Mechanical response of osteoblasts in 3D matrix

描述（由申请人提供）： 该项目的长期目标是阐明机械传导在骨适应中的作用。成骨细胞在体外培养体系中的生长发育因基质的几何形状不同而不同。与在二维表面上培养的细胞相比，在三维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。所观察到的差异提出了一个问题，基板的几何形状在成骨细胞对机械刺激的反应的作用。我们目前在体外的力学转导知识主要是基于成骨细胞生长在一个平坦的基板上。我们最近开发了一种新型的机械加载器与压电致动器适用于施加应变和流体流动的成骨细胞生长在三维多孔胶原基质。机械加载器能够在基质中的管状胶原孔隙中产生任何波形的应变和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转导中的作用，特别是间隙连接和肌动蛋白丝的作用。 拟议项目的三个具体目标是（i）确定最有效地诱导mechanotransduction的特定波形（ii）比较应变，应变诱导的流体流动，和全球的成骨细胞在2D和3D培养系统中生长的化学运输的反应，和（iii）检查间隙连接和肌动蛋白丝在mechanotransduction在3D矩阵的作用。在测定对机械刺激的反应时，将使用在狗行走期间测量的应变波形来确定对应变/应力敏感的基因的mRNA水平、蛋白质水平和蛋白水解活性。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。基质金属蛋白酶是一种对机械刺激有反应的蛋白水解酶家族，其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。这些研究的完成将为成骨细胞在3D环境中的机械反应提供重要的见解，并有助于开发。q负荷诱导的体外结缔组织工程。

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.