A bone to pick…

Almost 9 million people suffer from osteoarthritis in the UK – yet we know remarkably little about the disease. A multidisciplinary team of experts at the University of Manchester and Royal Veterinary College plan to change that – here they tell us about their recent collaboration combining osteoarthritis research with 3D synchrotron imaging

Have you ever experienced an aching pain in your joints when you stand up? Or stiffness after periods of rest? Throughout life, our joints change continuously and adapt to the varying and often huge demands placed upon them as we walk, run, lift and jump. However, as we get older our joints undergo structural changes that may lead to the aching and stiffness that is so often associated with ageing.

Osteoarthritis is the most important ageing-related disease, affecting almost 9 million people in the UK. It is estimated that more than 33% of the UK population aged over 45 have sought treatment for osteoarthritis. Joints, most often knees and hips, undergo structural deterioration with the loss of the articular cartilage that normally covers the ends of bones to allow pain free joint movement. This exposes the underlying bone which itself undergoes changes causing it to become thicker and stiffer, and this causes its sufferers much pain, disability and subsequent impairment of normal activities and quality of life.

Current osteoarthritis treatments are limited and largely consist of the use of pain-killers and physiotherapy

For this reason, osteoarthritis is a major financial, social and healthcare burden. Current osteoarthritis treatments are limited and largely consist of the use of pain-killers and physiotherapy. In some individuals, osteoarthritis progresses to the extent that total joint replacement is required. Currently we are unable to identify the more vulnerable individuals, to prevent the progression of their disease or to treat those at earlier disease stages to slow disease momentum. To change this, we must identify the genes, molecules and processes which cause osteoarthritis.

We know from veterinary studies that canine hip dysplasia, a hereditary predisposition to osteoarthritis, is more common in larger breeds of dogs which tend to grow more rapidly. Whilst no direct link has been made between growth dynamics and osteoarthritis, recent murine and human studies have prompted us to speculate that genetic abnormalities and subsequent deformities resulting from altered mechanical loading may contribute to disease onset and progression. Prior studies have suggested that cells in the articular cartilage undergo uncharacteristic changes, which may result in the deterioration of this tissue. However, little is known of the involvement of a further type of cartilage critical for bone lengthening, known as growth plate cartilage.

Our joint collaboration has combined our expertise in osteoarthritis research and synchrotron X-ray computed tomography – advanced image processing and computational modelling – to investigate whether there is a correlation between 3D high resolution images of growth plate cartilage topology and osteoarthritis onset. For this we used a mouse model, the STR/Ort mouse, which naturally develops knee osteoarthritis with ageing (at around 40 weeks of age) and shows similar disease symptoms to the human condition. Little is known about why these mice develop osteoarthritis and answering this will help address problems in treating osteoarthritis in humans. The growth plate cartilage topology was compared in these osteoarthritis-prone STR/Ort mice to their closest available parental strain, the CBA mouse which, in contrast, displays very low spontaneous osteoarthritis susceptibility.

Using synchrotron X-ray microtomography on the Diamond Manchester Imaging Branchline (I13-2) of the Diamond Light source – we visualised, and subsequently quantified, bony bridges forming across the length of the growth plate cartilage indicating fusion and the cessation of growth.

Imaging of entire intact knee joints in three dimensions and at high resolution (~3 ?m) is of great importance in enabling comparable longitudinal studies of bony bridge formation and building anatomy specific finite element models. The most common investigation techniques are based on optical microscopy, electron microscopy or atomic force microscopy – which restrict the exploration volume and rely on damaging sample preparation and serial sectioning. The Diamond-Manchester branchline can deliver in line phase contrast and high flux monochromatic X-rays over a large field of view, in the 5-35 keV energy range, which is ideal for scanning entire intact joints while producing good contrast. A typical 3D reconstruction of the joint and 2D slice through the image volume is shown in Figure 1A revealing the intricate details of the growth plate cartilage (Figure 1B) and bony bridges (Figure 1C).

[caption id="attachment_58329" align="alignnone" width="620"] Figure 1. A) 3D representation of the 40+ week STR/Ort joint; B) 3D representation of the growth plate cartilage (yellow) underneath the tibial joint surface (grey, a); C) 3D representation of bridges crossing the growth plate underneath the tibial joint.[/caption]

To quantify the bone bridge local number density, a method for projecting them onto the joint surface was developed. After projection, the distribution of the areal density of bridges was calculated and superimposed on the tibial joint surface, allowing location and number to be mapped in a single 3D image (Figure 2). The areal density, d, is defined as the number of bridges per 256 ?m x 256 ?m window and the histograms presented here were obtained with a class interval size of ?d=1 (Figure 2).

[caption id="attachment_58330" align="alignnone" width="620"] Figure 2. Location and areal density of bridges across the growth plate projected on the tibial joint surface: A) STR/ Ort 8 weeks; B) CBA 8 weeks; C) STR/Ort 40+ weeks; D) CBA 40+ weeks; E and F, Number of bridges per tibia in CBA and STR/Ort mice at 8 weeks of age (E) and ?40 weeks of age (F). The lateral and medial segments and anterior and posterior segments were split in order to examine whether bridging is balanced during fusion. G, Areal density (d) of bridges, defined as the number of bridges per 256 ?m × 256 ?m window. Bars in E-G show the mean?±?SEM (n?=?3 mice per group). ?
?=?P < 0.05; ???=?P < 0.01; ????=?P < 0.001, versus CBA mice except where indicated otherwise.[/caption]

These results show an accelerated growth phenotype in the mice that could contribute to their disease pathology

This method allowed us to directly test whether longitudinal growth, growth plate fusion and osteoarthritis exhibit inter relationships in the STR/Ort mice. Detailed 3-dimensional scanning revealed that the growth plate cartilage began to close prematurely in STR/Ort mice by forming bony bridges across its length prior to the onset of osteoarthritis disease. This bridging was excessive and was more common in areas in which the osteoarthritis was at its most severe in the aged mouse. These results show an accelerated growth phenotype in the mice that could contribute to their disease pathology. The clustering of bony bridges found in late osteoarthritis suggests that their formation is driven by mechanical factors likely influenced by the local topology of the growth plate, which gives us an important insight into how this disease progresses.

These methods provide an alternative to 2D destructive techniques and a powerful tool for the study of complex biological tissues, allowing us to discriminate osteoarthritic disorders. Our novel method for 3D quantification of bony bridging and our finite element models will also no doubt advance understanding of growth plate closure mechanisms. This work has also enabled us to examine the molecular signalling pathways which may underpin the accelerated growth plate bridging and osteoarthritis susceptibility seen. Targeting of both mechanical and molecular signalling pathways may provide future strategies upon which therapies for osteoarthritis can be developed, a growing need in our ageing-population.

Authors:

Dr Katherine Staines is a postdoctoral researcher at the Roslin Institute, University of Edinburgh. She is also the New Investigator representative for the Bone Research Society.

Dr Kamel Madi is a postdoctoral scientist at the University of Manchester.

Professor Andrew Pitsillides is Professor of Skeletal Dynamics at the Royal Veterinary College, London

Professor Peter Lee is Professor of Material Imaging at the Research Complex at Harwell, University of Manchester

Contact details  katherine.staines@roslin.ed.ac.uk

Further information  www.diamond.ac.uk/Home/Corporate-Literature/Annual-Review.html. Staines et al., ARTHRITIS & RHEUMATOLOGY Vol. 68, No. 4, April 2016, pp 880–891