansion of the cartilaginous callus (Xie et al, 2009). Administration of a COX-2 inhibitor in the early phase of healing compromised fracture repair (Simon et al, 2002).…”

Fracture healing and lipid mediators

“…42,43 When fractures in COX-2-null mice were treated with an EP2 (CP-463755) or an EP4 (CP-734432) agonist, the EP4 agonist rescued fracture healing in the COX-2-null mice better than did the EP2 agonist. 14 The effects of montelukast treatment on fracture healing have also been studied. Montelukast antagonizes cysteinyl leukotriene receptor 1 and is commonly used to treat asthma.…”

“…21,32 COX-2 mRNA levels at the fracture site increase immediately after fracture but peak during the regenerative, rather than inflammatory, phase of healing. 13,14,33 Few studies have actually measured lipid mediator levels during fracture healing. Dekel et al 34 measured prostaglandin E and F levels from the bone and surrounding muscle of rabbit tibial fractures.…”

“…13 Similar effects on type X collagen mRNA levels also have been observed in COX-2-null mice. 14 It was hypothesized that the failed chondrocyte hypertrophy stops endochondral bone formation, which leads to fracture nonunion. 13 In humans, NSAIDs, including COX-2-selective NSAIDs, are used therapeutically to limit heterotopic bone formation following hip fracture repairs.…”