Molecular profiling of a simple rat model of open tibial fractures with hematoma and periosteum disruption

Abstract

Bone fractures are a worldwide public health concern. Therefore, improving understanding of the bone healing process at a molecular level, which could lead to the discovery of potential therapeutic targets, is important. In the present study, a model of open tibial fractures with hematoma disruption, periosteal rupture and internal fixation in 6-month-old male Wistar rats was established, in order to identify expression patterns of key genes and their protein products throughout the bone healing process. A tibial shaft fracture was produced using the three-point bending technique, the hematoma was drained through a 4-mm incision on the medial aspect of the tibia and the fracture stabilized by inserting a needle into the medullary canal. Radiographs confirmed that the induced fractures were diaphyseal and this model was highly reproducible (kappa inter-rater reliability, 0.82). Rats were sacrificed 5, 14, 21, 28 and 35 days post-fracture to obtain samples for histological, immunohistochemical and molecular analysis. Expression of interleukin-1β ( Il-1β), transforming growth factor-β2 ( Tgf-β 2), bone morphogenetic protein-6 ( Bmp-6), bone morphogenetic protein-7 ( Bmp-7) and bone γ-carboxyglutamic acid-containing protein ( Bglap) genes was determined by reverse transcription quantitative polymerase chain reaction and protein expression was evaluated by immunohistochemistry, while histological examination allowed characterization of the bone repair process. Il-1β showed a biphasic expression, peaking 5 and 28 days post-fracture. Expression of Tgf-β 2, Bmp-6 and Bmp-7 was restricted to the period 21 days post-fracture. Bglap expression increased gradually, peaking 21 days post-fracture, although it was expressed in all evaluated stages. Protein expression corresponded with the increased expression of their corresponding genes. In conclusion, a clear and well-defined expression pattern of the evaluated genes and proteins was observed, where their maximal expression correlated with their known participation in each stage of the bone healing process.