Role of Angiogenesis in Distraction Osteogenesis

牵引成骨(DO)是一种广泛应用的骨科治疗方法，用于矫正肢体长度差异、先天性畸形、骨不连，以及因创伤而丢失的大量骨再生。这是机械刺激最戏剧性的应用之一，作为一种诱导长骨长度高达20%的再生的手段。虽然DO手术已经在临床上使用，手术技术也已经得到了改进，但这种手术促进新骨形成的基本机制并不是 很好理解。牵张成骨的主要特征之一是，它通过膜内突起诱导新骨形成，而膜内突起缺乏大量软骨。然而，最耐人寻味的观察是，在再生过程中，大量骨骼的再生伴随着修复组织的强大血管。这一建议的假设是，牵张成骨过程中的骨再生是由血管生成过程驱动的。这其中的一个组成部分 假说是，在牵张成骨过程中伴随着再生过程的血管生成事件是在没有软骨生成的情况下指导骨形成的主要调节机制。为了验证这一假说并检验定义骨形成分子机制的遗传模型，我们开发了一种DO小鼠模型。初步研究将确定在牵张成骨过程中驱动新骨形成的血管生成和骨形态发生信号的时间和空间表达 将这些信号的表达与那些在骨折愈合过程中驱动软骨内修复过程的信号进行比较(这些将作为项目4的一部分进行阐述)。在此之后，我们将进行实验，使用针对两种不同受体(Flk-1和Flt-1)的选择性阻断抗体来特异性阻断血管内皮生长因子(VEGF)介导的血管生成，并从功能上测试在牵张血管生成过程中，VEGF信号在多大程度上是血管生成和/或新骨形成的中介。我们会 在体内和体外实验中，骨形态发生蛋白-2促进分化与血管内皮生长因子表达的关系(S)以及血管内皮生长因子对骨形态发生蛋白表达的反作用(S)。然后，我们将确定在DO过程中BMP活性的选择性增减如何影响血管生成，以及这些变化如何影响该系统中的骨形成。

Distraction osteogenesis (DO) is a widely used orthopedic treatment for the correction of limb length discrepancies, congenital deformities, non-unions, and the regeneration of large amounts of bone that have been lost due to trauma. It is one of the most dramatic applications of mechanical stimulation as a means of inducing regeneration of up to 20% of the length of a long bone. While DO procedures are used clinically and the surgical techniques have been refined, the basic mechanisms by which this procedure promotes new bone formation are not well understood. One of the primary descriptive features of distraction osteogenesis is that it induces new bone formation through an intramembranous processes devoid of extensive amounts of cartilage. However, the most intriguing observation is that the regeneration of abundant amounts of bone is accompanied by robust vascularity of the repair tissue throughout the regenerative process. The hypothesis of this proposal is that bone regeneration during distraction osteogenesis is driven by an angiogenic process. A component of this hypothesis is that the angiogenic events that accompany the regenerative process in distraction osteogenesis are the primary regulatory mechanisms that direct bone formation in the absence of chondrogenesis. In order to test this hypothesis and examine genetic models that will define the molecular mechanisms regulating bone formation, we have developed a murine model of DO. Initial studies will define the temporal and spatial expression of the angiogenic and bone morphogenetic signals that drive new bone formation during distraction osteogenesis and compare the expression of these signals to those that drive the endochondral repair processes that occur during fracture healing (these will be elucidated as part of Project 4). This will be followed with experiments in which we specifically block vascular endothelial growth factor (VEGF)-mediated angiogenesis using selective blocking antibodies to two different receptors of VEGF (Flk- 1 and Flt- 1), and functionally test the degree to which VEGF signals are mediators of both angiogenesis and/or new bone formation during distraction angiogenesis. We will test, in both in vivo and in vitro experiments, the relationship of BMP-2 stimulated differentiation to the expression of VEGF(s) and the inverse effects of VEGF on the expression of BMP(s). We will then determine how selective gain or loss of BMP activity during DO affects angiogenesis and how these alterations affect the formation of bone in this system.