Joint Surgery and Cell Therapies

CARTILAGE RESEARCH GROUP

Directors: Professor Sally Roberts and the late Professor James Richardson

Members of the Research Team:

Karina Wright, Claire Mennan, Jan Herman Kuiper, Helen McCarthy, Johanna Wales, Charlotte Hulme, Sharon Owen,  John Garcia, Mike Williams,  Barbara Linklater-Jones, Paul Harrison, Nikki Kuiper and PhD students, Jessica Sykes, Jade Perry, Tim Hopkins and Jingsong Wang (Kobe).

Clinical Support:

Messrs Peter Gallacher, Paul Jermin, Drs Bernhard Tins, Andrea Bailey, Professor Iain McCall and all surgeons in RJAH

The sudden and unexpected death of Professor Richardson in February this year has impacted hugely on everyone in the group. We have tried to work through the huge sense of loss by throwing ourselves into continuing to work towards his vision of using science to improve our understanding of joint health and disease and continuing trialling and attempting to improve cell therapy as a treatment regime.  He would have been very pleased to see some of the work he was involved with, with one of his PhD students, Tim Hopkins, and Jan Herman Kuiper, be awarded a prize in Macau at the International Society of Cartilage Repair and Joint Preservation in March (see below). 

PREDICTION OF CLINICAL OUTCOME FOLLOWING AUTOLOGOUS CHONDROCYTE IMPLANTATION BY MAGNETIC RESONANCE IMAGING

Helen McCarthy, Iain McCall, Claire Mennan, Mike Williams, James Richardson and Sally Roberts

Funded by Arthritis Research UK and the MRC

Since 2010, we have been collating data from a retrospective study of autologous cell therapy (REACT), using a postal questionnaire completed by patients who have previously received this treatment for chondral/osteochondral defects of the knee here at this hospital.  We have used this group of patients to assess the quality of the repair tissue formed in the treated defect and to see how it correlates with clinical outcomes. The ability to predict long-term success of any surgical or clinical treatment is invaluable, particularly in clinical trials. Therefore such analyses may help us learn more about the success of this and other treatments.  Magnetic resonance images (MRIs) and histology of biopsies of the repair tissue which forms after treatment were used to determine the structure and make-up of the repair tissue.  The MRIs were ‘scored’ by assessing different parameters. We found that 6 features correlated with clinical outcome 12month after cell implantation and 3 of these features at 12months correlated with long term clinical outcome (up to 17 years).  This highlights the potential for MRIs to be used to predict the success of cartilage repair therapy and a patients’ long-term clinical outcome. The repair tissue biopsies themselves were very variable, with the majority being fibrocartilage. Despite no correlation between either histological score with overall clinical outcome, a poor quality cartilage or poor integration with the underlying subchondral bone was associated with increased pain levels.      Further analyses of the significant features found in all of these scores may actually provide an insight into the mechanisms of the cartilage repair process and yield information to further understand sources of pain not only in knees with focal cartilage defects, but also in more generalised osteoarthritis and ultimately lead to improved treatments. This work has just been accepted for publication.

BIOMARKER DISCOVERY IN ORTHOPAEDICS: MATCHING PATIENTS TO THE BEST TREATMENT

Charlotte Hulme, Heidi Fuller, Emma Wilson, Sally Roberts, Peter Gallacher, James Richardson, Sharon Owen, Karina Wright.

Funded by Arthritis Research UK

As in all treatments, a few patients who have their osteoarthritis treated by biological treatments, such as cell therapies, osteotomy or microfracture, do not respond so well as others, though we do not know why. Our previous work has identified particular molecules in synovial fluid or blood known as ‘biomarkers’ that have the potential to identify which patients are unlikely to improve clinically following the cell therapy known as autologous chondrocyte implantation (ACI). These investigations focussed on protein markers whose biology is known to relate to poor repair of cartilage or general inflammation e.g. an enzyme, aggrecanase-1, and CD14. Our recent work, however, has used a highly specialised technique called proteomics to identify larger numbers of candidate marker proteins which are altered within the synovial fluid of patients who either do or do not respond well to ACI. The marker proteins identified using this approach include many that have never been investigated before and which therefore provide attractive new candidates. To date, we have confirmed that four of these proteins, S100A13, MMP1, Complement C1S and MMP3, have the potential to inform us of whether or not a patient is suitable to continue through the ACI procedure. We have also used ‘bioinformatic’ analysis, using complex computer programmes, to inform us of how the ACI procedure may be effecting different biological mechanisms, such as ‘acute phase response signalling’ in individuals who improve following ACI compared to those who do not (Figure 1) and these studies may help us identify ways to improve ACI outcomes for patients in which it would not currently be successful.

