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

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.

Fibrotic conditions are a significant global disease burden. While some therapies delay disease progression, none reverse fibrosis. To understand how fibrosis might resolve, we developed a comparative single-cell atlas of capsule tissues from foetal, adult comparator & frozen shoulder, an unusually self-limiting chronic inflammatory fibrotic human disease affecting the shoulder joint. We identified a population of MERTK+ macrophages (Macs) enriched for negative regulators of inflammation, supporting the hypothesis that MERTK+Macs provide a resolving fibrotic niche in the shoulder capsule. Micro-cultures of patient-derived cells identified cell-matrix interactions between MERTK+Macs, DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Single-cell profiling & spatial analysis of human foetal shoulder tissues identified MERTK+Macs, DKK3+ and POSTN+ fibroblast populations analogous to those identified in adult shoulder capsule, suggesting that the template to resolve fibrosis is established during development. Having identified the cell types & molecules active in a resolving fibrotic niche, this project will determine if this mechanism is generalizable to other capsular tissues and if absent, could render them susceptible to persistent arthrofibrosis such as occurs in the knee. To replace animal models, this project will use well-phenotyped human tissues to construct a single-cell & spatial atlas to identify the cell types comprising the knee capsule during human embryonic development & in adult comparator & fibrotic knee capsule tissues. We will compare the cell types comprising the knee capsule where fibrosis persists relative to the resolving fibrotic niche in the shoulder capsule. Finally, we will develop in vitro functional assays comprised of patient-derived cells to confirm the cellular & molecular circuits causal to inflammatory fibrosis in the knee & test small molecules to resolve knee arthrofibrosis.