Developing patient-derived organoids to dissect the cellular & molecular mechanisms underpinning resolving and persistent forms of arthrofibrosis.

关节纤维化是由于过度瘢痕组织形成导致的关节病理性硬化，称为纤维化。肩周炎是一种影响肩关节的关节纤维化，通过构成肩关节的韧带（囊）的深度僵硬导致疼痛和残疾。肩周炎是一种独特的关节纤维化形式，因为这种情况会随着时间的推移而成功地消退。相反，其他关节的纤维化疾病持续存在，例如在患者接受膝关节置换术后的膝关节。该项目将利用纤维化如何在肩关节成功解决的知识来理解为什么纤维化在其他关节（如膝关节）持续存在。我们收集了来自非患病比较者和冷冻肩关节患者的组织样本，以生成包含肩关节囊的细胞类型图谱。该图谱显示，肩囊由成纤维细胞组成，成纤维细胞在产生构成胶原组织（如韧带）的蛋白质方面具有重要功能。我们还发现了巨噬细胞，这些免疫细胞具有调节炎症和纤维化的重要功能，两者都发生在冻疮期间。研究人员利用来自冻肩患者的成纤维细胞和巨噬细胞，发现溶解性巨噬细胞和成纤维细胞之间的相互作用可以抑制炎症并促进纤维化消退，这支持了我们的假设，即成纤维细胞和巨噬细胞是介导纤维化消退的关键细胞类型。我们还证实，这些与解决肩周炎有关的细胞在胎儿发育过程中也存在，这表明解决纤维化的模板可能在发育过程中被“印记”。控制纤维化是否消退或持续存在的生物学过程仍有待确定。从纤维化如何在肩关节成功解决的知识有可能告诉我们如何推动像膝关节纤维化这样的持续性纤维化疾病走向解决的轨道。该项目的总体目标是确定驱动膝关节囊持续性关节纤维化的关键细胞类型和分子，并了解与肩关节相关的任何差异。该项目将创建人类膝关节囊在发育、未患病和纤维化状态下的细胞图谱，这样细胞群就可以直接与纤维化消退的肩关节中的细胞群进行比较。该项目还将利用由患者来源的细胞组成的已建立的组织培养模型，以确认导致纤维化和分解的细胞类型和分子，并测试小分子以缓和纤维化。小鼠和啮齿动物模型经常用于研究关节疾病，包括骨关节炎和关节纤维化。这些动物模型并不能准确地概括等效的人类疾病，其中一些动物模型会引起相当大的疼痛、不适和痛苦。该项目将利用从人类患者身上收集的组织和细胞来代替动物模型来研究骨关节炎和关节纤维化。该项目将率先开发被称为类器官的3D模型来研究膝关节纤维化，利用无动物产品来构建我们将使用的类器官模型。随着时间的推移，随着研究人员采用本项目中使用的方法，预计本研究的结果将进一步减少用于研究影响关节及其他部位的纤维化疾病的动物数量。本研究将取代和减少对动物关节纤维化模型的需求，确定新的治疗方法来促进关节纤维化的消退，解决患者未满足的临床需求。有效的新型关节纤维化治疗将减少与活动能力降低相关的合并症的发展，并减少与关节置换术相关的医疗保健经济负担。

Arthrofibrosis is the pathologic stiffening of a joint due to exaggerated scar tissue formation, referred to as fibrosis. Frozen shoulder is a form of arthrofibrosis affecting the shoulder causing pain and disability through profound stiffening of the ligaments (capsule) that comprise the shoulder joint. Frozen shoulder is a unique form of arthrofibrosis because the condition successfully resolves over time. In contrast, fibrotic diseases of other joints persist, for example in the knee joint after patients undergo surgical knee replacement. This project will harness the knowledge of how fibrosis successfully resolves in the shoulder joint to understand why fibrosis in other joints such as the knee persist. We collected tissue samples from non-diseased comparator and frozen shoulder patients to generate an atlas of the cell types comprising the shoulder joint capsule. This atlas revealed that the shoulder capsule is comprised of cells called fibroblasts which have important functions in producing the proteins that comprise collagenous tissues like ligaments. We also identified macrophages, these are immune cells with important functions regulating inflammation and fibrosis which both occur during frozen shoulder. Using fibroblasts and macrophages derived from frozen shoulder patients, we discovered that crosstalk between resolving macrophages and fibroblasts dampen down inflammation and promote resolution of fibrosis, supporting our hypothesis that fibroblasts and macrophages are key cell types mediating fibrosis resolution. We also established that these cells implicated in resolving frozen shoulder are also present during foetal development, suggesting that the template to resolve fibrosis could be 'imprinted' during development. The biological processes that govern whether fibrosis resolves or persists remain to be identified. Knowledge from how fibrosis successfully resolves in the shoulder joint has the potential to inform how we could push persistent fibrotic diseases like knee arthrofibrosis towards a resolving trajectory. The overarching aim of this project is to identify the key cell types and molecules that drive persistent arthrofibrosis in the knee joint capsule and understand any differences relative to the shoulder joint. This project will create a cellular atlas of the human knee capsule during development, non-diseased and fibrotic states such that the cell populations can be directly compared with those in the shoulder joint where fibrosis resolves. The project will also utilise established tissue culture models comprised of patient-derived cells to confirm the cell types and molecules causal to fibrosis and resolution and test small molecules to moderate fibrosis. Murine and rodent animal models are frequently used to study joint diseases including osteoarthritis and arthrofibrosis. These animal models do not accurately recapitulate the equivalent human disease, some of these animal models cause considerable pain, discomfort and suffering. This project will utilise tissues and cells collected from human patients to replace the use of animal models to study osteoarthritis and arthrofibrosis. The project will pioneer the development of 3D models called organoids to study knee arthrofibrosis, utilising animal free products to construct the organoid models we will work with. Over time, as researchers adopt the approaches utilised in this project, it is anticipated that the findings from this research could further reduce the number of animals used to study fibrotic disease affecting the joint and beyond.This research will replace and reduce the requirement for animal arthrofibrosis models, identify new therapies to promote arthrofibrosis resolution, addressing an unmet clinical need for patients. Effective new arthrofibrosis treatments will reduce the development of co-morbidities associated with reduced mobility and reduce the healthcare financial burden for costs associated with joint replacement.