Omic investigation of chondrocytes response to varying mechanical load - assessment of aging and impact stress
Lead Research Organisation:
University of Manchester
Department Name: School of Medical Sciences
Abstract
The population is living longer and the burden this has on our health and wellness is ever increasing. As we age we are subjected to age-related pathophysiology, where cells are exposed to an increasing number of intrinsic and extrinsic pro-apoptotic stimuli and stresses. While this genetic, programmed cell death plays an essential role in cell and tissue homeostasis, an excess of these stimuli have been linked to an age related decline in cell number and ultimately impaired physiological function. We have developed bone/ cartilage impact models that allow us to apply physiological or pathophysiological loads to cartilage discs in vitro. We have demonstrated that cell death is a programed physiological response and have begun to elucidate some specific cellular signalling pathways based upon lipid and calcium signalling and Ca2+ overload. However, our preliminary investigations have demonstrated that a wide range of redundant pathways may be involved in chondrocyte physiological or pathophysiological response(s) to impact and this limits the utility of reductive, targeted investigative approaches. Therefore, a more systematic and thorough 'omic-based approach is necessary to elucidate the mechanisms underpinning chondrocytes response to impact load stress, especially when elucidating the difference(s) between physiological or pathophysiological response(s) to impact. Due to the dynamics of the response we are particularly interested in investigating cell signalling that takes place both before and after changes in gene expression have had a chance to occur (e.g. intracellular signalling events such as phosphorylation), thus ahead of transcriptional modulation.
This project will help our understanding of key regulatory pathways in cartilage homeostasis and chondrocyte survival, important components of locomotor physiology and ageing. This knowledge is key to understanding what maybe referred to as 'scientific wellness' or indeed healthy ageing, a key BBSRC remit. Our project also offers the opportunity to develop a detailed understanding of cell survival in response to extrinsic, or injurious, pro-apoptotic stimuli, which will have significant impacts across a range of cell and tissue types.
Crucially, the underpinning knowledge gained through this study will have wider implications and the potential to stimulate further studies, both in terms of molecular regulation of cell fate and potentially more clinically in terms of therapeutic intervention and lifelong health. Thus, this vital project will offer the student a unique opportunity both for high-impact outputs, state of the art scientific skills and enhanced employability.
The student will be exposed to a wide range of state-of-the-art techniques and equipment, with the knowledge and facilities to enable this project available in the partner laboratories. Both academic supervisors offer expertise on the core techniques involved. Professor Townsend brings expertise in cellular stress and survival mechanisms, and using 'omics to interrogate such pathways; whist Dr Graham and our industrial partner, Waters, bring in untapped levels of mass spectrometry sophistication that will help explore chondrocyte stress loading. The student will also be offered a unique opportunity to learn basic modelling and coding skills in order to simulate mechanical force using the custom build DACS system.
This project will help our understanding of key regulatory pathways in cartilage homeostasis and chondrocyte survival, important components of locomotor physiology and ageing. This knowledge is key to understanding what maybe referred to as 'scientific wellness' or indeed healthy ageing, a key BBSRC remit. Our project also offers the opportunity to develop a detailed understanding of cell survival in response to extrinsic, or injurious, pro-apoptotic stimuli, which will have significant impacts across a range of cell and tissue types.
Crucially, the underpinning knowledge gained through this study will have wider implications and the potential to stimulate further studies, both in terms of molecular regulation of cell fate and potentially more clinically in terms of therapeutic intervention and lifelong health. Thus, this vital project will offer the student a unique opportunity both for high-impact outputs, state of the art scientific skills and enhanced employability.
