During fracture healing, there is a sequential series of highly linked events that include mesenchymal proliferation, chondrogenesis, chondrocyte maturation and terminal differentiation, vascularization, primary bone formation, and remodeling.(37
) Because these events are tightly linked, cellular and molecular alterations that occur early during the healing response may result in an alteration in the timing of subsequent events. Thus, the early cell, tissue, and molecular events that occur immediately after bone injury have particular importance.
The phenotype of fracture repair in aged mice is consistent with altered early gene expression. Compared with young mice, aged mice have delayed formation of callus and chondrogenesis, suggesting a decrease in the rate of proliferation and differentiation of mesenchymal chondroprogenitors. Maximal expression of col2a1
occurred later, and gene expression persisted for a longer duration. ColX
, a marker of mature chondrocytes,(38
) was maximal at 7–10 days in fractures from young mice and at day 10 in fractures at aged mice. Furthermore, fractures in aged mice had reduced peak levels of colX
expression and expression persisted through 18 days, showing failure to efficiently complete endochondral bone formation. Mature, calcified cartilage acts as a template for bone formation and remodeling. Consistent with the delay and prolongation of the cartilage phase of fracture repair, osteocalcin
gene peak expression was reduced and expression also persisted in aged callus. Finally, vascularization and fracture union were delayed and the rate of remodeling was reduced in aged fractures. These findings are consistent with previous descriptions of delayed fracture healing in older mice and rats.(7
COX-2 has been shown to be a critical regulator of bone repair in multiple animal models.(19
expression is rapidly induced in fractures, consistent with our observation of expression in 3 day calluses, with peak expression by 5 days in young mice. COX-2–deficient mice have a fracture healing phenotype that is similar to aged mice, including a slower rate of chondrogenesis, chondrocyte maturation, delayed vascularization, and reduced bone formation. In COX-2−/−
mouse fractures, there is persistence of undifferentiated mesenchyme, suggesting that COX-2 is necessary for normal bone and cartilage differentiation in healing tissues.(19
In vitro studies have confirmed that COX-2 and its metabolite, PGE2
, enhance the differentiation of MSCs into both cartilage and bone.(19
) Addition of PGE2
to limb bud MSCs in high-density cultures enhances chondrogenesis.(23
) This has been observed in primary avian and murine cultures and mesenchymal cell lines and involves signaling through PKA.(23
) Similarly, PGE2
has been shown to stimulate osteoblast differentiation.(19
) Bone marrow MSC cultures isolated from COX-2−/−
mice have reduced osteoblast differentiation. However, this can be compensated by the addition of PGE2
to the cultures, suggesting that PGE2
is a key metabolite in osteoblast differentiation.(19
Whereas a role for COX-2 in bone repair has been extensively described, the cell populations responsible for COX-2 expression in fracture callus have not been defined. In situ hybridization experiments showed co-localization of COX-2 and col2a1 in the early chondroprogenitor population. Morphologically, these were flattened fibroblastic-appearing mesenchymal progenitors in the process of becoming embedded within a chondroid matrix. COX-2 expression was also present in immature chondrocytes but was absent in cells with a hypertrophic phenotype. Interestingly, COX-2 expression is absent in the growth plate, consistent with lack of a developmental phenotype in COX-2−/− mice. Thus, the expression of COX-2 seems to be unique to reparative cartilage. COX-2 expression was also present in the osteoblast population, although gene expression studies suggest that the highest levels of expression were associated with the initial cartilage phases of endochondral bone repair. Whereas prior work has established that osteoblasts express COX-2, these are the first experiments to show that cartilage is a major source of COX-2 during fracture repair. COX-2 expression was reduced in the fracture callus of aged mice, suggesting that the delayed healing in these mice may be caused by a functional decrease in this enzyme.
PGE-2 is the major metabolite of COX-2 in most tissues and activates one of four receptors, EP1, EP2, EP3, and EP4, that collectively are associated with the protein kinase C (PKC) and PKA signaling pathways. Numerous genes have been shown to be activated by PGE2
, including BMP-2
, two critical factors in bone repair.(20
) In young fractures, BMP-2
both have peak expression during the early cartilage period of fracture repair and are expressed in phase with COX-2
. EP2 and EP4 agonists have been shown to enhance bone formation in several bone repair models and may have anabolic effects when combined with BMP-2 therapy.(24
) In an ectopic bone formation model, BMP-2 and selective EP4 agonists had a synergistic effect.(56
) In rat growth plate chondrocytes, combined activation of EP2 and EP4 receptors resulted in enhanced proliferation and induction of col2a1
) The observation that both anabolic and remodeling genes have peak expressions during the early chondrogenic period of fracture repair further supports the notion that initial cellular and molecular events drive the overall repair process.