We have now assessed whether any of these ‘biomarkers’ can help us predict what the outcome might be following osteotomy or microfracture. We have shown that amount of S100A13 and the total amount of all protein within the synovial fluid before an operation relates to the clinical functional scores that patients achieve a year after their osteotomy. We hope that this work will help us to identify a reliable panel of molecular markers which can be used to help the surgeon and patient decide on what is the best treatment for that particular person. This would not only save the health service money, but most importantly get people pain free and back to their normal lives quicker.

Figure 1: Acute Phase Signalling is an example of a biological pathway that is altered people who do poorly following ACI. Proteins in red were increased and proteins in green were decreased following initial ACI surgery compared to pre-operative levels. This signalling pathway is therefore likely to be a good target for new drugs to improve the outcome for individuals who currently do not benefit from ACI.

 

HUMAN MESENCHYMAL STROMAL CELLS FROM BONE MARROW AND UMBILICAL CORD EXPANDED IN THE QUANTUM® HOLLOW-FIBRE BIOREACTOR SYSTEM.

Claire Mennan* Karina Wright*, John Garcia, Charlotte Hulme & Sally Roberts (*Both authors contributed equally to the work

Funded by Arthritis Research UK and MRC

Human umbilical cord (UC) and bone marrow (BM) stromal cells (MSCs) have shown potential for use in cell therapies such as cartilage repair and may be immune privileged. For these cells to be used successfully, large numbers need to be grown up and stored in cell banks to provide an ‘off the shelf’ treatment for patients.  The Quantum® bioreactor (Fig 2; Terumo BCT) is an automated hollow fibre system, which has an internal surface area of 2.1m2 (equivalent to 120 T-175 tissue culture flasks), allowing large scale expansion of cells.  We have been using the Quantum® to assess the characteristics of cells from human BM and UC after expansion in the system compared to the standard tissue culture plastic technique (TCP), with the aim of using these cells in cell therapies for example, for cartilage repair.  We have found that the Quantum® system was capable of producing up to 200 Million cells in ~14 days for BM and ~8 days for UC.  Our results suggest that these cells have similar characteristics to those grown on TCP and that the Quantum® system can successfully be used to expand large numbers of potentially therapeutic MSCs from BM and UC tissues.

John Garcia and Karina Wright have also been testing chondrocytes in the Quantum® system, the first time that this has ever been attempted. So far cells from 3 samples obtained from adult donors (total knee replacements) have been cultured in the Quantum® and the results are currently being analysed. Techniques for chondrocyte growth and harvest using the system have been optimised and further work will use juvenile sources of chondrocytes (from limb amputations) to assess whether these cells are more therapeutically potent compared to adult chondrocytes.  

Figure 2: The Quantum® cell expansion system. A. Computerised incubator and control panel. B. Disposable hollow fibre bioreactor module composed of ~11,500 fibres.

 

 THE EFFECT OF BONE HEALTH AND ACTIVITY LEVELS ON THE HEALTH OF THE HUMAN KNEE.

Timothy Hopkins, Nicola Kuiper, Karina Wright, Sally Roberts, James Richardson, Jan Herman Kuiper

Funded by the Orthopaedic Institute Ltd

Osteoarthritis (OA) is the most common joint disease in the world, but its onset and progression are still not fully understood. OA was traditionally regarded as a disease of the articular cartilage (AC) as this is the tissue in which the degenerative changes associated with the disease are most prominent. However, there has been a shift in attitudes towards OA in more recent times to treat OA as an ‘organ-level failure’ that affects and involves all tissues of the joint rather than the AC alone. As a result of this, the involvement of the other tissues in the joint on the progression of OA has gained interest. Of these tissues, the subchondral bone (SB) is of particular interest given the close physical interaction and shared signalling pathways between the two. In this project, a series of experiments will be performed to test the effect of cells taken from healthy and unhealthy areas of the SB on the growth and activity of chondrocytes (cartilage cells), to try and characterise the relationship between the two tissues (Figure 3), increasing our understanding of OA and possibly highlighting new areas for treatment.

Figure 3: Isolating chondrocytes from the cartilage and subchondral bone cells from the subchondral bone.

