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

项目总结 摘要颞下颌关节盘退变和穿孔是导致TMJ衰弱的主要原因。 人们的疾病，而目前的治疗方法充其量仍是姑息治疗。使用新的组织工程方法 同种异体软骨细胞可以在生物学和机械性能方面产生与天然软骨相似的组织。 然而，很少有人注意到工程化组织受体的免疫学方面。 界面。了解和定义影响此界面的关键参数在快速 组织工程和TMJ盘再生的发展领域。 MHC-I类相容性是确定组织植入物接受度的主要参数之一。 软骨细胞组成性地表达MHC I，因此可以被识别为非自我并随后被破坏 CD8细胞毒性T细胞。后一个前提挑战了传统的免疫特权地位的概念 软骨组织。因为MHC在组织移植中的兼容性还没有在 TMJ或其椎间盘，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。我们假设组织工程化的结构来源于 MHC I相合的软骨细胞在修复TMJ关节盘缺陷方面将有更好的长期结果 与来自不匹配的软骨细胞的构建相比。此外，通过剖析主机的类型 MHC-I配型与失配小型猪对TE软骨构筑的炎症反应 受者，我们将确定免疫反应的类型是否可以预测移植的长期结果 TMJ光盘。 在原代软骨细胞中还没有尝试过MHC I沉默或敲除，但已经实现了 在其他类型的原代细胞中成功。我们的特定目标二将产生MHC I-空的初级软骨细胞 通过使用CRISPR/Cas9敲除β2巨球蛋白基因。我们假设同种异体 与WT相反，与MHC I缺失的软骨细胞共培养时，淋巴细胞的增殖能力会降低 软骨细胞，机械坚固和具有生物功能的植入物可以从 MHC I阴性软骨细胞。如果成功，这一战略将为制造 免疫万能TMJ软骨植入物。 我们建议在小型猪动物模型上进行拟议的实验，这是一种很好的- 建立了用于人类TMJ翻译研究的大动物模型。我们的团队有丰富的经验和 具有使用此模型的专业知识。总而言之，这个项目将提供关于 TMJ隔室的免疫学，并将促进再生模式的临床翻译 TMJ光盘。

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.