Tissue Engineering of Neotrachea

描述(申请人提供)：目前，对于大于6厘米的气管节段的替换，尚无适当的治疗方法。这些研究的目标是利用组织工程学原理在兔模型中开发和制造具有功能的气管替代物，并在无菌大鼠模型中使用人类细胞制造人类大小的新气管。该实验室开发了一种生产大型无支架软骨片的方法，当这种软骨片植入体内并被筋膜或肌肉包围时，会产生一种血管化的新气管结构。将对兔耳和肩软骨细胞进行测试和优化，以生产软骨片，并将在气管节段性缺陷模型中测试其长期稳定性和功能。这些片材首先在定制的双扩散生物反应器中制造，然后将其植入未来气管节段性修复部位的近端，在那里，在节段性气管重建之前，它被允许成熟大约12周。将新生气管沿其血管移植到节段性气管缺损处，分别于移植后4、8、12周取材。将测试三种不同的手术形式，包括单独的直接移植，T管移植(帮助呼吸和促进修复)，以及T管移植和面颊粘膜游离移植。通过组织学和免疫组织化学方法(包括糖胺多糖染色、I型、II型和X型胶原蛋白和弹性蛋白的免疫组织化学)对收获的新生气管进行评估，组织学检查结构、组织整合、上皮化，并评估生物力学强度。将特别注意纤维化、粘膜的形成，以及新气管内是否有骨形成。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学，生产一种功能性气管来修复兔的节段性缺陷，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软骨细胞，并将首先使用软骨形成聚集体培养系统测试一组生长因子和细胞因子，从而优化软骨细胞的形成。然后，将在无菌大鼠模型中测试人类软骨细胞衍生的片材在体内形成人类大小的新气管的能力，并将通过组织学、生化和机械分析进行评估。

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.