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

描述（由申请人提供）：本设计驱动提案的目的是优化和采用无支架共培养物，以产生仿生纤维软骨，用于修复或置换颞下颌关节半月板和下颌软骨表面。使用纤维软骨细胞和关节软骨细胞的共培养物，我们最近产生了具有临床相关尺寸的大型结构，其外观类似纤维软骨，并且由暗示纤维软骨的细胞外基质（ECM）组成。此外，类似于天然组织的结构用纤维软骨细胞实现。受这些发现的启发，我们假设可以使用生物活性剂和机械刺激来优化共培养物以形成仿生纤维软骨构建体。为了验证这一假设，我们提出了以下具体目标：1）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）在无支架纤维软骨共培养中的使用，使用含血清培养基或化学成分确定的培养基;和3）检查生物活性剂和机械刺激的协同效应。初步研究发现软骨素酶ABC可显着提高结构的拉伸性能。我们的研究小组还证明，TGF-1和IGF-I显着增加纤维软骨结构中ECM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具有有益的影响，我们将单独和组合应用这些刺激以进一步增强结构。将对构建体进行糖胺聚糖（GAG）和胶原蛋白的组织学检查，以及胶原蛋白I和II的组织化学检查。将定量GAG、胶原蛋白和DNA含量，然后通过ELISA测量胶原蛋白I和II。生物力学评价将包括压缩、拉伸和蠕变压痕试验。此外，微阵列分析将被用来研究潜在的协同作用，可能会出现在使用外源性刺激。最后，将工程化构建体植入裸鼠中以检查存活力和稳定性。 公共卫生相关性：仿生纤维软骨的成功产生将是治疗TMJ半月板和下颌骨软骨表面损伤或退化的一大步，但在实现这一目标之前还需要完成很多工作。天然纤维软骨的组成和机械性质的异质性，进一步反映在其不同的功能，需要开发一系列组织再生。在确定最佳的软骨素酶ABC，生长因子和机械刺激治疗的过程中，三个细胞比例组中的每一个，该建议将产生洞察到的过程中产生的纤维软骨的频谱具有不同的机械和组成特性，biomirobiology重建的TMJ纤维软骨中看到的属性范围。此外，微阵列分析将阐明这些外源性刺激的作用。还将在免疫缺陷动物模型中研究工程化构建体的活力和稳定性。然后，由该提议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，而且可以应用于其他纤维软骨系统（例如，膝关节半月板（knee meniscus）。

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.