Generating durable and resilient repair of cartilage defects using tissue-specific adult stem cells - a systematic, therapeutic approach

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

Osteoarthritis is the single largest cause of physical disability in the world, and yet, there are no clinically effective treatments to restore normal, pain-free movement of affected joints. Osteoarthritic lesions begin as small isolated cartilage defects that spread across the joint, if these defects can be repaired, an excellent opportunity exists to restore function and prevent further disease.

For the past two decades tissue engineers have used a trial and error approach to repair cartilage defects, using an increasing and ingenious variety of scaffolds and cells with little regard to the normal growth mechanisms of articular cartilage. The sum of this approach is that this field of research has not moved significantly forward since its inception some 25 years ago. The method of transplanting cells into joints has inherent weaknesses that cannot be improved. The National Institute of Clinical Excellence UK guidelines state current transplantation methods do not provide long-term clinical effectiveness for patients nor are they cost-effective solution for short-term benefit. Our approach will break the status quo as it addresses and provides concrete solutions to the major problems that have thus far hindered progress.

The major stumbling block to progress has been the lack of suitable cells to restore normal cartilage in joints. Stem cells are critical components in any solution to repair damaged joints, and the discovery of adult stem cells in articular cartilage and their ability to make new permanent cartilage has been an important advance.

A problem when using stem cells is that the tissue they produce is immature - immature cartilage does not have the same properties as adult cartilage and is consequently prone to failure. A major recent achievement has been to understand what factors are required to convert immature cartilage into one that has adult properties.

The critical question is, can we make new cartilage using stem cells, then transform it so that it is as stiff and durable as adult cartilage to provide a fully functional implant to restore pain-free movement in damaged joints?

Our solution is to use 3D bio-printing in combination with cartilage stem cells to make new cartilage. Bioprinting allows us to make complex structures precisely, quickly and reproducibly, maximising the growth potential of stem cells to produce larger and thicker implants. We will then transform the engineered tissue so that it functions like adult cartilage before implanting it into the patient for preclinical testing. This approach is simple and emulates the same biological processes that occur in the body to produce durable and resilient articular cartilage in a highly accelerated and controlled manner. It also provides a clear pathway to systematically translate recent advances in cartilage biology and biofabrication into patient benefits.

Given the scope of the challenge we have tailored our approach to be automated and scalable, offering the real potential to treat thousands rather than the few. The team assembled to accomplish this goal is multidisciplinary, highly focused and leading experts in each particular aspect of the solution.

Technical Summary

This programme of research has a single, clear objective, to bioengineer mature adult articular cartilage for durable repair of isolated cartilage lesions in patients. Our approach emulates the natural developmental growth of articular cartilage in two fundamental respects; we will use tissue-specific stem cells to generate hyaline cartilage and induce this cartilage to undergo in vitro maturation so that it becomes as stiff and resilient as adult tissue.

To generate the hundreds of millions of stem cells needed for tissue engineering cartilage we will use bioreactors and gelatin-based macroporous beads. The phenotypic status, potency and genetic stability of cells will be monitored during the expansion phase. 3D bioprinting will be used to generate biphasic osteochondral constructs. Cells and biomaterials will be deposited as bio-ink in defined structural configurations having pore sizes that maximise soluble oxygen diffusion, waste removal, growth and differentiation of embedded stem cells. Novel bioprinting methods, including the direct use of expanded stem cells on macroporous beads that will reduce handling, minimising the risk of infection and increasing automation of processes, will be tested.

Tissue engineered cartilage will be subjected to growth factor-induced in vitro maturation such that it adopts the functional, structural and biochemical properties of adult articular cartilage. Optimisation of the phenotypic change in cartilage constructs will be monitored by gene expression of maturation-specific biomarkers, by biomechanical testing and imaging using atomic force microscopy, and by using Fourier transform infrared spectroscopy and X-ray imaging to quantify changes in collagen density and orientation.

We will conduct a controlled trial to assess the efficacy of tissue engineered in vitro matured allograft cartilage in an approved equine preclinical model of cartilage repair.

Planned Impact

We are developing the next generation of therapies to heal cartilage defects in order to give patients fully functional and long-term repair. Restoring pain-free movement of diseased or damaged joints represents an achievable but currently unmet clinical need for thousands of patients. The solution we propose is based on concrete concepts that emulate the normal growth and development of articular cartilage in a highly controlled and accelerated manner. Our key outputs will be;

- the methodology to expand stem cells for manufacturing scale tissue engineering applications,
- the rapid, reproducible and large-scale biofabrication of articular cartilage implants,
- the methods to mature biofabricated implants so that they are fully functional,
- a preclinical study in a FDA-approved large animal model to determine the efficacy of the solution and determine the parameters for use.

These outputs will be highly relevant to our academic colleagues including orthopaedic surgeons who will want to utilise our experience in generating this solution and translate it into direct patient benefit through a clinical trial. This will be achieved through early consultation with academic colleagues based in clinical research centres where experience of clinical trials for cartilage repair and the facilities to biofabricate articular cartilage implants under GMP conditions exist. In the UK, for example, the Robert Jones and Agnes Hunt Orthopaedic Hospital in Shrewsbury would be one such centre.

We are using an equine preclinical model of cartilage repair. The horse is both a model and target for therapy itself; joint disorders, as in humans, are the main cause of physical disability in horses. Unlike any other preclinical model, we will be able to use our research outputs to directly benefit this species by quickly developing treatment modalities that heal cartilage defects and prevent lameness. The advantages of this particular preclinical pathway is that ongoing advances in theraputic strategies using the equine model will directly inform orthopaedic surgeons developing the same procedures for humans.

