Controlling chondrocyte matrix degradation and repair in 3-D culture.

描述（由申请人提供）：本提案的长期目标是：1)提供关于如何操纵炎症信号通路影响软骨细胞形成新基质的力学特性的基本知识，以及2)创建组织工程手术解决方案来修复骨软骨缺损。这些目标将通过两个具体目标来实现：利用1)敲除MKK/MEK3基因的转基因小鼠软骨细胞构建和分析组织工程软骨；2)对组织工程软骨中调节蛋白聚糖和胶原沉积的关键信号通路（p38和JNK MARK）的药理学抑制。分析将包括机械特性、胶原蛋白、交联和GAG含量、多光子显微镜和组织的数学建模。本提案的目标与NIH和NIAMS的使命相关。功能性工程软骨将大大减轻影响约10%人口的软骨损伤(1)。获得的关于软骨新基质形成的遗传决定因素的信息将增加对风湿性关节炎和骨关节炎中软骨退化的理解。该提案包含组织工程、多光子成像和数学建模组件，是一项多学科的努力，旨在推进疾病和修复过程中软骨细胞遗传编程的基础知识。来自tnf - α和il -1 - β信号元件敲除和突变的小鼠的软骨细胞将被收获并植入海藻酸盐支架中，以允许新基质的发育。组织力学特性、胶原蛋白、蛋白多糖和交联含量将在正常和转基因表型下的培养过程中进行评估，并在已知影响细胞外基质产生的相同信号通路中进行药物抑制或不进行药物抑制(2)。多光子成像将用于评估胶原沉积和网络结构（使用二次谐波产生的信号）和胶原交联形成（通过双光子自身荧光信号）。来自组织工程软骨的数据将适用于软骨生长混合物模型，该模型先前开发用于评估天然软骨(3)。软骨损伤每年影响近100万美国人，导致20万例手术。损伤常常导致骨关节炎(1)。组织工程软骨是研究软骨细胞控制的软骨降解和修复的一种很有前途的方法。了解这些体外过程将有助于临床医生通过药物、遗传和外科操作来控制和修复软骨。

DESCRIPTION (provided by applicant): This proposal's long-term objectives are: 1) to provide basic knowledge about how manipulation of Inflammatory signaling pathways can affect the mechanical properties of chondrocyte created neo-matrix, and 2) to create a tissue-engineered surgical solution to restore osteochondral defects. These objectives will be through two specific aims: construction and analysis of tissue-engineered cartilage using 1) genetically-modified mouse chondrocytes with knockout of MKK/MEK3; and 2) pharmacologic inhibition of key signaling pathways (p38 and JNK MARK) that regulate proteoglycan and collagen deposition by chondrocytes in tissue-engineered cartilage. Analysis will encompass mechanical characterization, collagen, crosslink, and GAG content, multiphoton microscopy, and mathematical modeling of the tissue. The goals of this proposal are relevant to the mission of the NIH and NIAMS. Functional engineered cartilage would greatly alleviate cartilage damage which affects ~10% of the population (1). Information gained about the genetic determinants of cartilage neo-matrix formation would increase understanding of cartilage degradation in rheumatoid and osteoarthritis. This proposal has tissue engineering, multiphoton imaging, and mathematical modeling components, and is a multidisciplinary effort to advance basic knowledge of chondrocyte genetic programming in disease and repair processes. Chondrocytes from mice with knockouts and mutations of TNF-alpha and IL-1beta signaling elements will be harvested and seeded within alginate scaffolds to allow neo-matrix development. Tissue mechanical properties, collagen, proteoglycan, and crosslink content will be assessed during culture under normal and transgenic phenotype, and with or without pharmacologic inhibition of the same signaling pathways, known to affect extracellular matrix production (2). Multiphoton imaging will be used to assess collagen deposition and network structure (using signal from second harmonic generation) and collagen crosslink formation (through two-photon autofluorescence signal). Data from the tissue-engineered cartilage will be fit to a cartilage growth mixture model, previously developed to assess native cartilage (3). Every year, cartilage Injury affects nearly 1 million Americans resulting in 200,000 procedures. Injury often leads cryptically to osteoarthritis (1). Tissue-engineered cartilage is a promising method to examine chondrocyte-controlled cartilage degradation and repair. Understanding these processes in vitro would help clinicians control and repair cartilage through pharmaceutical, genetic, and surgical manipulations.