Interstitial Fluid Flow in Bone Remodeling

描述(申请人提供)：在过去的二十年里，已经有大量的证据表明，骨骼间质液体流动在骨骼的存活、维持以及对骨的加载和卸载的反应中起着关键的作用。我们的目标是利用分子、细胞和体内基因敲除和转基因模型来阐明间质液体流动(IFF)刺激骨细胞的机制。IFF的特点是由血管压力和机械载荷驱动的稳态和动态分量。尽管骨细胞对这两种流动成分都有反应，但我们已经证明，机械转导途径以及随后的细胞和体内对这两种机械刺激的反应是不同的。最重要的假设是，动态或脉动的血流导致成骨细胞的有丝分裂反应，而稳定的血流诱导抗吸收的骨骼反应和成骨细胞的分化。为了研究这一假说，我们提出了以下具体目标：1)检验体外振荡流诱导成骨细胞生长(有丝分裂)反应，而倾斜恒定流诱导成骨细胞分化的假说。将测量成骨细胞和骨细胞的有丝分裂指数(BrdU掺入)、转录激活物(EGR-1和c-fos)和分化指数(骨涎蛋白、CBFA-1和p57Kip2)。2)确定成骨细胞和成骨细胞中与一氧化氮相关的脉动流和斜坡流的机械力化学信号转导途径的机制。我们将确定哪些一氧化氮合酶(NOS)亚型介导了流动反应，并确定了NOS活性的调节机制。3)使用我们的新型植入式微流控振荡压力装置，我们将确定在后肢悬吊大鼠中，振荡流是否诱导合成代谢反应，而增加的稳定流是抗吸收的。4)利用成骨细胞特异性条件性基因敲除小鼠，确定一氧化氮合酶和小窝蛋白在介导骨骼细胞对动态间质液体流动的反应中的作用。本研究将阐明间质液流作用于骨的基本机制，并为基于间质液流调节抗废用性骨量减少的治疗提供依据。与公共卫生相关。越来越多的证据表明，间质流体流动(IFF)调节骨骼对加载和卸载的反应。最重要的假设是，动态IFF导致成骨细胞有丝分裂反应，而稳定的血流诱导成骨细胞分化。利用体外和体内模型，本研究将阐明IFF作用于骨的基本机制，并为基于IFF调节抗废用性骨量减少的治疗提供依据。

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.