Purinergic Stimulation of Bone Regeneration

描述（由申请人提供）：美国每年治疗超过200万例长骨骨折。虽然大多数骨折会自发愈合，但在骨折不愈合或由于创伤或感染而存在临界尺寸的节段性骨缺损的情况下，没有促进骨再生的“黄金标准”，破坏性的医疗问题导致严重残疾。最近开发的定制打印的生物材料支架，可以适应和填充大的骨缺损，可以提供一种新的解决方案，并与设计用于促进更快速和完整的骨愈合的试剂涂层这些支架可以增加修复支架在愈合节段性骨缺损的功效。虽然目前用于促进骨生成，但生长因子如rh-BMP 2（BMP 2）的功效值得怀疑，并存在显著的安全性问题。我们最近报道，腺苷A2 A受体（A2 AR）刺激增加成骨细胞的数量，并调节成骨细胞的功能，在小鼠模型的炎性骨质溶解，A2 AR刺激减少破骨细胞分化，抑制NFκB激活和核转位。此外，A2 AR在体外和体内刺激血管生成和血管发生。因此，我们建议测试的假设，三维打印的支架与代理，潘生丁，增加局部腺苷水平和间接刺激A2 AR可以进一步促进骨再生在关键尺寸的节段性骨缺损，并确定这种现象的细胞和分子机制。因此，我们提出以下目标：涂层生物活性陶瓷支架治疗关键节段性骨缺损的研究。我们将确定是否植入三维打印的三磷酸钙/羟基磷灰石支架涂有双嘧达莫，一种代理，阻止细胞腺苷摄取和增加腺苷浓度的细胞外液，促进骨再生的兔桡骨模型的节段性骨缺损。我们将进一步最大限度地提高支架设计和双嘧达莫剂量在体外和小鼠颅骨模型的骨再生。二.确定A2 AR刺激促进骨再生的细胞机制。使用A2 AR的全局和细胞选择性敲除，我们将确定A2 AR介导的小鼠颅骨模型中的骨再生的细胞基础。三. A2 AR刺激促进成骨细胞中骨再生的分子机制的检查。我们将测试这一假设，即A2 AR信号与关键的细胞内信号级联相互作用，以促进骨再生，使用信号通路的药理学抑制剂，并在原代细胞和细胞系中的关键信号分子的靶向敲低。这个高度转化项目的目标是建立靶向A2 AR刺激骨再生的分子和细胞基础，并将这些发现快速转化为临床。

DESCRIPTION (provided by applicant): Over 2 million long bone fractures are treated in the United States every year. Although most bone fractures heal spontaneously there is no "gold standard" for promoting bone regeneration in those settings in which either fractures do not heal or there is a critical sized segmental bone defect due to trauma or infection, devastating medical problems leading to significant disability. The recent development of custom printed biomaterial scaffolds that can fit and fill large bone defects may provide a novel solution and coating these scaffolds with agents designed to promote more rapid and complete bone healing may increase the efficacy of prosthetic scaffolds in healing segmental bone defects. Although currently used to promote bone generation, growth factors such as rh-BMP2 (BMP2) are of questionable efficacy and present significant safety issues. We have recently reported that adenosine A2A receptor (A2AR) stimulation increases osteoblast number and regulates osteoblast function in a murine model of inflammatory osteolysis and that A2AR stimulation diminishes osteoclast differentiation by inhibiting NFκB activation and nuclear translocation. Moreover, A2ARs stimulate angiogenesis and vasculogenesis in vitro and in vivo. Thus, we propose to test the hypothesis that 3- dimensional printed scaffolds coated with an agent, dipyridamole, that increases local adenosine levels and indirectly stimulates A2ARs can further promote bone regeneration at critical sized segmental bone defects and to determine the cellular and molecular mechanisms for this phenomenon. We therefore propose the following aims: I. Development of coated bioactive ceramic scaffolds to treat critical segmental bone defects. We will determine whether implanting 3-dimensionally printed calcium triphosphate/hydroxyapatite scaffolds coated with dipyridamole, an agent which blocks cellular adenosine uptake and increases adenosine concentration in extracellular fluids, promotes bone regeneration in a rabbit radius model of segmental bone defect. We will further maximize scaffold design and dipyridamole dosing in vitro and in a murine calvaria model of bone regeneration. II. Determination of the cellular mechanism by which A2AR stimulation promotes bone regeneration. Using global and cell-selective knockouts of A2AR we will determine the cellular basis for A2AR-mediated bone regeneration in the murine calvaria model. III. Examination of the molecular mechanisms by which A2AR stimulation promotes bone regeneration in osteoblasts. We will test the hypothesis that A2AR signaling interacts with critical intracellular signaling cascades to promote bone regeneration using pharmacologic inhibitors of signaling pathways and by targeted knockdown of critical signaling molecules in primary cells and cell lines. The goals of this highly translational project are to establish the molecular and cellular basis for targeting A2ARs to stimulate bone regeneration and to rapidly translate these findings to the clinic.