Fate and Regulation of Fracture-induced Prx1 Cells

项目摘要 骨折愈合是一个精心策划的再生过程，在很大程度上仍然未知。揭示了细胞 控制骨折修复的分子机制将有助于确定新的治疗靶点， 这是一个临床相关的问题，每年影响美国60万人。 自体骨移植是治疗骨不连的金标准，但也有很多缺点：侵入性强，成本高， 有风险，有时也是无效的。因此，对替代性和新型疗法存在迫切且未满足的需求。 治疗骨不连该建议的最终目的是获得关于关键机制驱动的新知识 骨折修复和设计它们来促进愈合。骨折后骨骼的高度再生能力 这意味着存在有助于修复过程的成年祖细胞。然而， 这些祖细胞的表达模式仍然是难以捉摸的。我们发现了一个离散的血管周围 细胞表达Prx 1（Prx 1+），驻留在公认的再生壁龛。通过研究功能，我们 发现骨折促进了Prx 1表达和Prx 1+表达细胞，这些细胞有助于骨折修复 过程，但细胞失去Prx 1表达分化。我们还发现，在骨折修复过程中，Prx 1 + 细胞共表达BMP 2和CXCL 12。在进一步探索这种串扰的过程中，我们发现， 在Prx 1骨软骨祖细胞中缺乏完整BMP 2的小鼠中发现骨折愈合受损， 的特征在于异常持续增加的Prx 1和细胞因子CXCL 12。这些异常 用CXCL 12受体拮抗剂AMD 3100（可恢复愈合）纠正。最后，通过使用体内和 在体外方法中，我们已经表明BMP 2通过CXCL 12信号传导调节Prx 1表达。电流 知识和这些令人兴奋的新观察为中心假设设定了科学前提， 这一建议，即Prx 1表达细胞是骨折修复的十字路口， Prx 1的表达模式受BMP 2和MMP 3之间适时的相互作用的调控。 CXCL12提出了两个具体的目标来测试这个新的假设。目标1旨在确定 骨折修复过程中Prx 1和Prx 1表达细胞的需求及其命运和性质。Aim 2设计 以确定Prx 1表达和Prx 1+细胞命运受以下因素调节的机制： BMP 2和CXCL 12在骨折修复过程中的相互作用。将采取综合办法， 通过结合特定基因工程小鼠的产生、细胞示踪、 受教育的动物模型使用;药理学和细胞移植研究;和体外研究。一队 已召集专家研究人员以确保该项目的成功实现。是 预计这项研究产生的新发现将具有重大的生物医学意义， 影响：a）理解骨折愈合的细胞和分子机制;和B） 为开发治疗骨不连的药理学和基于细胞的临床试验奠定基础。

PROJECT SUMMARY Fracture healing is a well-orchestrated regenerative process that remains largely unknown. Revealing the cellular and molecular mechanisms governing fracture repair will help identify novel therapeutic targets to treat patients that suffer of non-unions, a clinically relevant problem that affects annually 600,000 people in the United States. Bone autografts, the gold-standard treatment for non-unions, have multiple drawbacks: they are invasive, costly, risky, and sometime ineffective. Therefore, there is an urgent and unmet need for alternative and novel therapies to treat non-unions. The ultimate goal of this proposal is to gain new knowledge on pivotal mechanisms driving fracture repair and to devise them to promote healing. The highly regenerative ability of bones after fracture implies the existence of adult progenitors that contribute to the reparative process. However, the nature and the expression pattern of these progenitors is still elusive. We have discovered a discrete population of perivascular cells expressing Prx1 (Prx1+) that reside in recognized regenerative niches. By investigating functionality, we have found that facture elicits Prx1expression and Prx1+ expressing cells that contribute to the fracture repair process, but cells lose Prx1 expression with differentiation. We have also found that during fracture repair, Prx1+ cells co-express BMP2 and CXCL12. On the way to further explore this crosstalk, we discovered that the impaired fracture healing found in mice lacking a full complement of BMP2 in Prx1 osteochondroprogenitors, was characterized by an abnormally persistent increase of Prx1 and the cytokine CXCL12. These abnormalities were corrected by AMD3100, a CXCL12 receptor antagonist that restored healing. Lastly, by using in vivo and in vitro approaches, we have indicated that BMP2 through CXCL12 signaling regulates Prx1 expression. Current knowledge and these exciting novel observations set the scientific premise to the central hypothesis of this proposal, namely that Prx1 expressing cells are a crossroad in fracture repair and their commitment to regeneration and their Prx1 expression pattern is regulated by a well-timed interplay between BMP2 and CXCL12. Two specific aims are proposed to test this novel hypothesis. Aim 1 is designed to determine the requirement of Prx1 and Prx1 expressing cells and their fate and nature during fracture repair. Aim 2 is designed to determine the mechanisms by which the expression of Prx1 and the fate of Prx1+ cells is regulated by the interplay between BMP2 and CXCL12 during fracture repair. A comprehensive approach will be applied to accomplish the proposed aims, by combining generation of ad hoc genetically engineered mice; cell-tracing; educated use of animal models; pharmacological and cell transplant studies; and in vitro studies. A team of expert research investigators has been assembled to ensure successful achievement of the project. It is expected that the novel findings generated from this research will have major biomedical relevance and implications for: a) understanding the cellular and molecular mechanisms governing fracture healing; and b) laying the groundwork for the development of pharmacological and cell-based clinical trials to treat non-unions.