 Another part of this project is based in the clinic. A patient’s knee function is known to vary over their lifetime. However, there also appears to be variation over a more short-term basis, week-to-week or even day-to-day. One factor thought to play a part in this variability is the patient’s activity levels. However, the precise effect of activity levels on the reported function of the knee, remains largely uncharacterised. This poses a problem for the clinical evaluation of knee function, as currently available validated scoring systems (such as the Lysholm score) do not accurately take activity levels into account and are therefore prone to unwanted variation on a short-term basis. Therefore understanding the relationship between knee function and activity levels may enable the creation of improved knee function scoring systems with an appreciation for different levels and types of activity. We also hypothesise that the relationship between activity levels and knee function may also depend on the individual patient’s attitude to pain and pain management strategies. In order to shed some light on these areas, we plan to use wearable activity trackers (e.g. FitBit) to monitor patients’ activity levels alongside validated scoring systems for knee function and attitudes to pain.

Further to these areas of study, we also carried out a Delphi consensus study to investigate the levels of agreement on some of the important factors involved in cartilage repair. There are a number of surgical techniques that are employed to repair or regenerate cartilage lesions in the human knee. However, there is currently no set of established, consistent criteria used to assess the repair cartilage resulting from these techniques. From a panel of experts, working in the field of cartilage, we obtained a preliminary collection of items that were agreed upon to be important in assessing and predicting the structural quality of cartilage repair. This collection can be used to predict the success of cartilage repair and to guide how it is assessed, as well as to influence the direction of future clinical and fundamental scientific research. We were lucky enough to be awarded the Young Investigator’s Award by the International Cartilage Repair and Joint Preservation Society (ICRS) at their biannual congress in Macau, China. 

Timothy Hopkins (left, presenting Author) and Dr Jan Herman Kuiper (right, supervisor) with the ICRS Young Investigator’s Award.

 

GROWING CHONDROCYTES IN THE LABORATORY FOR TREATING PATIENTS – WHAT HAPPENS IF WE MAKE IT MORE LIKE THE BODY?

Claire Mennan, John Garcia, Sharon Owen, Helen McCarthy, Karina Wright, James Richardson, Rob Banerjee and Sally Roberts

Chondrocytes (cartilage cells) destined for autologous chondrocyte implantation (ACI) of the knee are extracted from articular cartilage existing in a low oxygen (~2%, hypoxia) environment.  Growing these cells up in the laboratory requires their culture under much higher oxygen tensions (~21%, normoxia) that the cells are not used to.  It is well known that these cells change their morphology when grown in normoxia and begin to lose some of the properties associated with being able to form cartilage.  We hypothesised that culturing of chondrocytes under hypoxic conditions would maintain the properties associated with good cartilage formation and chondrogenic potency.  We used a fully hypoxic workstation to grow chondrocytes isolated from total knee replacements and used sister populations of these cells grown up in the usual way in normoxia with the aim of comparing the two conditions and their effect on the cells.  We hoped to find the best environment for culturing cells destined for treating patients to repair damaged cartilage and to try to understand their mechanism of action when returned to the patient.  Our results showed that whilst chondrocytes grown in hypoxia grew relatively slowly compared to normoxia, they retained the ability to produce collagen II (a major building block of cartilage) and retained their chondrocyte morphology.  The cells grown in normoxia proliferated much faster but could not produce collagen II and did not behave like chondrocytes.  The normoxic cells actually behaved much more like Mesenchymal Stromal/Stem Cells (MSCs) and produced molecules that ‘dampen’ down an immune response, that are normally associated with MSCs, whereas the hypoxic cultures did not.  We currently do not know what effect these two conditions might have on cartilage repair if these cells were to be used in vivo, but further work from the ASCOT clinical trial, which uses MSCs and chondrocytes might help us answer this question.

CAN CELLS FROM UMBILICAL CORDS BE USED TO TREAT PATIENTS WITH OSTEOARTHRITIS? Preclinical studies

Jade Perry, Claire Mennan, Helen McCarthy, George Bou-Gharios*, Rob Van‘T Hof*, Peter Milner*, Cosimo De Bari#, Anke Roelofs#, Karina Wright, James Richardson & Sally Roberts.  *Liverpool University; #Aberdeen University

Funded by Keele University & Arthritis Research UK

Osteoarthritis (OA) is a complex degenerative joint disease, characterised by degradation and loss of articular cartilage (causing the joint space to narrow) as well as a lot of changes in the bone, such as  abnormal bony spurs or ‘osteophytes’ forming. Currently, there are no effective pharmaceutical or non-surgical therapies to reverse osteoarthritis. Researchers around the world are seeking new approaches, including seeing if cell therapy could be useful. Here in Oswestry we have been obtaining mesenchymal stromal or stem cells (MSCs) from human umbilical cords (UC-MSCs) for some years now. These are an attractive source of cells for regenerative medicine since they are fairly easy to grow and they also appear to have anti-inflammatory properties which may be useful and make an immune reaction less likely if used in a different individual.  We are currently working with collaborators in other universities to see if the cells prepared here in Oswestry could delay osteoarthritis in their models.

