Purinergic Stimulation of Bone Regeneration

描述（申请人提供）：美国每年治疗超过200万例长骨骨折。虽然大多数骨折都是自发愈合的，但对于那些骨折无法愈合或由于创伤或感染而存在严重骨缺损的情况，没有促进骨再生的“金标准”，这是毁灭性的医疗问题，导致严重的残疾。近年来，定制打印生物材料支架的发展可以适应和填充较大的骨缺损，这可能提供了一种新的解决方案，并且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提高假体支架在愈合节段性骨缺损方面的功效。虽然目前用于促进骨生成，但生长因子如rh-BMP2 （BMP2）的有效性值得怀疑，并且存在重大的安全性问题。我们最近报道了腺苷A2A受体（A2AR）刺激在小鼠炎症性骨溶解模型中增加成骨细胞数量并调节成骨细胞功能，并且A2AR刺激通过抑制NFκB激活和核易位来减少破骨细胞分化。此外，在体外和体内，A2ARs还能刺激血管生成和血管生成。因此，我们提出验证一种假设，即涂有双吡啶达摩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尺寸的节段性骨缺损的骨再生，并确定这一现象的细胞和分子机制。因此，我们提出以下目标：1 .开发包被生物活性陶瓷支架治疗关键节段性骨缺损。我们将确定是否在兔桡骨节段性骨缺损模型中植入涂有双嘧达莫的三维打印三磷酸钙/羟基磷灰石支架，双嘧达莫是一种阻断细胞腺苷摄取并增加细胞外液中腺苷浓度的药物。我们将进一步在体外和小鼠颅骨骨再生模型中最大化支架设计和双嘧达莫剂量。2。确定A2AR刺激促进骨再生的细胞机制。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基础。3。A2AR刺激促进成骨细胞骨再生的分子机制研究。我们将测试A2AR信号与关键的细胞内信号级联相互作用的假设，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。这个高度转化项目的目标是建立靶向A2ARs刺激骨再生的分子和细胞基础，并迅速将这些发现转化为临床。

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.