Study of early pathological changes in calcified cartilage in osteoarthritis and alkaptonuria

We are seeking a motivated individual to work on this exciting project investigating the role that the calcified cartilage has in rare and common joint osteoarthritis, using novel approaches such as gene silencing and establishment of a 3D chondrocyte cell culture model.

OA, a world-wide healthcare burden, has no effective disease modifying treatment available. There is a need to understand the mechanism(s) by which cartilage destruction occurs in OA to pinpoint new therapeutic targets. Studying human OA is difficult and limited to imaging modalities and donated tissue with end-stage disease in which it is too late to unravel a mechanism. Researching rare forms of OA however can uncover disease mechanisms amplified or accelerated by genetic defects. Trabecular excrescences and high-density mineralised protrusions are example of osteoarthritic features first identified in the rare disease alkaptonuria (AKU)^[1]. AKU is a degenerative disorder characterised by tissue pigmentation (ochronosis) leading to severe and early onset OA. In AKU, homogentisic acid (HGA) is not metabolised by the liver and enters the circulation, and overtime causes pigmentation of connective tissues, particularly cartilage.

By studying AKU, this project aims to understand the pathophysiological changes that articular cartilage undergoes with natural ageing that firstly allows pigment to bind, and secondly how this pigmentation leads to severe OA. This project will use murine models of OA such as STR/Ort in collaboration with Prof Pitsillides lab^[2], and AKU mice known as Hgd^-/- generated by us here in Liverpool^[3], alongside in vitro work. The calcified cartilage, which is very understudied, is an area of focus in this project, as both AKU and STR/Ort mice both show early pathological changes, such as pigmentation and hypertrophy respectively, before changes in non-calcified articular cartilage are observed ^[4,5].

Objectives

1. Investigate the role of calcified cartilage chondrocytes in OA initiation and progression by combining the AKU and STR/Ort phenotypes in mice.
2. Further characterise the calcified cartilage of AKU mice using high resolution synchrotron CT imaging, nanoscale atomic force microscopy (AFM) and mechanical testing.
3. Establish 3D chondrocyte cell culture and an ex vivo cartilage degradation model mimicking AKU via addition of HGA, to investigate the process of pigmentation and subsequent effect on the transcriptome, proteome and biomechanical properties.

Novelty

• This is an exciting opportunity to combine two murine OA models with calcified cartilage pathology, using a novel siRNA approach to knockdown Hgd expression to induce the AKU phenotype in STR/Ort mice.
• We will use imaging techniques that have not been used before in AKU, such as synchrotron CT (Prof Pitsillides lab) and atomic force microscopy here at Liverpool. Early cartilage changes are nearly impossible to study in living AKU patients.

Experimental approach

A Hgd gene silencing method will be used to induce a metabolic AKU phenotype in STR/Ort mice to determine if genetic predisposition to greater levels of chondrocyte hypertrophy will increase susceptibility to a more marked ochronotic phenotype and increased severity of OA. Histological analysis of pigmentation and OA severity will be carried out.

This project will involve working with mouse models of disease as mentioned above, cell culture and ex vivo cartilage degradation assays, using techniques such as enzymatic degradation assays, qPCR, histology, mechanical testing and various omics methodologies. Training will be provided.

Potential impact

It is estimated that AKU alone costs the NHS/economy approximately £680,526 per patient in the UK across their lifetime. This work will inform new treatments to prevent/treat AKU osteoarthropathy, in addition to providing mechanistic insights into the pathophysiology of general OA which is a major age-related chronic disease and global healthcare burden for which no disease modifying treatment is currently available.

Research environment and opportunities

The successful candidate will join the wider AKU research group at the University of Liverpool, where we have close links to the National Alkaptonuria Centre within the Royal Liverpool University Hospital and the AKU Society (a patient charity). Opportunities will arise such as attending international scientific AKU meetings and AKU patient workshops, bursary applications to travel to another lab to learn new techniques, and collaboration with the Royal Veterinary College (London) via Professor Pitsillides Lab who have expertise of STR/Ort mice.

Applications and enquiries

For informal enquiries and/or to apply for this position, please send a CV and cover letter directly to Dr Juliette Hughes via email – [email protected].