Validation of pre-clinical models of musculoskeletal healing following trauma

创伤后肌肉骨骼愈合的临床前模型的验证

• 批准号: 10392328
• 负责人: Jeffry Stephen Nyman
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10392328
• 关键词: Activities of Daily Living; Adult; Affect; Age; Aging; Architecture; Area; Assisted Living Facilities; Bioluminescence; Blood; Blood specimen; Body Weight; Bone Density; Bone Tissue; Bone callus; Burn injury; CCL2 gene; Cartilage; Clinical; Cognition; Cognitive; Collagen; Contralateral; Deposition; Direct Lytic Factors; Dual-Energy X-Ray Absorptiometry; Euthanasia; Evaluation; Failure; Femoral Fractures; Femur; Fibrin; Fibrosis; Finite Element Analysis; Fluorescent Probes; Foundations; Fracture; Future; Goals; Hindlimb; Histology; Homeostasis; Hyperalgesia; Hypersensitivity; Image; Impaired cognition; Impairment; Individual; Inflammation; Inflammatory Response; Injections; Injury; Interleukin-10; Interleukin-13; Interleukin-6; Intervention; Lead; Life; Ligaments; Limb structure; Literature; Luciferases; Measurement; Measures; Memory; Memory impairment; Metabolic dysfunction; Minerals; Modeling; Monitor; Movement; Multiple Fractures; Multiple Trauma; Mus; Muscle; Musculoskeletal; Musculoskeletal Diseases; Natural regeneration; Organ; Osteoporosis; Outcome; Pain; Pathologic Processes; Pharmacology; Phenocopy; Plasma; Pre-Clinical Model; Quality of life; Regulatory Element; Reporter; Reproducibility; Resistance; Running; Severities; Short-Term Memory; Site; Skin injury; Sleep; Sleep Wake Cycle; Soft Tissue Injuries; Structure; TNF gene; Testing; Therapeutic; Thick; Time; Tissue imaging; Tissues; Transgenic Mice; Trauma; Traumatic injury; Validation; Veterans; allodynia; biomechanical test; bone; bone fracture repair; bone fragility; bone loss; bone mass; bone quality; bone strength; calcification; chronic pain; cognitive function; density; ex vivo imaging; experience; functional decline; functional disability; gait examination; healing; improved; in vivo; in vivo imaging; injured; mechanical stimulus; microCT; military service; mouse model; multiplex assay; muscle regeneration; musculoskeletal injury; novel; object recognition; pain perception; pain sensitivity; placebo group; pre-clinical; productivity loss; prognostic indicator; repaired; response; response to injury; severe injury; sex; skin burn; soft tissue; spine bone structure; therapeutic target; tissue repair; vehicular accident; vertebra body

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.

随着衰老，日常生活活动（ADL）的精确损失，功能和维修的下降 肌肉骨骼（MSK）组织是这种损失的重要原因。退伍军人特别容易受到影响 ADL的降低是因为经历严重创伤受伤多个MSK组织的可能性是 服兵役高于平民生活。尽管这些严重的创伤性伤害现在威胁生命 比过去，MSK再生的并发症出现并导致以后辅助生活 生活。这是强烈需要多发性损伤的临床前模型，以促进研究 将较差的MSK修复与更少的ADL连接的机制。文献中存在几种这样的模型，但没有 在与MSK再生受损相关的关键临床问题方面已得到验证， 即延迟骨折愈合，肌肉纤维化或软组织钙化，骨质疏松症，认知障碍， 疼痛敏感性和过度炎症。因此，该项目的目的是验证 损伤的结合 - 皮肤燃烧，下后肢的肌肉损伤以及股骨中轴的断裂 - 目标1，我们将比较康复反应 成人3个损伤组中的转基因小鼠：i）较低的单股骨骨折或单肌损伤 后肢，ii）股骨骨折加单肌损伤，iii）股骨骨折，肌肉损伤和皮肤 烧伤。当NFκB调节元件活跃时，这些转基因小鼠表达荧光素酶，从而允许 我们通过生物发光图像注射组织。为了验证模型，我们将确定骨折是否愈合 在多发性小鼠中，受伤后28天延迟（DPI）和肌肉纤维化持续在42-DPI中。 到单损伤小鼠。在AIM 2中，在AIM 1中使用相同的老鼠，但添加假同窝仔（无伤害），我们将 比较5组损伤对变性的影响：i）假控制，ii）单股骨断裂，iii）单个 下后肢的肌肉损伤，iv）骨和肌肉双重损伤，v）多发性损伤与 皮肤燃烧。从基线开始（受伤前）和伤害后的间隔，面积骨矿物质密度（双能量） 未受伤的股骨，全身体重（质量），认知功能（新物体）的X射线绝对测定法） 识别测试），疼痛敏感性（合金和痛苦刺激的痛苦），睡眠持续时间（笼子 活动）和活动性（自愿车轮运行加步态分析）将被评估，直到42-DPI。另外， 对侧，未受伤的股骨和腰椎身体将通过μCT进行成像，并进行 三分弯曲（股​​骨）或压缩（椎骨）中的负载到失败测试，​​以确定皮质是否是 结构，小梁微体系结构，体积骨矿物质密度和强度不同 组。相对于变性，骨质和骨骼强度，验证多发性多龙的模型 在联合伤害组中，将需要比无伤害组的假手术组更高和更低。在 此外，我们的另一个验证目标是证明MSK对骨骼和肌肉的损伤与皮肤损伤 燃烧还会损害记忆，导致疼痛过敏，减少睡眠时间并减少运动 因为这些是多发性多元的临床并发症。在AIM 3中，将从安乐死收集血液样本 AIM 1（28-DPI和42-DPI）中的小鼠以及早期的NFκB报告者小鼠（1-） DPI，3-DPI，7-DPI和14-DPI），以便我们可以量化超炎性反应的临时变化 受伤。在进行安乐死之前，我们将在体内对所有小鼠进行成像，以评估组织感染，以及安乐死之后， 我们将在体内映像单个器官。为了验证模型，全身性超炎和组织 在多发性组合组中，炎症将比单损伤组发生更长的时间。 有了经过验证的多发性鼠模型，该模型具有许多与差有关的临床问题 肌肉骨骼组织的再生，未来的研究可以识别和研究机制，治疗 策略和预测者将随着年龄的增长而帮助退伍军人保持日常生活的活动。