Tissue Engineering of Immuno-Universal Cartilage Implants for Temporomandibular Joint Disc Regeneration

项目摘要 颞下颌关节盘退变和穿孔是导致颞下颌关节衰弱的主要原因 然而，目前的治疗充其量只是治标不治本。新的组织工程方法， 同种异体软骨细胞可以产生在生物学和机械性能方面类似于天然的组织。 然而，很少有人关注工程组织受体的免疫学方面 接口.理解和定义影响该接口的关键参数对于快速 为组织工程和颞下颌关节盘再生开辟了新的研究领域。 MHC I类相容性是定义组织植入物可接受性的主要参数之一。 软骨细胞组成型表达MHC I，因此可以被鉴定为非自身的，随后被破坏 CD 8细胞毒性T细胞。后一个前提挑战了免疫豁免状态的传统概念。 软骨组织由于组织移植中的MHC相容性尚未在以下背景下探索： 我们的第一个目标是确定MHC I类匹配在TMJ椎间盘修复中的相关性， 同种异体细胞来源的组织工程软骨。我们假设组织工程构建物来源于 从MHC I匹配的软骨细胞将有更好的修复颞下颌关节盘缺损的长期结果 与来源于错配软骨细胞的构建体相比。此外，通过剖析宿主的类型， MHC I匹配与不匹配小型猪对TE软骨构建体的炎症反应 接受者，我们将确定免疫反应的类型是否可以预测植入的长期结果， 颞下颌关节盘 MHC I沉默或敲除尚未尝试在原代软骨细胞中进行，但已实现 在其他类型的原代细胞中成功。我们的具体目标二将产生MHC I无效的原代软骨细胞 通过使用CRISPR/Cas9敲除β 2巨球蛋白基因。我们假设同种异体 与WT相比，在与MHC I缺失的软骨细胞共培养中，淋巴细胞增殖将降低 软骨细胞，并且机械上坚固的和生物学功能的植入物可以由 MHC I无效软骨细胞。如果成功，这一战略将为制造 TMJ软骨植入物。 我们建议在小型猪动物模型中进行所提出的实验，这是一个很好的- 建立了人颞下颌关节转化研究的大型动物模型。我们的团队经验丰富， 专业知识来使用这个模型。总之，该项目将提供关于 TMJ区室的免疫学，并将促进再生模式的临床翻译， 颞下颌关节盘。

PROJECT SUMMARY Temporomandibular joint (TMJ) disc degeneration and perforation are the leading causes of debilitating TMJ disorders in people, while current treatments remain palliative at best. Novel tissue-engineering methods using allogeneic chondrocytes can produce tissue akin to native in terms of biological and mechanical properties. Little attention, however, has been given to the immunological aspects of the engineered tissue-recipient interface. Understanding and defining key parameters that affect this interface are critical in the rapidly developing field of tissue-engineering and TMJ disc regeneration. MHC class I compatibility is one of the major parameters defining acceptance of the tissue implant. Chondrocytes constitutively express MHC I and thus can be identified as non-self and subsequently destroyed by CD8 cytotoxic T cells. The latter premise challenges the conventional concept of immunoprivileged status of cartilage tissue. Because MHC compatibility in tissue transplantation has not been explored in the context of TMJ or its disc, our first aim will determine the relevance of MHC class I matching in TMJ disc repair using allogenic cell-sourced tissue-engineered cartilage. We postulate that tissue-engineered constructs sourced from MHC I-matched chondrocytes will have better long-term outcomes in the restoration of TMJ disc defects as compared to constructs sourced from mismatched chondrocytes. Further, by dissecting the type of host inflammatory response towards TE cartilage constructs in MHC I matched versus mismatched minipig recipients, we will determine if the type of immune response can predict long-term outcomes of implantation in the TMJ disc. MHC I silencing or knockout has not been attempted in primary chondrocytes but was achieved successfully in other types of primary cells. Our specific aim two will generate MHC I-null primary chondrocytes by knocking out the beta 2 macroglobulin gene using CRISPR/Cas9. We hypothesize that allogeneic lymphocyte proliferation will be decreased in coculture with MHC I-null chondrocytes as opposed to WT chondrocytes and that mechanically robust and biologically functional implants can be manufactured from MHC I-null chondrocytes. If successful, this strategy will provide a novel platform for the manufacturing of immunouniversal TMJ cartilage implants. We propose to carry out the proposed experiments in the minipig animal model, which is a well- established large animal model for translational studies of human TMJ. Our team has ample experience and expertise in working with this model. In summary, this project will deliver essential information on the immunology of the TMJ compartment and will boost the clinical translation of regenerative modalities of the TMJ disc.