The goal of this current study is to examine the role of COX-2 in the initiation of cortical bone healing. We took the advantage of a segmental bone graft transplantation model, which allows a mix-and-match approach to examine the phenotype of WT or COX-2-/- donor periosteal progenitors in a WT or COX-2 deficient host injury environment during the early phase of healing. Our data provides strong evidence to show that COX-2, produced by infiltrating inflammatory cells and early periosteal progenitors, functions as an essential modulator in host injury milieu directing the efficient initiation of cortical bone healing.
COX-2 is critically implicated in fracture healing (30
). In COX-2-/-
mice, fracture healing is delayed due to impaired mesenchyme differentiation and delayed transition of cartilage to bone. High incidence of non-union is found in these mice due to impaired chondrogenesis and osteogenesis. Compared to fracture healing, segmental bone graft transplantation represents a more challenging endochondral bone repair model, in which a large critical defect must be repaired. As expected, when graft transplantation was performed in the KO mice, a more severe phenotype was observed. Histologic analyses showed that donor progenitor cell-initiated bone formation was nearly abolished in KO-to-KO transplantation. As a result, more than 90% of the cortical bone junctions failed to reach histological union in COX-2 knockout host as compared to only 22% in WT-to-WT transplantation. These results underscore the importance of COX-2 in cortical bone healing and provide strong evidence to show that COX-2 plays a key role during the initiation phase of cortical bone healing.
To further determine the host vs. donor contribution to the healing, we performed graft transplantation between WT and KO mice. This approach allows mutant progenitor cells to be placed in a WT host or vise versa. Interestingly, our data demonstrated that when a live WT graft with intact periosteum was transplanted into COX-2-/- mice, donor periosteal progenitor cell initiated chondrogenesis and bone formation were abolished. On the contrary, when a COX-2-/- graft was placed into WT mice, donor periosteum initiated bone formation and angiogenesis were markedly enhanced. These data strongly suggest that host plays a predominant role in activation and initiation of periosteal endochondral and intramembraneous bone formation. COX-2, likely functions through its metabolites such as prostaglandins, is required in early host injury milieu for effective initiation of chondrogenesis and osteoblastogenesis.
The early blockade of progenitor cell differentiation in the COX-2-/-
host suggests that COX-2 produced during inflammatory phase of healing by host infiltrating cells and progenitors plays a key role. RT-PCR analyses showed that COX-2 was induced as early as day 5 and peaked on day 7, paralleled with Col2a1
expression. Using immunohistochemical staining for COX-2, we were able to localize COX-2 protein in infiltrating inflammatory cells at the cortical bone junction as early as day 3. More intensified staining was found in the cells surrounding the hematoma. In addition to sporadic staining in soft tissue callus, we also found intense staining of COX-2 in chondroprogenitor cells at day 5, which correlated with the induction of COX-2 mRNA expression. These data corroborate with our previous finding in fracture healing model, in which COX-2 was found in infiltrating cells as well as in chondroprogenitors and proliferating chondrocytes in the early fracture callus (25
). The localization of COX-2 in early infiltrating cells and mesenchymal progenitors during inflammatory phase of healing show that COX-2 plays a key role in modulating early inflammatory response and coordinating the activation and initiation of progenitor cell-dependent endochondral and intramembraneous bone formation.
The high percentage of fibrotic tissue and high incidence of fibrotic nonunion in COX-2-/-
mice indicate a potential role of COX-2 in modulating fibrotic tissue formation and inflammatory response during early stage of bone healing. Experiments transplanting Rosa-LacZ mice into COX-2-/-
mice showed that the fibrotic tissues surrounding donor bone graft was derived from the host (data not shown) suggesting that COX-2 produced from the surrounding inflammatory tissues in the host is required for suppression of fibrotic tissue formation during healing. PGE2 has been known to suppress the growth of fibroblasts (4
mice develop fibrosis in multiple organs (5
). In COX-2-/-
mice, fibrotic response following lung injury is increased accompanied by an enhanced and persistent inflammatory response to injury (17
). The studies using COX-2-/-
mice further show that COX-2 derived prostaglandins promote early microphage infiltration but also restrain the inflammatory response at the late stage thereby are involved in the resolution of exacerbated inflammation (5
). Additionally, one recent study show that COX-2 is involved in modulating early inflammatory response during muscle healing by counteracts TGF-β induced fibrotic response (29
). Based on these findings, we deduce that COX-2 plays an intricate role modulating the inflammatory response during the initiation of bone healing. On one hand, COX-2/PGE2 stimulates bone progenitor cell proliferation and differentiation. On the other hand, COX-2/PGE2 suppresses fibrotic response associated with injury and healing. Lack of COX-2 in the host disrupts the intricate balance in host injury milieu, resulting in detrimental effects on activation and differentiation of donor periosteal stem/progenitor cells. Restoration of balanced host injury milieu re-establishes the initiation of donor graft healing and repair.
Aside from deficiency in the initiation of graft healing, careful histomorphometric studies by quantifying percent bone and cartilage formation in graft transplantation experiments also revealed that COX-2-/-
donor graft displayed a delayed mineralization and neovascularization in a wild type host, similar to what we have observed in the COX-2-/-
fracture callus (30
). Interestingly, the delayed cartilage ossification was incompletely restored in wild type host (KO-to-WT transplantation), as evidenced by higher percentage of cartilage tissue on the bone graft and relatively lower vessel volume when compared to WT-to-WT transplantation. This data argue that COX-2 and its metabolites play a role in endochondral ossification and neovascularization in donor chondrocytes, presumably through an autocrine mechanism of prostaglandins. The effect of COX-2 on cartilage tissue turnover may involve a cell autonomous mechanism that cannot be completely corrected by WT host environment. Further studies are needed to address the mechanisms involved in cartilage turnover and neovascularization.
Induction of COX-2 is the hallmark of inflammatory response following tissue injury (36
). Anti-inflammatory drugs NSAIDs suppress production of prostaglandins via suppression of COX-2. NSAIDs have been shown to delay or impair skeletal healing in human and animal models (2
). Several recent studies further show that COX-2 inhibitor when administered at the inflammatory phase significantly perturbs fracture healing in rats (11
). Previous study from our laboratory, in which we used a similar allograft transplantation model in mice, have demonstrated that the use of NSAIDs, including inhibitor of COX-2 at the early stage of healing leads to increased incidence of nonunions (27
). Using COX-2-/-
mice, our current study provides further evidence for a critical role of COX-2 in the early inflammatory phase of healing. Taken together, our data argue against the use of high dose of NSAIDs or COX-2 inhibitors during the initiation stage of skeletal repair and reconstruction.
In summary, we have demonstrated that COX-2 in injury milieu is critical for initiation of donor bone graft healing. In the absence of COX-2, the activation, proliferation and differentiation of donor periosteal progenitor cells are markedly impaired, leading to high incidence of fibrotic nonunion in bone graft transplantation. This study suggests that suppression of COX-2 at the initiation stage of healing could lead to negative effects on bone healing and repair.