Date Published: July 31, 2007
Publisher: Public Library of Science
Author(s): Yan Chen, Heather C Whetstone, Alvin C Lin, Puviindran Nadesan, Qingxia Wei, Raymond Poon, Benjamin A Alman, William Horton
Abstract: BackgroundDelayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The β-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of β-catenin signaling in bone repair.Methods and FindingsWestern blot analysis showed significant up-regulation of β-catenin during the bone healing process. Using a β-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that β-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/β-catenin pathway) inhibited β-catenin signaling and the healing process, suggesting that WNT ligands regulate β-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized β-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific β-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of β-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated β-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture.ConclusionsThese results demonstrate that β-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of β-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, β-catenin positively regulates osteoblasts. This is a different function for β-catenin than has previously been reported during development. Activation of β-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage.
Partial Text: Fracture healing is a complex regenerative process initiated in response to injury, which in the optimal case results in restoration of skeletal function. In the initial phase of fracture repair, undifferentiated mesenchymal cells aggregate at the site of injury, proliferate, and differentiate, presumably in response to growth factors produced by the injured tissues . This process involves both intramembranous and endochondral ossification. Intramembranous ossification involves the formation of bone directly from committed osteoprogenitor cells and undifferentiated mesenchymal cells that reside in the periosteum, resulting in hard callus formation [2,3]. During endochondral ossification, mesenchymal cells differentiate into chondrocytes, producing cartilaginous matrix, which then undergoes calcification and eventually is replaced by bone. The formation of primary bone is followed by extensive remodeling until the damaged skeletal element regains original shape and size. These processes are reminiscent of embryonic bone development, suggesting that fracture repair recapitulates normal bone development [4–6]. When fracture healing is impaired, osteoblastic differentiation is inhibited, and undifferentiated mesenchymal tissue remains at the fracture site. In patients, this outcome results in delayed union, or nonunion, usually requiring additional surgery for successful fracture healing. In certain surgeries, such as spinal fusion surgery or total joint replacement, a lack of bone ingrowths results in a failed outcome and the need for additional surgery. A pharmacologic adjunct to improve bone healing has the potential to avoid the need for additional surgery in these cases, improving patient outcomes.
In this study, we demonstrated that β-catenin signaling plays a crucial role in fracture healing. We observed that precise regulation of β-catenin is important in the early phases of fracture healing to allow differentiation of mesenchymal cells into osteoblast and chondrocyte lineages. Our findings also indicate that β-catenin plays a disparate role in undifferentiated mesenchymal cells and in committed osteoblasts, and as such acts differently during different phases of fracture repair. These findings are important not only for understanding the role of β-catenin in different cell types, but also has a practical implication for therapy—pre-fracture lithium treatment inhibits the repair process, but post-fracture treatment enhanced bone healing. As such, lithium will enhance fracture healing, but only if started after cells have become committed to the osteoblast lineage.