Aged mice have reduced and temporally prolonged expressions of both BMP-2
is regulated by COX-2 and PGE2
) Undifferentiated human MSCs constitutively express more COX-2, PGE2
, and BMP-2 than mature osteoblasts.(49
) When treated with selective COX-2 and EP4 inhibitors, the induction of BMP-2 in these cells was suppressed.(49
) Similarly RANKL
is also regulated by PGE2
) Osteoblasts, stromal cells, and fibroblasts all express increased RANKL
after treatment with PGE2
) This effect is primarily caused by activation of the EP4 receptor, which activates the PKA signaling pathway. Gain of EP4 function stimulated RANKL
expression, whereas loss of function prevents RANKL
expression in PGE2
has been shown to be expressed in chondrocytes, and our laboratory recently has established that BMP-2 signaling is a potent inducer of RANKL
expression in chondrocytes.(60
) However, direct induction of RANKL
has not been examined in a chondrocyte population. Altogether, regulation of BMP-2
is consistent with an early gene that regulates critical subsequent steps including cell differentiation and subsequent remodeling.
The likely source of the mesenchymal precursor population is the periosteum. Prior work from our laboratory using a murine bone/periosteal cell transplant model clearly established that periosteal cells undergo proliferation and subsequent chondrogenesis in response to injury.(25
) In a murine model in which PGE2
was delivered to the periosteal surface through an Alzet pump, periosteal bone formation occurred in wildtype and EP1, EP2, and EP3 knockout mice, but not in EP4-deficient mice.(26
) This suggests that the periosteal stem cell population is particularly responsive to EP4 signaling. For this reason, we examined whether local delivery of an EP4 receptor agonist could compensate for the reduced rate of fracture repair observed in aged mice.
Local injection of an EP4 agonist to the fracture site of aged mice compensated for the reduced fracture repair observed with aging. Day 14 fractures were selected for detailed analysis because this is the time point in which fractures in young animals are completing the final stages of endochondral ossification and only the final vestiges of hypertrophic cartilage remain. In contrast, aged mice continued to have abundant immature cartilage. Fractures receiving the EP4 agonist had a significant reduction in both immature and hypertrophic cartilage and more efficient completion of endochondral ossification. This effect was readily observed at day 14, when endochondral bone formation is being completed in young mice and fracture union is occurring. As a result, EP4 agonist-treated fractures in aged mice had increased bone formation and developed a histological phenotype that was similar to that observed in young mice. Our gain of function findings are consistent with prior work showing that EP4 knockout mice have delayed endochondral bone formation and fracture healing.(36
A second finding observed in aged mice with fractures treated with the EP4 agonist was that the total amount of fracture callus was increased in mice treated with CP73 for 21 days. Unlike the observations at 14 days with cartilage maturation, the increase in callus area and bone formation occurred in comparison with both vehicle-treated young and aged mice with fractures. One possible explanation for these findings is that, in the later phases of fracture repair, CP73 may have additional effects on the osteoblast population that results in increased total bone formation and callus area. Thus, once endochondral bone has formed, EP4 may have an additional role in the proliferation, recruitment, or retention of osteoblastic cell populations involved in fracture repair. Prior work has established that EP4 stimulates osteoblast differentiation and matrix production.(26
) Furthermore, EP4 knockout mice were observed to have reduced bone with aging and decreased osteoblastogenesis, consistent with an important role in bone formation.(36
This study focused on the expression pattern of COX-2 in fractures, the reduction in COX-2 expression in fractures in a model of aging, and the potential of COX-2/EP4 gain of function to compensate for and accelerate fracture repair in the setting of aging. The work did not address the issue of whether an EP4 receptor agonist has the potential to stimulate repair in young mice, and this remains an important issue. The molecular events associated with nonunions in young subjects are not understood and it is possible the EP4 receptor gain of function may have a role in promoting normal and delayed fracture repair in young individuals.
The experiments used only a single dose of the EP4 agonist, CP73. CP73 has previously been shown to restore trabecular bone mass and strength in ovariectomized rats.(63
) Based on the doses used in rats, we chose a daily injection of 20 mg/kg/d, which represents the maximal effects in these animal models. Because the goal of these experiments was to show the concept that a EP4 gain of function can compensate for the reduced rate of fracture healing observed in aged mice, experiments designed to determine the relative potency of different concentrations of CP73 were not completed. Similarly, we did not study the relative potential of other factors, such as BMP-2 or PTH, to compensate for reduced fracture healing in the aging model. PTH and BMP-2 are important and perhaps overlapping or integrated pathways that have been shown to regulate bone repair.(29
) Similar to EP4, the PTH receptor is a G-coupled protein receptor that activates the protein kinase A signaling pathway.(65
) Whereas these agents may have an important role in fractures in aging, the current studies focused on the role of EP4 receptor signaling. Future studies will need to determine the relative effectiveness of these and other agents.
Altogether, the experiments showed that the impaired fracture healing with aging involves essentially all stages of the process and suggest that altered expression of early genes involved in fracture repair affect the entire healing cascade. These findings define COX-2/EP4 signaling as an important potential therapeutic target to improve fracture healing in the aging population.