Tissue Engineering of Neotrachea

描述（由申请人提供）：目前，没有足够的治疗方法可用于置换大于6厘米的气管段。这些研究的目标是利用组织工程原理在兔模型中开发和制造功能性气管替代物，并在胸腺模型中使用人类细胞制造人类大小的新气管。本实验室开发了一种生产大型无支架软骨片的方法，当植入体内并被筋膜或肌肉包围时，产生血管化的新气管结构。兔耳和兔肩软骨细胞将在气管节段性缺损模型中测试和优化其软骨片的生产，并测试其长期稳定性和功能。首先在定制的双扩散生物反应器中制作薄片，然后将其植入气管近端未来节段修复部位，在此让薄片成熟约12周，然后进行节段气管重建。新气管沿其脉管系统移植至节段性气管缺损，并于移植后4、8和12周切除。将测试三种不同的手术形式，包括直接移植，t型管移植（辅助呼吸和促进修复），t型管移植和无粘膜移植。采集的新气管通过组织和免疫组织化学方法进行评估，包括糖胺聚糖染色，I型、II型和X型胶原和弹性蛋白的免疫组织化学染色，从组织学上检查结构、组织整合、上皮化，并评估生物力学强度。特别要注意的是纤维化，粘膜的形成，以及新气管内是否形成骨。可以采用将上皮细胞应用于工程组织的替代方法。长期目标是开发一种方法来制造功能性气管来修复兔子的节段性缺陷，并将这项技术转化为人类来源的细胞。为此，在对兔软骨细胞进行优化研究的同时，将从气管、耳朵、鼻子和关节表面获得人类软骨细胞，并将通过使用软骨形成聚合培养系统首先测试一组生长因子和细胞因子来优化软骨片的形成。人类软骨细胞衍生的薄片将在无胸大鼠模型中进行测试，以测试它们在体内形成人类大小的新气管的能力，并将通过组织学、生化和力学分析进行评估。

DESCRIPTION (provided by applicant): Currently, there is no adequate treatment available for the replacement of tracheal segments larger than 6 cm. The goal of these studies is to develop and fabricate a functional trachea replacement in a rabbit model using tissue engineering principles, and to also fabricate a human-sized neotrachea using human cells in an athymic rat model. This laboratory has developed the methodology to produce large scaffold-free cartilage sheets, which when implanted in vivo and surrounded by fascia or muscle, produces a vascularized neotracheal construct. Rabbit ear and shoulder chondrocytes will be tested and optimized for cartilage sheet production and will be tested for their long-term stability and function in a trachea segmental defect model. The sheets are first fabricated in a custom double diffusion bioreactor and then implanted proximal to the future segmental repair site in the trachea where it is allowed to mature for approximately 12 weeks prior to segmental tracheal reconstruction. The neotrachea is transplanted along its vasculature into segmental tracheal defect and will be harvested at 4, 8 and 12 weeks postimplantation. Three distinct surgical formats will be tested including the direct transplant alone, transplant with a T-tube (to assist breathing and promote repair), and transplant with T-tube along with cheek mucosal free transplant. The harvested neotracheas are assessed by histologic and immunohistochemical methods including staining for glycosaminoglycans, and immunohistochemistry for collagens type I, II, and X, and elastin, examined, histologically, for structure, tissue integration, epithelialization, and are evaluated for biomechanical strength. Particular attention will be paid to the fibrosis, the formation of a mucous membrane, and also whether bone forms within the neotrachea. Alternative approaches to apply epithelial cells to the engineered tissue may be employed. The long-term goals are to develop the methodology to produce a functional trachea to repair segmental defects in rabbits, and to translate this technology to human derived cells. To this end, in parallel with the optimization study of rabbit chondrocytes, human chondrocytes will be obtained from the trachea, ear, nose and from articular surfaces and will be optimized for cartilage sheet formation by first testing a bank of growth factors and cytokines using a chondrogenesis aggregate culture assay system. Human chondrocyte-derived sheets will then be tested in an athymic rat model for their ability to form a human-sized neotrachea in vivo, and will be assessed by the histologic, biochemical and mechanical assays.