Regenerating the fibrocartilage spectrum of the TMJ: from disc to condyle

描述(申请人提供)：这项设计导向的方案的目标是优化和使用无支架共培养，以生成仿生纤维软骨，用于修复或替换颞下颌关节半月板和下颌软骨表面。利用纤维软骨细胞和关节软骨细胞的共培养，我们最近产生了临床相关维度的大结构，这些结构在外观上类似纤维软骨，由暗示纤维软骨的细胞外基质(ECM)组成。此外，纤维软骨细胞也获得了类似于天然组织的结构。受这些发现的启发，我们的假设是，利用生物活性物质和机械刺激可以优化共培养，以形成仿生纤维软骨结构。为了解决这一假设，我们提出了以下具体目标：1)在纤维软骨的无支架共培养中，优化软骨素酶ABC和生长因子转化生长因子-1(TGF-1)和胰岛素样生长因子-1(IGF-1)的使用；2)通过机械刺激增强纤维软骨仿生结构；3)检测生物活性物质和机械刺激的协同效应。软骨素酶ABC已在初步研究中被发现可显著提高结构的拉伸性能。我们的研究小组还证明，转化生长因子-1和胰岛素样生长因子-I显著增加纤维软骨结构中细胞外基质的产生。我们的实验室已经证明，使用静水压力和直接压缩刺激对关节软骨和纤维软骨结构有有益的影响，我们将单独和联合应用这些刺激来进一步增强结构。组织学检查糖胺聚糖(GAG)和胶原蛋白，免疫组织化学检查I型和II型胶原。GAG、胶原蛋白和DNA含量将被量化，然后用ELISA法测定I型和II型胶原。生物力学评估将包括压缩、拉伸和蠕变压痕试验。此外，微阵列分析将被用来研究在使用外部刺激时可能出现的潜在协同效应。最后，工程化的构造将被植入裸鼠体内，以检查其生存能力和稳定性。 公共卫生意义：仿生纤维软骨的成功生成将是向治疗TMJ半月板和下颌软骨表面的损伤或退变迈出的一大步，但在实现这一目标之前，还需要完成许多工作。天然纤维软骨的成分和力学性能的异质性，进一步反映在它们不同的功能上，需要开发一系列组织来再生。在确定最佳软骨素酶ABC、生长因子和机械刺激处理的过程中，三种细胞比例组各一种，这项建议将深入了解产生一系列具有不同机械和组成特性的纤维软骨的过程，从生物仿生学上重建TMJ纤维软骨中所见的特性范围。此外，微阵列分析将阐明这些外源刺激的作用。工程构建的可行性和稳定性也将在免疫缺陷动物模型中进行研究。然后，该方案产生的使能技术不仅可以应用于TMJ半月板或下颌软骨的一个特定区域的组织工程，而且还可以应用于其他纤维软骨系统(例如，膝关节半月板)。

DESCRIPTION (provided by applicant): The objective of this design-driven proposal is to optimize and employ scaffold-free co-cultures for the generation of biomimetic fibrocartilages for the repair or replacement of the temporomandibular joint meniscus and the mandibular cartilage surfaces. Using co-cultures of fibrochondrocytes and articular chondrocytes, we recently generated large constructs of clinically relevant dimensions that were fibrocartilage-like in appearance and composed of extracellular matrix (ECM) suggestive of fibrocartilage. Moreover, constructs similar to native tissue were achieved with fibrochondrocytes. Motivated by these findings, it is our hypothesis that the co- cultures can be optimized using bioactive agents and mechanical stimuli to form biomimetic fibrocartilage constructs. To address this hypothesis, we propose the following specific aims: 1) to optimize the use of chondroitinase ABC and the growth factors transforming growth factor -1 (TGF-1) and insulin-like growth factor 1 (IGF-I) using serum-containing medium or chemically defined medium in the scaffold-free co-culture of fibrocartilage; 2) to enhance the biomimetic fibrocartilage constructs with mechanical stimulation; and 3) to examine synergistic effects of bioactive agents and mechanical stimuli. Chondroitinase ABC has been found in preliminary studies to significantly increase construct tensile properties. Our group has also demonstrated that TGF-1 and IGF-I significantly increase ECM production in fibrocartilage constructs. The use of hydrostatic pressure and direct compression stimulation has been shown by our laboratory to have beneficial effects on articular cartilage and fibrocartilage constructs, and we will apply these stimuli individually and in combination to further enhance the constructs. Constructs will be examined histologically for glycosaminoglycan (GAG) and collagen, and immunohistochemically for collagen I and II. GAG, collagen, and DNA content will be quantified, followed by ELISA to measure collagen I and II. Biomechanical evaluation will include compression, tension, and creep indentation testing. Furthermore, microarray analysis will be used to study potential synergisms that may arise in the use of the exogenous stimuli. Finally, the engineered constructs will be implanted in the nude mouse to examine viability and stability. PUBLIC HEALTH RELEVANCE: Successful generation of biomimetic fibrocartilage would be a great stride toward the treatment of injuries or degeneration of the TMJ meniscus and mandibular cartilage surfaces, but much needs to be accomplished before this goal can be reached. The compositional and mechanical property heterogeneity of native fibrocartilages, further mirrored in their varying functions, necessitates development of a range of tissues for regeneration. In the course of identifying the optimal chondroitinase ABC, growth factor, and mechanical stimulation treatments, one for each of three cell ratio groups, this proposal will yield insight into the process of generating a spectrum of fibrocartilages with varying mechanical and compositional properties, biomimetically recreating the range of properties seen in the TMJ fibrocartilages. Furthermore, microarray analysis will elucidate the roles of these exogenous stimuli. The viability and stability of the engineered constructs will also be investigated in an immunodeficient animal model. The enabling technologies generated by this proposal can then be applied not only to tissue engineering one particular region of the TMJ meniscus or mandibular cartilage, but also to other fibrocartilaginous systems (e.g., knee meniscus) as well.