Wnt signaling in skeletal tissue regeneration

危害或削弱骨骼功能的疾病构成了美国最普遍的慢性损伤，每年的直接和间接费用为8490亿美元。随着婴儿潮一代的年龄增长，这一数字预计将在未来10-20年内上升。因此，迫切需要刺激骨骼修复和再生的新策略。目前的文献表明，Wnt途径是增强骨骼修复和再生的最有吸引力的候选者之一。Wnt信号通路的中断与多种人类肌肉骨骼疾病有关，Wnt信号传导是身体对骨骼损伤的自然反应的一部分。因此，本提案的目的是检查Wnt信号传导影响骨再生的基本机制，并使用此信息制定骨修复的治疗策略。在目标1中，我们将研究Wnt抑制剂Axin 2的缺失如何影响细胞中的Wnt信号传导。我们假设Axin 2的缺失将导致Wnt反应的持续时间和幅度增加。我们将检查从Axin 2突变小鼠分离的原代骨祖细胞中的Wnt应答，所述Axin 2突变小鼠已经用Wnt报告基因慢病毒转染，其中荧光素酶活性是Wnt信号传导的读数。在目标2中，我们将在体外和体内检测Axin 2突变对骨髓细胞成骨潜能的影响。我们假设Axin 2的缺失增加了骨髓基质细胞（BMSC）的成骨潜能。在我们的第一组实验中，我们将评估野生型和Axin 2突变BMSC与或不与纯化Wnt 3a蛋白的成骨细胞分化。此外，我们将通过将Axin 2小鼠的骨髓细胞移植到颅骨的临界大小缺损中来测试体内Axin 2小鼠的骨髓细胞的成骨潜力。我们将检测细胞增殖、成骨分化和损伤部位的基质沉积。在目标3中，我们将使用Wnt蛋白本身作为治疗剂，并确定其对骨愈合和再生的影响。与目标2类似，将Wnt 3a或PBS脂质体处理的全骨髓移植到颅骨中的临界尺寸缺损中。我们将使用原位杂交、u-CT、组织学和免疫组织化学来表征治疗对愈合组织中细胞增殖、成骨细胞分化和基质沉积的影响。总之，这些实验将阐明Axin 2在Wnt信号传导中的功能，并将直接测试使用Wnt刺激骨再生的可行性。我们设想，这些信息将是相当有价值的治疗方法，以治疗肌肉骨骼条件。

DESCRIPTION (provided by applicant): Diseases that jeopardize or weaken skeletal function constitute the most prevalent chronic impairments in the U.S with associated annual direct and indirect costs of $849 billion. As the baby boomer generation ages, this figure is expected to escalate in the next 10-20 years. Therefore, new strategies that stimulate skeletal repair and regeneration are badly needed. The current literature indicates that the Wnt pathway is among the most attractive candidates for enhancing skeletal repair and regeneration. Disruptions in the Wnt pathway are associated with multiple human musculoskeletal diseases and Wnt signaling is part ofthe body's natural response to skeletal damage. Thus, the objective of this proposal is to examine the basic mechanism(s) by which Wnt signaling effect bone regeneration and to use this information to formulate therapeutic strategies for bone repair. In Aim 1, we will examine how the loss of the Wnt inhibitor, Axin2, affects Wnt signaling in a cell. We hypothesize that the loss of Axin2, will result in an increased in the duration and magnitude of Wnt response. We will examine the Wnt response in primary osteoprogenitor cells isolated from Axin2 mutant mice that has been transfected with Wnt reporter lentivirus where luciferase activity is the readout of Wnt signaling. In Aim 2, we will examine the effect of Axin2 mutation on the osteogenic potential of bone marrow cells both in vitro and in vivo. We hypothesize that loss of Axin2 increases the osteogenic potential of bone marrow stromal cells (BMSC). In our first set of experiments we will evaluate osteoblast differentiation of wild-type and Axin2 mutant BMSCs with or without purified Wnt3a protein. In addition, we will test the osteogenic potential of bone marrow cells from Axin2 mice in vivo by transplanting them into critical size defects in calvaria. We will examine cell proliferation, osteogenic differentiation, and matrix deposition at the injury site. In Aim 3, we will use Wnt protein itself as a therapeutic agent and we will determine its affect on bone healing and regeneration. Similar to Aim 2, Wnt3a or PBS liposomes treated whole bone marrow will be grafted into critical size defects in the calvaria. We will use in situ hybridization, u-CT, histology and immunohistochemistry to characterize the effects of the treatment on cell proliferation, osteoblast differentiation, and matrix deposition in the healing tissue. In summary, these proposed experiments will clarify the function of Axin2 in Wnt signaling, and will directly test the feasibility of using Wnt to stimulating bone regeneration. We envision that this information will be of considerable value in therapeutic approaches to treating musculoskeletal conditions.