Self-Assembling Process in Tissue Engineering of Articular Cartilage

描述(由申请人提供)：该申请的长期目标是使用全面的生物工程方法成功地解决关节软骨再生问题。为此，最近开发了一种自组装工艺来组织工程关节软骨，使用高密度种植的天然软骨细胞在琼脂糖制成的模具中。这项研究的主要假设是，自组装过程可以通过添加生长因子和机械刺激来协同增强。为了验证这一假设，我们提出了以下具体目标：1)确定体外自组装关节软骨再生的最佳生长因子条件。2)利用机械刺激加强这一过程。3)将最佳的生长因子和力学条件相结合，使关节软骨具有自组装能力。在自组装过程中将以不同的浓度和剂量频率应用四种生长因子(转化生长因子-31、转化生长因子-1、骨形态发生蛋白-2和胰岛素样生长因子-1)。将确定每种生长因子的最有利浓度和剂量频率，并检查这些组合以选择最佳生长因子条件。静水压力和直接压缩的影响也将在不同的幅度、频率和应用时间下进行检查。每种机械刺激及其组合的最佳条件将从多种处理中选择。最后，将评估优化的生长因子条件和最优机械刺激条件之间的相互作用对自组装过程的影响。在不同的时间点，形态、生化和生物力学特性将被量化，并与自然组织值进行比较，从而能够选择最佳条件。更具体的假设包括：1)生长因子的组合将通过改善自组装结构的功能特性而显示出协同效应，接近天然组织的三分之二。2)与单个机械刺激下未加载的对照或结构相比，静水压力和直接压缩之间的相互作用将增强再生过程。3)在生长因子和机械刺激条件的协同作用下，可以构建出综合性能接近天然关节软骨的关节软骨结构。

DESCRIPTION (provided by applicant): The application's long-term objective is to use a comprehensive bioengineering approach to successfully address articular cartilage regeneration. To this end, a self-assembling process was recently developed to tissue engineer articular cartilage using high density seeding of native chondrocytes in molds made of agarose. The study's main hypothesis is that the self-assembling process can be enhanced synergistically by the addition of growth factors and mechanical stimulation. To test this hypothesis, we propose the following specific aims: 1) To determine optimal growth factor conditions for the self-assembling process for in vitro articular cartilage regeneration. 2) To enhance this process using mechanical stimuli. 3) To engineer articular cartilage combining optimal growth factor and mechanical conditions for the self-assembling process. Application of four growth factors (TGF-(31, TGF-(33, BMP-2, and IGF-I) will be implemented in the self-assembling process at varying concentrations and dosage frequencies. The most favorable concentrations and dosage frequencies of each growth factor will be determined and combinations of these will be examined to select the optimal growth factor conditions. The effects of hydrostatic pressure and direct compression will also be examined at varying magnitudes, frequencies, and application times. The best conditions for each mechanical stimulus and their combinations will be selected from the multiple treatments. Finally, the effects of interactions between the optimized growth factor conditions combined with the optimal mechanical stimulation conditions on the self-assembling process will be assessed. At various time points, morphological, biochemical, and biomechanical properties will be quantified and compared to native tissue values, enabling the selection of optimal conditions. More specific hypotheses include: 1) The combination of growth factors will exhibit a synergistic effect by improving the self-assembled constructs' functional properties approaching two-thirds of native tissue. 2) Interactions between hydrostatic pressure and direct compression will enhance the regeneration process when compared to unloaded controls or constructs under a single mechanical stimulus. 3) The synergistic effects of combined growth factor and mechanical stimulation conditions will yield articular cartilage constructs with combined functional properties approaching those of native articular cartilage.