MECHANICAL LOADING AND BONE

描述(申请人提供)：拟议研究的长期目标是阐明骨中机械转导的机制。我们目前以生物工程为导向的项目开发了一种高分辨率的压电机械加载器，并利用培养的成骨细胞评估了机械刺激在骨中的作用。结果表明：(A)三维胶原基质的变形可以诱导应变诱导的流体流动；(B)应变诱导的流体流动，而不是应变本身，主要激活成骨细胞中的应力反应基因；(C)三维胶原基质的结构建立了应变诱导的流体流动和分子运输的模式。动物研究中的许多证据支持1000-2000微应变的应变促进骨重建。我们的体外研究和这些动物研究之间的一个不清楚的联系是应变和液体流动在骨重建中的作用。在体外成骨细胞培养，包括我们目前的研究，使用2D基质或3D基质，很难模拟体内应变诱导的液体流动。体外和体内数据之间的这种差异使得很难评估应变和液体流动在骨重建和抗炎中的作用。首先，骨骼中的微观应变可能高于用应变计测量的宏观应变。因此，高于1000-2000微应变的局部微观应变可能会驱动骨中的液体流动。第二，骨骼中的骨陷窝网络可以以一种载荷频率依赖的方式放大应变诱导的液体流动。最后，骨间质液流动可能是由原位应变引起的，也可能是远端应变引起的，导致相对较软的骨骺变形引起骨干皮质骨的液体流动。 这一更新建议将使用小鼠尺骨体外以及小鼠体内加载来检验上述数据差异的可能解释。具体目标包括：(1)制造用于体外和体内使用的压电式机械加载器；(2)使用电子散斑干涉技术以及使用光漂白后荧光恢复的分子传输来量化体外宏观和微观应变；(3)进行骨组织形态计量学以评估体外数据；以及(4)检测负载驱动的不良反应与基因表达和酶活性(例如基质金属蛋白酶)的关系。机械载荷将在尺骨加载(轴向加载)和肘部加载(横向加载)模式下施加。这两种模式已被证明以不同的应变分布模式促进骨干的骨重建。成功完成建议的更新建议，将提供有关诱导骨骼内液体流动的基本知识，并为制定强骨和预防骨质流失的治疗策略建立一个研究平台。

DESCRIPTION (provided by applicant): The long-term objective of the proposed studies is to elucidate the mechanism of mechanotransduction in bone. Our present bioengineering-oriented project developed a high-resolution piezoelectric mechanical loader and evaluated the role of mechanical stimulation in bone using cultured osteoblasts. The results reveal that (a) deformation of 3D collagen matrix can induce strain-induced fluid flow; (b) strain-induced fluid flow, and not strain itself, predominantly activates the stress-responsive genes in osteoblasts; and (c) architecture of 3D collagen matrix establishes a pattern of strain-induced fluid flow and molecular transport. Many lines of evidence in animal studies support enhancement of bone remodeling with strain of 1000 - 2000 microstrains. An unclear linkage between our in vitro studies and these animal studies is the role of strain and fluid flow in bone remodeling. In vitro osteoblast cultures including our current studies use 2D substrates or 3D matrices that hardly mimic the strain-induced fluid flow in vivo. This difference between in vitro and in vivo data makes it difficult to evaluate the role of strain and fluid flow in bone remodeling and anti-inflammation. First, microscopic strain in bone might be higher than the macroscopic strain measured with strain gauges. A local microscopic strain higher than 1000 - 2000 microstrains may therefore drive fluid flow in bone. Second, the lacunocanalicular network in bone could amplify strain-induced fluid flow in a loading-frequency dependent fashion. Lastly, interstitial fluid flow in bone might be induced by in situ strain as well as strain in a distant location, such that deformation of relatively soft epiphyses induces fluid flow in cortical bone in diaphyses. This renewal proposal will use mouse ulnae ex vivo as well as mouse in vivo loading to examine the above possible explanations for the data divergence. Specific aims include: (1) fabricating a piezoelectric mechanical loader for ex vivo and in vivo use; (2) quantifying ex vivo macroscopic and microscopic strains using electronic speckle pattern interferometry as well as molecular transport using fluorescence recovery after photobleaching; (3) conducting bone histomorphometry to evaluate ex vivo data; and (4) examining load-driven adverse effects with gene expression and enzyme activities (e.g., matrix metalloproteinases). Mechanical loads will be given in the ulna-loading (axial loading) and elbow-loading (lateral loading) modes. These two modes have been shown to enhance bone remodeling in the diaphysis with different patterns of strain distribution. Successful completion of the proposed renewal proposal will provide basic knowledge about induction of fluid flow in bone and establish a research platform for devising therapeutic strategies for strengthening bone and preventing bone loss.