Validation of pre-clinical models of musculoskeletal healing following trauma

Veterans Health Administration, Decatur PA

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Abstract

With aging, there is a precipitous loss in activities of daily living (ADLs), and a decline in the function and repair of musculoskeletal (MSK) tissues is a significant contributor to this loss. Veterans are particularly vulnerable to a reduction in ADLs because the likelihood of experiencing severe trauma that injuries multiple MSK tissues is higher in military service than in civilian life. While these severe traumatic injuries are less life-threatening now than in the past, complications of MSK regeneration arise and lead to the requirement of assisted living later in life. Thus, there is a strong need for preclinical models of polytrauma injury that facilitate studies into the mechanisms connecting poor MSK repair to fewer ADLs. Several such models exist in the literature, but none have been validated with respect to the key clinical problems associated with impaired MSK regeneration, namely delayed fracture healing, muscle fibrosis or soft tissue calcification, osteoporosis, cognitive impairment, pain sensitivity, and hyper-inflammation. Therefore, the goal of this project is to validate a murine model of combined injuries – skin burn, muscle injury to the lower hindlimb, and fracture of the femur mid-shaft – for its ability to recapitulate many of the problems that reduce ADLs. In Aim 1, we will compare the healing response of adult, transgenic mice among 3 injury groups: i) single femur fracture or single muscle injury in the lower hindlimb, ii) femur fracture plus single muscle injury, and iii) combined femur fracture, muscle injury, and skin burn. These transgenic mice express luciferase when the NFκB regulatory element is active, thereby allowing us to image tissue inflammation by bioluminescence. To validate the model, we will determine if fracture healing is delayed at 28-days post injury (DPI) and muscle fibrosis persists at 42-DPI in the polytrauma mice compared to the single injury mice. In Aim 2, using the same mice in Aim 1 but adding sham littermates (no injury), we will compare the effect of injury on degeneration among 5 groups: i) sham control, ii) single femur fracture, iii) single muscle injury in the lower hindlimb, iv) dual injury to bone and muscle, and v) combined polytrauma injury with skin burn. Starting at baseline (before injury) and at post-injury intervals, areal bone mineral density (dual-energy X-ray absorptiometry) of the uninjured femur, whole-body weight (mass), cognitive function (novel object recognition test), pain sensitivity (allodynia and hyperalgesia to mechanical stimuli), duration of sleep (cage activity), and mobility (voluntary wheel running plus gait analysis) will be assessed until 42-DPI. Additionally, the contralateral, uninjured femur and the lumbar vertebral body will be imaged ex vivo by μCT and subjected to load-to-failure tests in three-point bending (femurs) or in compression (vertebra) to determine whether the cortical structure, trabecular micro-architecture, volumetric bone mineral density, and strength are different among the groups. To validate the model of combined polytrauma with respect to degeneration, bone loss and bone strength will need to be higher and lower in the combined injury group than in the no-injury, sham group, respectively. In addition, our other validation goal is to demonstrate that the combined MSK injury to bone and muscle with skin burn also impairs memory, causes hypersensitivity to pain, reduces duration of sleep, and reduces movement as these are clinical complications of polytrauma. In Aim 3, blood samples will be collected at euthanasia from the mice in Aim 1 (28-DPI and 42-DPI) as well as from additional NFκB reporter mice at earlier time points (1- DPI, 3-DPI, 7-DPI, and 14-DPI) so that we can quantify the temporal changes in the hyper-inflammatory response to injury. Prior to euthanasia, we will image all mice in vivo to assess tissue inflammation, and after euthanasia, we will image individual organs ex vivo. To validate the model, systemic hyper-inflammation and tissue inflammation will occur for a longer period time in the polytrauma combined group than in the single injury group. With a validated murine model of polytrauma that possesses many of the clinical problems associated with poor regeneration of musculoskeletal tissue, future studies can identify and investigate mechanisms, therapeutic strategies, and prognosticators that will help Veterans maintain activities of daily living as they age.