Mechanical response of osteoblasts in 3D matrix

3D 矩阵中成骨细胞的机械响应

• 批准号: 6773674
• 负责人: Hiroki Yokota
• 金额: $ 22.82万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-12 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6773674
• 关键词: biomechanics; cell cell interaction; cell proliferation; collagen; enzyme activity; extracellular matrix; fluid flow; gap junctions; mechanical pressure; messenger RNA; metalloendopeptidases; microarray technology; microfilaments; osteoblasts; polymerase chain reaction; shear stress; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The long-term objective of the proposed project is to elucidate the role of mechanotransduction in bone adaptation. Depending on their substrate geometries, osteoblasts grow and develop differently in the in vitro culturing system. Compared to the cells cultured on 2D surface, osteoblasts grown in a 3D matrix exhibit slower cellular proliferation and enhanced calcium deposition. The observed difference raises a question on the role of substrate geometries in the osteoblastic responses to mechanical stimuli. Our current in vitro knowledge on mechanotransduction is mostly based on the osteoblasts grown on a flat substrate. We recently developed a novel mechanical loader with a piezoelectric actuator suitable for applying strain and fluid flow to osteoblasts grown in the 3D porous collagen matrix. The mechanical loader is capable of generating strain in any waveform and strain-induced fluid flow in tubular collagen pores in the matrix. Using this mechanical loader, the proposed project aims to investigate the responses of osteoblasts in the 3D collagen matrix, and the role of intercellular contacts in mechanotransduction, especially the role of gap junctions and actin filaments. Three specific aims of the proposed project are to (i) identify the specific waveforms most effective in inducing mechanotransduction (ii) compare the responses to strain, strain-induced fluid flow, and global chemotransport of osteoblasts grown in the 2D and 3D culturing systems, and (iii) examine the role of gap junctions and actin filaments in mechanotransduction in the 3D matrix. In assaying the responses to mechanical stimuli, mRNA levels, protein levels, and proteolytic activities of the genes sensitive to strain/ stress will be determined using the strain waveform measured in dogs during walking. The mRNA level will be determined by cDNA array as well as RT-PCR, and the protein level will be determined by Western analysis. Activity of matrix metalloproteinases, a family of proteolytic enzymes responsive to mechanical stimuli, will be assayed using fluorescent fibril degradation and zymography. Pharmacological inhibitors and dominant negative mutants will be used to disrupt intercellular communications. Completion of these studies should provide significant insight into the mechanical responses of osteoblasts in a 3D environment, and contribute to developing. q load-induced in vitro connective tissue engineering.

描述（由申请人提供）： 该项目的长期目标是阐明力转导在骨适应中的作用。根据其基质几何形状，成骨细胞在体外培养系统中生长和发育不同。与在 2D 表面培养的细胞相比，在 3D 基质中生长的成骨细胞表现出较慢的细胞增殖和增强的钙沉积。观察到的差异提出了关于基质几何形状在成骨细胞对机械刺激的反应中的作用的问题。我们目前关于机械转导的体外知识主要基于在平坦基质上生长的成骨细胞。我们最近开发了一种带有压电致动器的新型机械加载器，适用于对 3D 多孔胶原基质中生长的成骨细胞施加应变和流体流动。机械加载器能够产生任何波形的应变，并在基质的管状胶原孔中产生应变诱导的流体流动。使用这种机械加载器，该项目旨在研究成骨细胞在 3D 胶原基质中的反应，以及细胞间接触在机械传导中的作用，特别是间隙连接和肌动蛋白丝的作用。 该项目的三个具体目标是 (i) 确定最有效诱导机械转导的特定波形 (ii) 比较在 2D 和 3D 培养系统中生长的成骨细胞对应变、应变诱导的流体流动和整体化学转运的响应， (iii) 检查间隙连接和肌动蛋白丝在 3D 矩阵中的力传导中的作用。在测定对机械刺激的反应时，对应变/应激敏感的基因的mRNA水平、蛋白质水平和蛋白水解活性将使用在狗行走时测量的应变波形来确定。 mRNA水平将通过cDNA阵列以及RT-PCR测定，蛋白质水平将通过Western分析测定。基质金属蛋白酶（一种对机械刺激有反应的蛋白水解酶家族）的活性将使用荧光原纤维降解和酶谱法进行测定。药理学抑制剂和显性失活突变体将用于破坏细胞间通讯。完成这些研究应该可以深入了解 3D 环境中成骨细胞的机械反应，并有助于发育。 q 负载诱导的体外结缔组织工程。