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Purpose The classic fracture model, described by Bonnarens and Einhorn in 1984, enlists a blunt guillotine to generate a closed fracture in a pre-stabilized rodent femur. However, in less experienced hands, this technique yields considerable variability in fracture pattern and requires highly-specialized equipment. This study describes a reproducible and low-cost model of mouse fracture healing using an open femoral osteotomy. Methods Femur fractures were produced in skeletally mature male and female mice using an open femoral osteotomy after intramedullary stabilization. Mice were recovered for up to 28 days prior to analysis with microradiographs, histomorphometry, a novel μCT methodology, and biomechanical torsion testing at weekly intervals. Results Eight mice were excluded due to complications (8/193, 4.1%), including unacceptable fracture pattern (2/193, 1.0%). Microradiographs showed progression of the fracture site to mineralized callus by 14 days and remodelling 28 days after surgery. Histomorphometry from 14 to 28 days revealed decreased cartilage area and maintained bone area. μCT analysis demonstrated a reduction in mineral surface from 14 to 28 days, stable mineral volume, decreased strut number, and increased strut thickness. Torsion testing at 21 days showed that fractured femurs had 61% of the ultimate torque, 63% of the stiffness, and similar twist to failure when compared to unfractured contralateral femurs. Conclusions The fracture model described herein, an open femoral osteotomy, demonstrated healing comparable to that reported using closed techniques. This simple model could be used in future research with improved reliability and reduced costs compared to the current options., Highlights • This study characterized a simple and reproducible model of fracture healing in mice using an open femoral osteotomy. • Analysis by x-ray, histomorphometry, µCT, and biomechanical testing demonstrated healing comparable to current models. • This simple model could be used to increase investigation into fracture healing, delayed union, and non-union.

Publisher Summary The protein kinase inhibitor (PKI) family consists of three proteins (PKIα, PKIβ, and PKIγ), all of which are potent and specific inhibitors of cAMP-dependent protein kinase (PKA). In response to a stimulus that increases the intracellular cAMP level, PKA-C is released from the regulatory subunit (PKA-R), translocates into the nucleus, and regulates gene expression by phosphorylating transcription factors such as CREB. PKI also translocates into the nucleus, inactivates PKA-C by binding to it, and then translocates PKA-C out of the nucleus. This translocation out of the nucleus is due to interactions between the nuclear export signal (NES) motif found in all three members of the PKI family and the leptomycin-sensitive nuclear export machinery. However, translocation out of the nucleus is not necessary for PKI to terminate PKA-C signaling in the nucleus, as binding to PKI is sufficient. It is important to note that PKI may also have important functions in regulating PKA-C activity in the cytoplasm of cells. Short-term knockdown of PKI, or other proteins, can be achieved by either siRNA or antisense approaches, either of which can be applied to cells as oligonucleotides or by expression of gene constructs. Activation of Gs-coupled receptors also induces a transient anti-apoptotic effect in osteoblasts that depends on PKA signaling. It demonstrates that endogenous levels of PKI are sufficient to terminate physiological effects of PKA-C in the nucleus following stimulation of cAMP levels by Gs-coupled receptors.