The student will be exposed to a wide range of state-of-the-art techniques and equipment, with the knowledge and facilities to enable this project available in the partner laboratories. Both academic supervisors offer expertise on the core techniques involved. Professor Townsend brings expertise in cellular stress and survival mechanisms, and using 'omics to interrogate such pathways; whist Dr Graham and our industrial partner, Waters, bring in untapped levels of mass spectrometry sophistication that will help explore chondrocyte stress loading. The student will also be offered a unique opportunity to learn basic modelling and coding skills in order to simulate mechanical force using the custom build DACS system.
| Description | I have developed a novel mechanical loading instrument used to mimic the compressive strains experienced by cartilage tissue present in the knee joint. This has allowed the examination of specific signalling pathways and downstream biological readouts relevant to cartilage health and damage. Cyclic loading, similar to that experienced during our normal physiological gait has been shown to promote cartilage health by reducing the expression of matrix enzymes such as ADAMTS-4, -5 and MMP-13. This, interestingly, is an effect which appears to occur even after a pathological impact event which typically is associated with post-traumatic OA progression. As such, we hypothesise that low magnitude cyclic loading, equivalent to low strain sporting activities may have potential for delaying the progression of OA. This is consistent with biomechanical studies which have also observed the beneficial effects of exercise in OA patients. |
| Exploitation Route | In collaboration with Waters, we aim to continue this work by examining the global cartilage proteome in response to mechanical stimulation. By using cutting edge technology such as liquid chromatography tandem mass spectrometry. Using this technique we are able to detect small changes in specific target proteins on a high throughput scale, allowing for pathway analysis to obseve which pathways are unregulated or down regulated in response to mechanical stimulation. Based on this data, these pathways can be targeted using agonists or antagonists to promote or regulate pathways of interest. The aim here is to either promote cartilage health and homeostasis or alternatively prevent the progression of OA following a sporting injury. Following the discovery proteomics study, we identified SUMOylation as a key post-translational modification that appeared to be mechanosensitive and respond significantly to mechanical stress in porcine cartilage cells. In my final year I will investigate the role of SUMOylation in the context of Osteoarthritis (OA) development and examine its downstream effects on chondrocyte cell viability and inflammation. |
| Sectors | Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Pharmaceuticals and Medical Biotechnology |
| Title | Novel Mechanical Loading instrument for cartilage tissue |
| Description | We have developed a custom-made loading rig incorporating an Electroforce BioDynamic 5110 test instrument which allows ex vivo examination of cartilage tissue in an enclosed regulated culture environment. This equipment will be available for other researchers to use in the University of Manchester at the Henry Royce Institute. |
| Type Of Material | Model of mechanisms or symptoms - in vitro |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | As a pre-clinical model this loading instrument will allow the examination of specific tissue types following dissection permitting multiple cartilage explants to be loaded at once and stored in a suitable culture environment. This will ultimately reduce the number of animals required for research as effective ex vivo expriements will be able to be conducted prior to more expensive and traumatic in vivo studies. |
| Description | Waters Corporation Industrial Partnership |
| Organisation | Waters Corporation |
| Department | Waters Corporation |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | The project was designed to work in collaboration with Waters and their investigative team at the Wilmslow, HQ in the UK. The partnership has allowed me to visit Waters regularly to have meetings with experts in the field of analytical chemistry, specifically proteomics mass spectrometry. I have used their cutting edge technology to demonstrate its real-life application and how their G2-Si high-definition MS instrument is used in basic research. |
| Collaborator Contribution | Waters have provided me with constant project supervision as well as access to cutting-edge MS technology, in both cases assisting me progress my project aims and the scope of my project and its findings. |
| Impact | This collaboration has allowed me to identify a specific pathway that may play a key role in the development of Post-traumatic Osteoarthritis (PTOA). Following this finding, I plan to investigate this post-translational modification in more detail and assess whether inhibition or induction of this pathway results in decreases or increases in cell death and tissue inflammation. |
| Start Year | 2017 |
| Description | Manchester Science Festival 2018 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | In this public engagement event I helped run a stand helping children to think about electrical connections and how this effects our day-to-day lives. A colleague at the university has developed an app which visualises this premise and allow children to play around with nodes and electrical stimulation to complete tasks on the screen. The children were extremely engaged with the game which allowed us to describe the fundamental scientific principle associated within it and the rules that are common in our daily lives. |
| Year(s) Of Engagement Activity | 2018 |