One of these (called the PMM (partial medial meniscectomy) model) is very like end-stage osteoarthritis, with loss of cartilage and osteophyte formation (see Figure 4C) whilst the other is a small isolated injury to the cartilage, which can lead to secondary osteoarthritis. We are examining the effect of a single injection of UC-MSCs into these joints to see if they can help repair or regenerate damaged joints. If successful, it is likely they could be an excellent allogenic (i.e. from another person) source of cells for the treatment of OA.

We are currently analysing the histology of the joints from these two models as well as CT X-ray images of the PMM model.  From these studies we can clearly see that no-adverse reactions to the injected human UC-MSCs arose.  Semi-quantitative assessments of the treated and untreated groups are currently being acquired to determine the influence of MSCs in ameliorating the development of OA. So far we have found that different results were yielded from the three different patients’ umbilical cords. The UC-MSCs from one patient significantly reduced joint space narrowing, indicating their potential for cartilage regeneration. Therefore we suggest that a complete characterisation of cells is carried out before choosing a particular population for allogeneic therapy. In addition to this, we observed that the UC-MSCs from two of the patients also reduced the number of bony spurs forming, compared with the control group and these joints were no longer statistically different to a normal knee. It was also demonstrated that while UC-MSCs may not ameliorate bony spurs formed post-PMM prior to injection, they may have the potential to prevent them progressing over time.  Currently we are in the process of analysing other knee joint structures including the synovium and menisci, since osteoarthritis is a whole joint organ failure and not limited to the articular cartilage or subchondral bone.

Figure 4: Histological changes in the PMM knees showing the medial femoral condyle (MFC) and medial tibial plateau (MTP). The red colour shows proteoglycan in the articular cartilage lining the knee joint. (A) Knee with cells added following PMM treatment, (B) Normal knee (no PMM surgery or cells), (C) Control PMM knee (no cells).

 

CELL-BASED MENISCUS TISSUE ENGINEERING: COMPARING MENISCUS CELLS, CHONDROCYTES, BMSCS AND CO-CULTURES ON SYNTHETIC SCAFFOLDS

Jingsong Wang, Sally Roberts, James Richardson, Karina Wright

Funded by the Orthopaedic Institute Ltd

Menisci play an important role in force transmission, shock absorption, joint stability, lubrication, and proprioception of the knee joint. Meniscus tears are the most commonly observed intra-articular knee injury. In the UK, there are 60-70 meniscal injuries each year per 100,000 people. When the irreparable meniscus (the avascular zone with no blood vessels in) is damaged, the articular cartilage is exposed to excessive loading, which eventually increases the risk of early osteoarthritis. Currently, two scaffolds are used in clinic in a cell-free approach as replacement menisci: CMI® (collagen scaffold) and Actifit® (polyurethane scaffold). Though both scaffolds have shown promising results, there are still some limitations: the grafts shrink, do not integrate well with the patients’ own tissues and they have a fairly high failure rate. Therefore, we aim to see if adding cells to these scaffolds would improve things. Meniscal cells, cartilage cells (chondrocytes) and mesenchymal stromal/stem cells (MSCs) from bone marrow are being studied in this project as potential cell sources.  This work is forming a PhD for a Chinese orthopaedic surgeon (Jingsong Wang) who is in his first year. He aims to develop ways of seeding cells into the synthetic scaffolds and see how effective they will be at synthesising the molecules needed for a healthy meniscus. The result of this study will hopefully determine whether this approach can be applied in the clinic.

Jingsong, with other members of the team, is also looking at 2 patients who have previously been treated by Prof Richardson with a combination of cells (both autologous chondrocytes and MSCs from the patients’ bone marrow), 8 and 9 years ago. Interestingly both patients appear to have improved better and for longer than might be expected, with defects seen on MRI in their bone beneath the cartilage appearing less obvious with time.