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

Villafan‑Bernal,Jose  Rafael; Franco‑de la Torre,Lorenzo; Sandoval‑Rodriguez,Ana  Soledad; Armendariz‑Borunda,Juan; Alcala‑Zermeno,Juan  Luis; Cruz‑Ramos,Jose  Alfonso; Lopez‑Armas,Gabriela; Bastidas-Ramirez,Blanca  Estela; González‑Enríquez,Gracia  Viviana; Collazo‑Guzman,Emerson  Armando; Martinez‑Portilla,Raigam  Jafet; Sánchez‑Enríquez,Sergio

doi:10.3892/etm.2016.3758

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

Authors:
- Jose Rafael Villafan‑Bernal
- Lorenzo Franco‑de la Torre
- Ana Soledad Sandoval‑Rodriguez
- Juan Armendariz‑Borunda
- Juan Luis Alcala‑Zermeno
- Jose Alfonso Cruz‑Ramos
- Gabriela Lopez‑Armas
- Blanca Estela Bastidas-Ramirez
- Gracia Viviana González‑Enríquez
- Emerson Armando Collazo‑Guzman
- Raigam Jafet Martinez‑Portilla
- Sergio Sánchez‑Enríquez
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Affiliations: Young Scientist and Cathedras Department, National Council of Science and Technology (CONACYT), Mexico City 03940, Mexico, Molecular Biology and Genomics Department, University Center for Health Sciences, University of Guadalajara, Guadalajara, Jalisco 44340, Mexico, Institute of Molecular Biology in Medicine and Gene Therapy, University Center for Health Sciences, University of Guadalajara, Guadalajara, Jalisco 44340, Mexico, Cancer Institute of Jalisco, Guadalajara, Jalisco 44280, Mexico, Technical and Industrial Teaching Center (CETI) Colomos, Guadalajara, Jalisco 44638, Mexico, Methodological and Instrumental Disciplines Department, University Center for Health Sciences, University of Guadalajara, Guadalajara, Jalisco 44340, Mexico, Surgery Department, Health Science University Center, Autonomous University of Aguascalientes, Aguascalientes, Aguascalientes 20131, Mexico
Published online on: September 29, 2016 https://doi.org/10.3892/etm.2016.3758
Pages: 3261-3267
Copyright: © Villafan‑Bernal et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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‑β2), 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‑β2, 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.

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