Targeting collagen as an interventional approach to improve bone material properties

项目摘要 机械负荷和药物干预都能改善骨的力学性能，但存在一个 我们对胶原蛋白在介导这些效应中所起作用的理解存在重大差距。这种知识上的差距 胶原蛋白的加工、组织、矿化和水合作用随着负荷和药物的组合而变化 治疗是发展增加抗骨折性的联合疗法的关键障碍 我们的长期目标是开发改变物理特性的方法， 以增加抗骨折能力。本申请的总体目标是阐明如何 机械载荷和RAL类似物（RALA）修饰新形成的和预先存在的骨骼，以降低脆性。 中心假设是，除了质量和矿物质的变化，胶原修饰作用存在于 加载和RALA，两者的结合交互地提高了机械完整性， 或者单一疗法。这一假设的前提源于申请人在申请过程中产生的初步数据。 laboratories.这项工作的基本原理是，成功地使骨骼更强壮，更有抵抗力， 结合RALA的水化作用和对骨量和椎弓根周围骨的机械调节， 基质活性可以为骨科界提供替代方法， 疾病在初步数据的指导下，这一假设将使用三个具体目标进行测试：1）定义 载荷对骨细胞骨陷窝周围基质活性和成骨细胞基质沉积的影响; 2）确定载荷对骨细胞骨陷窝周围基质活性和成骨细胞基质沉积的影响; RAL/RALA如何改变胶原质量和基质水合作用; 3）确定负载的相互作用效应 还有拉拉在第一个目标下，已经到位的技术将用于研究体外和体内负载 在健康细胞和动物中的作用，以及在破坏胶原合成的模型中的作用。体外加载将是 对于成骨细胞通过基底拉伸或对于骨细胞通过脉动流体流诱导。酶的基因表达 和分子伴侣以及与再吸收相关的分子将被定量。矩阵生产， 将评估组织、组成和机械完整性。对于体内负载实验，类似 技术将被用来评估胶原合成，翻译后修饰，和交联沿着 通过纳米级和全骨机械完整性、抗疲劳性和断裂韧性测试。在Aim中 2，目标1的结局指标将用于研究RAL/RALA作为疾病功能的影响 状态在目标3中，将评估联合负荷和基于药物的治疗的相互作用。的方法 是创新的，因为它的重点是胶原蛋白，除了质量和建筑。它也关注成骨细胞- 在表面产生胶原蛋白，并在整个骨骼中由骨细胞引起变化。这项工作意义重大 因为它将证明通过联合治疗的相互作用可以改善骨骼力学， 表型，不是通过纠正疾病的原因，而是通过影响胶原蛋白的合成，组装，矿化， 和组织水合作用。这些知识将为治疗与脆弱性有关的疾病提供新的途径。

PROJECT SUMMARY Mechanical loading and pharmaceutical interventions both improve bone mechanical properties, but there is a critical gap in our understanding of the role that collagen plays in mediating these effects. This gap in knowledge by which collagen processing, organization, mineralization, and hydration change with combined load and drug treatment is a critical impediment to the development of combination therapies that increase fracture resistance by targeting tissue moieties other than mineral. Our long-term goal is to develop ways to alter physical properties of bone tissue to increase fracture resistance. The overall objective in this application is to elucidate how mechanical loading and a RAL-analog (RALA) modify newly forming and pre-existing bone to decrease fragility. The central hypothesis is that in addition to changes in mass and mineral, collagen-modifying effects exist for both loading and RALA, the combination of which interactively improve mechanical integrity beyond the effects of either monotherapy. The premise of this hypothesis stems from preliminary data generated in the applicants' laboratories. The rationale for the proposed work is that successfully making bone stronger and more resistant to fracture by combining RALA's hydrating effects with mechanical regulation of bone mass and perilacunar matrix activity could provide alternative ways for the orthopaedic community to approach the treatment of bone diseases. Guided by preliminary data, this hypothesis will be tested using three specific aims: 1) to define influences of loading on osteocyte perilacunar matrix activity and osteoblast matrix deposition; 2) to determine how RAL/RALA modify collagen quality and matrix hydration; and 3) to determine interactive effects of loading and RALA. Under the first aim, techniques already in place will be used to investigate in vitro and in vivo loading effects in healthy cells and animals, as well as in models of disrupted collagen synthesis. In vitro loading will be induced by substrate stretching for osteoblasts or pulsatile fluid flow for osteocytes. Gene expression of enzymes and chaperones will be quantified, as well as molecules associated with resorption. Matrix production, organization, composition and mechanical integrity will be assessed. For in vivo loading experiments, similar techniques will be used to assess collagen synthesis, post-translational modifications, and crosslinking along with nanoscale and whole bone tests of mechanical integrity, fatigue resistance and fracture toughness. In Aim 2, outcome measures from Aim 1 will be used to investigate the effects of RAL/RALA as a function of disease state. In Aim 3, interactive effects of combined loading and drug-based treatment will be assessed. The approach is innovative because of its focus on collagen, in addition to mass and architecture. It also focuses on osteoblast- produced collagen on surfaces and changes induced by osteocytes throughout the bone. This work is significant because it will demonstrate that interactions through combination therapies can improve skeletal mechanical phenotypes, not by correcting the disease cause, but by impacting collagen synthesis, assembly, mineralization, and tissue hydration. Such knowledge will provide new ways to approach treatment of fragility-related diseases.