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

描述（由申请人提供）：这个设计驱动的提案的目的是优化和使用无支架共培养来生成仿生纤维软骨，用于修复或替换颞下颌关节半月板和下颌软骨表面。通过纤维软骨细胞和关节软骨细胞的共培养，我们最近生成了具有临床相关尺寸的大型结构，其外观呈纤维软骨样，由提示纤维软骨的细胞外基质（ECM）组成。此外，用纤维软骨细胞实现了与天然组织相似的结构。基于这些发现，我们假设可以使用生物活性剂和机械刺激来优化共培养，以形成仿生纤维软骨结构。为了解决这一假设，我们提出以下具体目标：1)在无支架的纤维软骨共培养中，使用含血清培养基或化学定义的培养基优化软骨素酶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-I）的使用；2)机械刺激增强仿生纤维软骨构建体；3)研究生物活性剂和机械刺激的协同效应。在初步研究中发现，软骨素酶ABC可显著提高结构物的拉伸性能。我们的团队还证明了TGF-1和igf -1显著增加纤维软骨结构中ECM的产生。我们的实验室已经证明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，我们将单独或联合应用这些刺激来进一步增强结构。构建体将进行糖胺聚糖（GAG）和胶原蛋白的组织学检查，以及I和II型胶原蛋白的免疫组织化学检查。测定GAG、胶原蛋白和DNA含量，ELISA法测定胶原蛋白I和II含量。生物力学评估将包括压缩、张力和蠕变压痕测试。此外，微阵列分析将用于研究在使用外源刺激时可能产生的潜在协同作用。最后，工程构建体将被植入裸鼠体内以检测其生存能力和稳定性。

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.