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.

随着年龄的增长，日常生活活动能力（ADL）急剧下降，功能和修复能力下降， 肌肉骨骼（MSK）组织的损伤是这种损失的重要原因。退伍军人特别容易受到 ADL降低，因为经历严重创伤损伤多个MSK组织的可能性是 在军队服役比在平民生活中更高。虽然这些严重的创伤现在不那么危及生命 与过去相比，MSK再生的并发症出现，并导致以后需要辅助生活。 生活因此，强烈需要有助于研究多发性创伤损伤的临床前模型。 将MSK修复不良与ADL减少联系起来的机制。文献中有几种这样的模型，但没有一种 已经在与受损的MSK再生相关的关键临床问题方面得到验证， 即骨折愈合延迟、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，本项目的目标是验证小鼠模型， 复合损伤-皮肤烧伤，下肢肌肉损伤和股骨中段骨折-对于其 能够概括许多降低ADL的问题。在目标1中，我们将比较 3个损伤组中的成年转基因小鼠： 后肢，ii）股骨骨折+单一肌肉损伤，以及iii）股骨骨折、肌肉损伤和皮肤损伤 节省大量每天往返于这些转基因小鼠在NFκB调节元件激活时表达荧光素酶，从而允许 让我们通过生物发光对组织炎症进行成像。为了验证模型，我们将确定骨折愈合是否 在多发性创伤小鼠中， 对单次损伤的小鼠。在目标2中，使用与目标1相同的小鼠，但添加假同窝小鼠（无损伤），我们将 比较5组中损伤对退变的影响：i）假手术对照，ii）单次股骨骨折，iii）单次股骨骨折， 下肢肌肉损伤，iv）骨和肌肉双重损伤，以及v）合并多发性创伤损伤， 皮肤灼伤。从基线（损伤前）和损伤后开始， 未受伤股骨的X线吸收测定法）、全身重量（质量）、认知功能（新对象 识别测试）、疼痛敏感性（对机械刺激的异常性疼痛和痛觉过敏）、睡眠持续时间（笼 活动）和移动性（自主轮跑加步态分析）将进行评估，直至42-DPI。另夕h 将通过μCT对侧未受伤的股骨和腰椎体进行离体成像，并进行 三点弯曲（股骨）或压缩（椎骨）载荷失效试验，以确定皮质骨 骨结构、骨小梁微结构、体积骨矿物质密度和强度在不同年龄组之间存在差异。 组验证多发伤复合伤模型的退变、骨丢失和骨强度 复合伤组的血药浓度应分别高于和低于非损伤假手术组。在 此外，我们的另一个验证目标是证明MSK对骨骼和肌肉的联合损伤与皮肤 烧伤还损害记忆，引起对疼痛的过敏，减少睡眠时间，减少运动 因为这是多发性创伤的临床并发症在目标3中，将在安乐死时采集血液样本， Aim 1中的小鼠（28-DPI和42-DPI）以及来自其他NFκB报告小鼠的小鼠在较早时间点（1- 24小时）。 DPI、3-DPI、7-DPI和14-DPI），以便我们可以量化过度炎症反应的时间变化 受伤。在安乐死之前，我们将对所有小鼠进行体内成像以评估组织炎症，并且在安乐死之后， 我们将在体外对单个器官成像。为了验证该模型，系统性过度炎症和组织 多发伤复合伤组炎症反应持续时间较单纯伤组长。 用一个经过验证的多发性创伤小鼠模型，该模型具有许多与不良反应相关的临床问题， 再生的肌肉骨骼组织，未来的研究可以确定和调查机制，治疗 策略，和辅助工具，这将有助于退伍军人保持日常生活活动，因为他们的年龄。