Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing

Abstract

Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1Rflox/flox/2.3-kb α1(1)-collagen-Cre (KO) and IGF1Rflox/flox (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by μCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation during fracture repair, but it plays an important role in coordinating chondrocyte, osteoclast, and endothelial responses that all contribute to the endochondral bone formation required for normal fracture repair.

Analysis of the mechanism by which nerve growth factor promotes callus formation in mice with tibial fracture

“…TRAP is an osteoclast characteristic enzyme. Degradation of the cartilage matrix is closely associated with the osteoclast absorption effect on the cartilage matrix (16). A large number of cartilage and mesenchymal tissues were evident in the middle and late stage of fracture healing because the COL2A1 expression of the chondrocyte marker gene in the wild group was significantly lower than that of the transgenic group (17).…”

The osteogenic potential of human bone callus

“…Osteogenic growth factors, mainly secreted by osteoblasts19, also reach peak concentrations at early time points202122. As osteoblasts and osteogenic factors peak, callus formation and bone healing is initiated232425. Specifically, once fracture occurs, mesenchymal stem cells are recruited to the wound site and differentiate into osteoblasts, which are responsible for secreting growth factors to induce osteogenic differentiation26.…”

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice

“…The downward middle test probe, positioned exactly at the midshaft position, was controlled by an automated actuator, which retrieved the test probe immediately after the bone was fractured (as detected by an accelerated downward probe movement) to minimize soft tissue damage. Using expected means and standard deviations derived from our previous studies (31) and an estimated power (1-β) of 80%, the sample sizes required to detect a difference in μCT, static, and dynamic skeletal parameters were estimated to be 8 mice/group for comparisons between KO versus Cont mice and 15 mice/group for comparisons among mice treated with or without PTH and/or R568. S2B).…”