Interstitial Fluid Flow in Bone Remodeling

描述（由申请人提供）：在过去二十年中，有大量证据证明骨骼间质液流动在骨的活力、维持和对骨加载和卸载的反应中起着关键作用。我们的目的是阐明间质液流（IFF）刺激骨细胞的机制，使用分子，细胞，体内敲除和转基因模型。IFF的特征在于由血管压力和机械载荷驱动的稳态和动态分量。尽管骨细胞对两种流动成分都有反应，但我们已经证明了机械转导途径和随后的细胞和体内对这两种机械刺激的反应不同。总体假设是，动态或脉动流导致成骨细胞有丝分裂反应，而稳定流诱导抗吸收骨骼反应以及成骨细胞分化。为了研究这一假设，我们提出了以下具体目标：1）测试假设，即振荡流在体外诱导成骨细胞生长（促有丝分裂）的反应，而斜坡稳定流诱导成骨细胞分化。将在成骨细胞和骨细胞中测量促有丝分裂指数（BrdU掺入）和转录激活因子（BNF-1和c-fos）以及分化指数（骨唾液酸蛋白、Cbfa-1和p57 Kip 2）。2)确定成骨细胞和骨细胞中脉动流和斜坡稳态流的一氧化氮相关机械化学信号转导途径的机制。我们将表征一氧化氮合酶（NOS）亚型介导的流量响应，并确定NOS活性的调节机制。3)使用我们的新型可植入微流体振荡压力装置，我们将确定振荡流是否会诱导合成代谢反应，而增加稳定流在后肢悬吊大鼠中是抗吸收的。利用成骨细胞特异性条件性基因敲除小鼠，研究NOS和小窝蛋白在介导骨细胞对动态间质液流动的反应中的作用。拟议的研究将阐明间质液流动作用于骨的基本机制，并为基于间质液流动调节的治疗提供基础，以对抗废用性骨质减少。公共卫生相关性。有越来越多的证据表明，间质液流（IFF）介导的骨骼反应的加载和卸载。最重要的假设是，动态IFF导致成骨细胞有丝分裂反应，而稳定的流动诱导成骨细胞分化。使用体外和体内模型，拟议的研究将阐明IFF作用于骨的基本机制，并为基于IFF调节的治疗提供基础，以对抗废用性骨质减少。

DESCRIPTION (provided by applicant): Over the past two decades, there has been an accumulation of evidence demonstrating the critical role of skeletal interstitial fluid flow in the viability, maintenance, and response to loading and unloading of bone. Our objective is to elucidate the mechanisms by which interstitial fluid flow (IFF) stimulates bone cells, using molecular, cell, and in vivo knockout and transgenic models. IFF is characterized by both steady and dynamic components driven by vascular pressure and mechanical loading. Even though bone cells respond to both flow components, we have demonstrated that the mechanotransduction pathways and the subsequent cellular and in vivo responses to these two mechanical stimuli differ. The overarching hypothesis is that dynamic or pulsatile flows result in an osteoblast mitogenic response while steady flow induces both an anti-resorptive skeletal response as well as osteoblast differentiation. To investigate this hypothesis, we propose the following the specific aims: 1) Test the hypothesis that oscillatory flow in vitro induces an osteoblast growth (mitogenic) response, while ramped steady flow induces osteoblast differentiation. Mitogenic indices (BrdU incorporation) and transcriptional activators (egr-1 and c-fos) and differentiation indices (bone sialoprotein, Cbfa-1, and p57Kip2) will be measured in osteoblasts and osteocytes. 2) Determine the mechanisms for the nitric oxide-related mechanochemical signal transduction pathways of pulsatile flow and ramped steady flow in osteoblasts and osteocytes. We will characterize which nitric oxide synthase (NOS) isoforms mediate the flow responses, and determine the mechanism of regulation of NOS activity. 3) Using our novel implantable microfluidic oscillatory pressure device, we will determine if oscillatory flow induces an anabolic response, while increased steady flow is anti-resorptive in the hindlimb suspended rat. And 4) using osteoblast-specific conditional knockout mice, determine the roles of NOS and caveolin in mediating the skeletal cellular responses to dynamic interstitial fluid flow. The proposed research will elucidate the basic mechanisms by which interstitial fluid flow acts on bone, and provide the basis for treatments based on the modulation of interstitial fluid flow to counter osteopenia of disuse. PUBLIC HEALTH RELEVANCE. There is mounting evidence that interstitial fluid flow (IFF) mediates the skeletal response to loading and unloading. The overarching hypothesis is that dynamic IFF results in an osteoblast mitogenic response while steady flow induces osteoblast differentiation. Using both in vitro and in vivo models, the proposed research will elucidate the basic mechanisms by which IFF acts on bone, and provide the basis for treatments based on the modulation of IFF to counter osteopenia of disuse.