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Role of c-Met in Abnormal Bone Homeostasis Leading to Fracture Non-union

  • Author / Creator
    Guoju Hong
  • Fracture healing involves a fragile continuum from inflammation to repair and finally ends in bone reestablishment. Fractures that fail to heal progress to a state of nonunion, causing sub-stantial morbidity for the individual. Nonunion is one of the most serious complications of frac-ture. In an updated investigation, the incidence of nonunion in fractures was found to be 1.9%; however, the value can be as high as 9% in specific fracture types, such as tibial and clavicular fractures. Nonunion results in major living and economic burdens on patients and seriously af-fects their mobility. Currently, no effective medical treatments are available to promote fracture repair, and the mainstay of fracture nonunion management is surgery. Infection, ageing, and poor blood supply have been implicated in nonunion. However, the exact mechanism of fracture nonunion is unknown.
    In recent studies, it has been shown that HGF/c-Met are closely related to the differentia-tion of osteoblasts and osteoclasts, which are the prominent cells responsible for callus for-mation and bone remodelling during fracture repair. Furthermore, hypoxia caused by poor vas-cularization in fracture healing interrupted osteogenesis in vivo and impaired osteoblast and oste-oclast differentiation in vitro. There is also evidence that hypoxia regulates c-Met in many cell types. We hypothesized that hypoxia modulates osteoblast and osteoclast differentiation via c-Met and ultimately causes fracture nonunion.
    To validate our hypothesis, we performed three types of experiments. First, human bone samples obtained from participants with fracture union and nonunion were collected to determine the expression of c-MET. The results show that c-MET expression is downregulated in fracture nonunion tissues such as callus and the greater trochanter. In serum and bone samples of pa-tients with fracture nonunion, key osteoblastic and osteoclastic markers were also suppressed.
    To further explore the mechanism of c-Met in the regulation of osteoblasts and osteoclasts, we generated c-Metfl/fl mice possessing loxP sites flanking exon 16 of the c-Met sequence. Re-moving exon 16 results in the loss of the c-Met catalytic domain and the mice are thus knockout mice. We then crossed the c-Metfl/fl mice with Prx1cre mice (to target expression in osteoblast progenitors) or Ctskcre mice (to target expression in osteoclast precursors) and investigated dif-ferences in fracture healing following a proximal femur osteotomy. Our findings suggest that conditional knockout of c-Met in both cell lines does not cause abnormal bone development. However, we observed impaired osteoblast and osteoclast differentiation after birth, subsequent-ly leading to osteopenia (Prx1cre c-Metfl/fl) and increased bone formation (Ctskcre c-Metfl/fl). Knockout of c-Met also delayed the process of fracture healing in the gene-edited mice following fracture surgery.
    Finally, cell models were established to explore if c-Met regulates osteogenesis under hy-poxic conditions or following treatment with cabozantinib (a c-Met kinase activity inhibitor). Our findings show that hypoxia increases c-Met phosphorylation during the differentiation of MC3T3 (osteoblast progenitors) and RAW264.7 (osteoclast precursors) cells. However, the dif-ferentiation of neither cell type was affected after treatment with cabozantinib. Thus we conclude that enhanced phosphorylation of c-Met in hypoxia is not related to the inhibition of osteoblast and osteoclast differentiation.
    To summarize, our findings indicate that c-Met is a molecular driver of osteoblast and os-teoclast differentiation and that the knockout of c-Met results in delayed fracture union in mice. In addition, we did not find evidence to link c-Met and hypoxia from tissue ischaemia to im-paired fracture repair.

  • Subjects / Keywords
  • Graduation date
    Spring 2022
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-51h5-2220
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.