Bone Abnormalities & Healing Defect in Muscular Dystrophy

描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种退行性肌肉疾病，其特征是肌营养不良蛋白缺乏表达，最终导致心脏或呼吸衰竭。DMD患者还会出现骨量减少、脆性骨折和脊柱侧弯，这表明DMD患者也存在骨骼系统动态平衡缺陷。据推测，这些骨骼异常很可能是肌肉丧失(骨质疏松症)的次要后果；然而，目前还不清楚它们是否可能是由于直接的内在原因。 骨骼缺陷。最近出现的证据表明，成人干细胞功能障碍与DMD的组织病理发生有关。从Dystrophin/utroin双基因敲除(DKO)小鼠(DMD的一种严重动物模型)分离的肌源性祖细胞(MPC)被发现其增殖和分化能力存在缺陷。我们和其他人已经报道，这些dKO小鼠的骨骼、关节软骨和椎间盘出现了一系列退行性变化，并经历了脊柱畸形、异位骨化、心肌病和寿命缩短，所有这些都支持在这个小鼠模型中出现肌肉骨骼过早衰老的表型。在这些小鼠中也观察到了骨愈合缺陷；然而，目前尚不清楚这种缺陷是内在的骨愈合问题还是与骨质疏松症的次要影响有关(目标1)。初步证据支持在这些小鼠的MPC和间充质干细胞(MSCs)中都存在成体干细胞缺陷，支持这样一种理论，即异常的骨愈合可能是成体干细胞室自主缺陷的结果。因此，该项目的第二个目标将是进一步验证在不同年龄分析的这些小鼠中的MPC和MSCs是否与从MDX和野生型(WT)小鼠分离的MPC和MSCs相比在增殖和成骨分化能力方面存在缺陷。最近有研究表明，降低成纤维细胞生长因子-2(FGF2)活性可防止干细胞耗尽/衰竭；因此，我们还建议确定FGF2抑制剂负载的仿生凝聚体是否可以修复这种自主的成人干细胞缺陷，并延缓dKO小鼠骨相关组织病理学的发生(目标2)。由于也有证据表明干细胞生态位也可能通过非自主机制对成年干细胞功能产生负面影响，我们建议进行实验，以确定dKO小鼠中观察到的骨缺陷是否可以通过共生配对来挽救，这将通过在dKO和年轻的WT动物之间创建共享循环来恢复营养不良的微环境(目标3)。我们有初步数据支持这样一个事实，即来自幼年动物的循环因子对dKO小鼠的骨骼形态和愈合能力有有益的影响。总之，这一创新的赠款申请将：1)确定dKO小鼠的骨异常和愈合是否代表固有的骨缺陷；2)表征在dKO小鼠观察到的进行性骨组织病理学是否主要由细胞自主和/或非自主机制驱动。

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is a degenerative muscle disorder characterized by a lack of dystrophin expression that ultimately results in cardiac or respiratory failure. DMD patients also acquire osteopenia, fragility fracture, and scoliosis indicating that a deficiency in skeletal system homeostasis also occurs in DMD patients. It is speculated that these skeletal abnormalities are likely a secondary consequence to muscle loss (sarcopenia); however, it remains unclear if they could be due to a direct intrinsic skeletal defect. Recent evidence has emerged implicating adult stem cell dysfunction in the histopathogenesis of DMD. Muscle derived progenitor cells (MPCs) isolated from dystrophin/utrophin double knock-out (dKO) mice (a severe animal model of DMD) have been found to be defective in their proliferation and differentiation capacities. We, and others, have reported that these dKO mice exhibit a spectrum of degenerative changes in their bone, articular cartilage, and intervertebral discs and experience spinal deformities, heterotopic ossification, cardiomyopathy and a decreased lifespan, all of which support a premature musculoskeletal aging phenotype in this mouse model. A defect in bone healing was also observed in these mice; however, it is still unclear whether this defect is an intrinsic bone healing problem or associated with the secondary effects of sarcopenia (Aim 1). Preliminary evidence supports the existence of an adult stem cell defect in both MPCs and mesenchymal stem cells (MSCs) in these mice, supporting the theory that abnormal bone healing could be the consequence of an autonomous defect in the adult stem cell compartment. Thus the second aim of this project will be to further validate whether the MPCs and MSCs in these mice, analyzed at different ages, are defective in their proliferation and osteogenic differentiation capacities compared to MPCs and MSCs isolated from mdx and wild type (WT) mice. It has recently been shown that reducing fibroblast growth factor-2 (FGF2) activity prevents stem cell depletion/exhaustion; therefore, we also propose to determine whether FGF2 inhibitor-loaded biomimetic coacervate could rescue this autonomous adult stem cell defect and delay the onset of bone related histopathologies in dKO mice (Aim 2). Since there is also evidence that the stem cell niche may also negatively impact adult stem cell function, via a non-autonomous mechanism, we propose experiments to determine if the bone defect observed in dKO mice can be rescued through parabiotic pairing which will rejuvenate the dystrophic microenvironment by creating a shared circulation between a dKO and a young WT animal (Aim 3). We have preliminary data that supports the fact that circulating factors from young animals have a beneficial effect on the bone morphologies and healing capacity of dKO mice. In summary, this innovative grant application will: 1) determine whether the bone abnormalities and healing in dKO mice represent an intrinsic bone defect and 2) characterize whether the progressive bone histopathology observed in the dKO mice, is primarily driven by cell autonomous and/or non-autonomous mechanisms.