Purinergic Stimulation of Bone Regeneration

描述(申请人提供)：在美国，每年有超过200万名长骨骨折患者接受治疗。尽管大多数骨折会自动愈合，但在骨折无法愈合或因创伤或感染导致严重节段性骨缺损、破坏性医疗问题导致严重残疾的情况下，并没有促进骨再生的“黄金标准”。最近开发的定制打印生物材料支架可以适应和填充较大的骨缺损，提供了一种新的解决方案，在这些支架上涂上旨在促进更快和更完整的骨愈合的药物，可能会增加人工支架在修复节段性骨缺损方面的疗效。虽然目前用于促进骨生成，但生长因子如rh-BMP2(BMP2)的疗效值得怀疑，并存在重大的安全性问题。我们最近报道，在炎症性骨溶解的小鼠模型中，腺苷A2A受体(A2AR)刺激增加成骨细胞数量并调节成骨细胞功能，A2AR刺激通过抑制核转录因子κB的激活和核转位而减少破骨细胞的分化。此外，A2ARs在体外和体内都能刺激血管生成和血管生成。因此，我们建议验证这样一种假说，即涂覆有双嘧达莫的三维打印支架可以增加局部腺苷水平并间接刺激A2ARs，从而进一步促进临界大小的节段性骨缺损的骨再生，并确定这种现象的细胞和分子机制。因此，我们提出了以下目标：1.开发涂层生物活性陶瓷支架，用于治疗关键的节段性骨缺损。我们将确定植入涂有双嘧达莫的三维打印三磷酸钙/羟基磷灰石支架是否促进了兔桡骨节段性骨缺损模型的骨再生。双嘧达莫是一种阻止细胞腺苷摄取并增加细胞外液中腺苷浓度的试剂。我们将在体外和小鼠颅骨再生模型中进一步最大化支架设计和双嘧达莫剂量。II.A2AR刺激促进骨再生的细胞机制的确定。利用A2AR的全局和细胞选择性敲除，我们将确定A2AR介导的小鼠颅骨模型骨再生的细胞学基础。研究A2AR刺激促进成骨细胞骨再生的分子机制。我们将验证这一假设，即A2AR信号与关键的细胞内信号级联反应，通过使用信号通路的药物抑制剂和靶向敲除原代细胞和细胞系中的关键信号分子来促进骨再生。这个高度翻译的项目的目标是建立靶向A2ARs的分子和细胞基础，以刺激骨再生，并将这些发现迅速转化为临床。

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.