Immediately following skeletal injury, a sequence of biochemical and cellular events commence to induce an inflammatory response. A myriad of factors including growth factors, cytokines, and prostaglandins are released. These factors are likely to play an essential role initiating the healing response that leads to new bone formation (34
). The crucial events in adult bone formation are the recruitment, proliferation, and differentiation of mesenchymal stem cells with endochondral and intramembranous bone formation at the injury site (35
). In endochondral ossification, mesenchymal cells first differentiate into chondrocytes, which subsequently undergo terminal differentiation and apoptosis, leading to calcification of the matrix. The calcified matrix then serves as a template for primary bone formation, whereby osteoblasts deposit bone directly onto calcified cartilage (36
). In intramembranous bone formation, mesenchymal cells differentiate directly into osteoblasts (36
). Thus, both endochondral and intramembranous bone formation are dependent upon osteoblast differentiation from mesenchymal stem cells. Although the mechanism of recruitment and stimulation of mesenchymal stem cell differentiation during adult bone regeneration is largely unknown, there is evidence suggesting that local inflammation plays an important role in the process (34
Prostaglandin production and COX-2
mRNA are increased in fracture callus during the first 2 weeks following injury (16
), suggesting a role in the early phase of bone healing. In this study we demonstrated that COX-2 knockout mice had persistence of mesenchymal cells at the fracture site as well as a significant delay of ossification of cartilage tissue. Most remarkable was the reduction in osteoblastogenesis, a finding that was confirmed by two independent in vivo models of intramembranous bone formation as well as by in vitro studies that demonstrated a critical requirement for COX-2 in osteoblastogenesis. These findings demonstrate that the production of COX-2 metabolites during the inflammatory phase is required for efficient bone healing and that mesenchymal cell differentiation is a major target of cyclooxygenase activity.
A more subtle phenotype was the persistence of uncalcified cartilage at the fracture site in COX-2–/–
mice. By evaluating the expression of the established markers of chondrocyte terminal differentiation (colX
), we were able to show that this process still occurs in the absence of COX-2 (Figure and data not shown), but primary bone formation on the cartilagenous template was absent. However, these experiments do not eliminate the possibility of an important effect of COX-2 on chondrocytes during endochondral bone formation in fracture repair. Similarly, the reduced number of osteoclasts in the fracture callus of COX-2–/–
mice suggests a role for prostaglandins in fracture remodeling as suggested by previous studies (4
), but this is a relatively late event in fracture healing that requires additional investigation.
The use of two separate in vivo models of intramembranous bone formation demonstrates that COX-2 plays a role in osteoblast differentiation and bone formation in response to diverse stimuli. In the titanium implantation model, there is an initial period of inflammatory bone resorption, followed by the deposition of new bone. In the second model, recombinant FGF-1 was injected onto mouse calvaria. FGF-1 has been shown to stimulate cellular proliferation, differentiation, and chemotaxis during bone repair (40
), and local and systemic injections have been shown to increase bone formation in both calvaria and long bones (32
). COX-2 was demonstrated to have an important role in bone formation in both of these models, suggesting that it has general importance for reparative events in bone.
Prior work has demonstrated that prostaglandins such as PGE2
induce bone nodules to form in bone marrow stromal cell cultures (6
) and in cultured rat calvaria osteoblasts (43
). Furthermore, systemic or local injection of PGE2
stimulates bone formation in vivo and appears to induce osteoblastogenesis from bone marrow precursors (7
). Ex vivo cultures of bone marrow stromal cells from rats injected with PGE2
for 2 weeks yield four times more mineralized bone nodules compared with cultures from vehicle-injected rats (47
). Finally, Scutt et al. demonstrated that PGE2
increases bone nodule formation in low-density cultures of rat bone marrow cells by recruiting osteoblast precursors from a population of nonadherent mesenchymal precursor cells present in the bone marrow (48
). Our work, using a genetic model based upon marrow cell cultures obtained from COX-1–/–
mice and wild-type littermates, confirms the prior findings and additionally implicates COX-2 as the critical isoform involved in this process. Altogether, the studies suggest that prostaglandins play a role in osteoblast recruitment and differentiation.
The finding that the COX-2 metabolite PGE2
can reverse the decrease in osteoblastogenesis observed in COX-2–/–
marrow cell culture is not surprising given the effect of PGE2
on bone formation both in vitro and in vivo (6
). However, the finding that BMP-2 complements the deficiency in COX-2 and enhances bone nodule formation to a degree similar to that observed in wild-type cultures is particularly interesting and suggest that BMP signaling events may be downstream of COX-2 activity and PGE2
effects. Furthermore, since induction of osteoblastogenesis by BMP-2, including bone nodule formation and cbfa-1
expression, was further enhanced by PGE2
, it appears that BMPs and PGE2
may also have independent and complementary effects. The latter observations are consistent with prior data demonstrating that PGE2
enhances BMP-2–mediated osteoblast differentiation in human periodontal ligament cells (49
Based on these findings, we propose a mechanism of action for COX-2 in bone repair (Figure ). Under basal conditions, COX-2 activity maintains a population of mesenchymal stem cells in a pre-osteoblast state responsive to additional osteoblastic signals. During injury, elevated COX-2 expression increases the osteoblastic potential of mesenchymal stem cells and supports their differentiation to osteoblasts in response to osteogenic signals. COX-2 may exert its effect through regulation of the transcription factors cbfa1 and osterix. Since cbfa-1
is upstream of osterix
, the COX-2–dependent regulation is likely at this earlier stage of differentiation, although the findings do not rule out a direct effect of cyclooxygenase metabolites on osterix
expression. Similarly, our findings showing BMP-2 complementation of COX-2 in vitro support the possibility that BMP signaling events could be downstream of cyclooxygenase metabolites, consistent with the recent demonstration of BMP-7 induction by PGE2
in the osteoblast cell line U2-OS (50
). However, the additive effects of PGE2
and BMP-2 on osteoblastogenesis in our experiments also provide evidence for an independent role of these factors. This model postulates a unique mechanism of COX-2 in bone repair that is significantly different from that in fetal skeletal development. This mechanism may be particularly important in injury or during inflammation where large amounts of PGE2
are produced by COX-2, as opposed to normal skeletogenesis where the role of COX-2 is limited.
Figure 10 Schematic model representing the potential mechanism of COX-2 regulation of mesenchymal cell differentiation in bone repair. In the proposed model, COX-2 is induced in the early phase of the bone reparative process and produces increased amounts of PGE (more ...)
In summary, we demonstrate that reparative bone formation is deficient in COX-2–/– mice and demonstrate a primary defect in osteoblastogenesis. The effect is associated with a decreased expression of two genes essential for osteoblastogenesis, cbfa1 and osterix. Both intramembranous and endochondral bone formation are affected by the absence of COX-2. We further demonstrate that this defect is associated with abnormal mesenchymal cell recruitment and differentiation into osteoblasts. Our study raises concerns regarding the use of COX-2 inhibitors in patients who suffer from bone fractures or who are undergoing other types of bone repair.