We have identified that there may be synergy between the human and veterinary fields of orthopaedic medicine in respect of using techniques developed for one species that may be benificial for another. This would be an important advantage of this proposal. Such a working principal is at the heart of the 'One Health' vision which states that human and animal health are inextricably linked and that there should be joint efforts in the development and evaluation of medicinal treatments.

The platform technologies that we are seeking to develop are highly transferable; new methods of biofabrication and maturation of tissues are of general interest to our academic colleagues. We are seeking to develop allograft technology of cartilage-based structures, and these structures form the basis of the nose, ears, jaws, ribs, trachea as well as joints. Our work will be directly relevant to plastic and reconstructive surgeons who require cartilage-based replacement parts for those suffering from disease, congenital deformities, injury, trauma and burns.

In terms of economic and social impact, it is generally acknowledged that 3D printing is a disruptive technology, i.e. one that will transform life, business and the global economy. 3D biofabrication will have a similar impact in the health sector, and it is a strategic priority that these technologies are developed and matured in the UK to go hand in hand with well developed stem cell technology. Our collaborative work with Utrecht University engages us with one of the leading groups in the world in tissue biofabrication and will result capacity building of experience and expertise. Building critical research capacity and engagement with small and medium enterprises to transfer and develop these exciting new technologies to stimulate greater enterprise will be a priority for us.

Publications

10 25 50

 
Description Growth and Remodelling in the Porcine Heart-- Pushing Mathematics through Experiments
Amount £400,000 (GBP)
Funding ID EP/S014306/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 04/2022
 
Title Biofabrication of cartilage sheets 
Description One of the limiting factors in producing large amounts of cartilage is the need for millions, if not billions, of cells for tissue engineering. Our idea, expressed as a specific objective in our proposal was to optimise the growth of chondroprogenitors on microbeads. Following their growth on microbeads we intended to depart from conventional methodology, which is to remove cells from the microbead carriers, and instead induce differentiation of the chondroprogenitors whilst on the beads. The beads are collagen-based and therefore provide the idealised scaffold for cartilage tissue engineering; the microcarriers are biocompatible and also biodegradable. Our innovation has been to devel;op the techniques to produce cartilage sheets made of chondroprogenitor-laden microbeads. This technology allows us to produce large sheets, limited only by the size of culture vessel. As cells are expanded on the microbeads and our data shows at optimal growth that they divide once a day, we can obtain large amounts of substrate for cartilage sheet formation within a week. The fact that microbeads have such a large diameter ~300 micrometers, means that the use of a relatively small amount of beads can generate large sheets. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We have been able to streamline the cartilage production process by seeding and expanded chondroprogenitor cells on the actual scaffold that will produce the final construct. This process will eliminate a step, the removal of cells from the microbead carriers, that can be inefficient and problematic. The biofabrication of cartilage sheets is the first step in scalable production of tissue engineered constructs in an efficient way and is conducive to a marketable solution to health agencies. 
 
Title Microcarriers for cartilage tissue engineering 
Description We have developed the methods to rapidly produce large cartilage constructs using microcarriers. The technological hurdles we have overcome include inhibition of celll migration resulting in acellular scaffolds, improved protocols for differentiation, and integrating with demineralised bone to produce osteochondral implants. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact It will allow us to easily produce shaped cartilage constructs. 
 
Title Use of BMP9 to induce chondrogenesis of articular chondroprogenitors 
Description There have been no significant developments of the culture medium used to induce chondrogenesis of mesenchymal cells for the past 25 years. As part of this study we undertook a systematic analysis of growth factors and small molecules that have been previously shown to influence chondrogenesis in cells and tissues and assayed their ability to fulfill this function in articular chondroprogenitors. We discovered that BMP9 showed a precocious ability to induce chondrogenesis which was significantly more potent than any method published. 
Type Of Material Biological samples 
Provided To Others? No  
Impact The problems associated with successful cartilage tissue engineering can be catagorised into four discrete parts; which cell to use, how to differentiate cells to produce cartilage, integration of cartilage into the implant site and lastly maturation of the implanted cartilage. We have shown that articular chondroprogenitors are the ideal cell line to use for tissue engineering, now through this study we have shown that BMP9 is the optimal factor for differentiation of these cells. The cartilage produced by BMP9 induced differentiation is better both quantitatively and qualitatively. We believe the activity of BMP9 is similar on all chondroprogenitors, so its application will be highly beneficial all areas of cartilage tissue engineering including; nasal, auricular, articular and tracheal reconstruction. 
 
Description Developing a new generation of cartilage implants 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution I have begun a strategic relationship with Dr Jing Yang, Nottingham University, who is a materials expert, in order to develop the next generation of biomaterials that will enable cartilage to grow rapidly and allow lateral and vertical integration into defect sites. I will be using all the considerable knowledge developed as a result of my previous grant award and Dr Yang as a recipient of UKRMP Hub funding will be using his extensive knowledge base to help develop biomaterials that can be used in patients. In respect of the current relationship I have provided articular cartilage chondroprogenitors to Dr Yang to test his novel bioconductive biomaterials.
Collaborator Contribution Dr Yang has acted as a consultant to our research, we have used his extensive knowledge of cartilage tissue engineering and biomaterials to try and modify chondroprogenitors so that they are able to better accommodate the current breed of biomaterials to generate useful implantable devices.
Impact The followiing manuscript has been submitted to Acta Biomaterilia and is under review: Three dimensional printed degradable and electroactive polymer scaffolds promote chondrogenic differentiation of chondroprogenitor cells Aruna Prasopthum1,4,¥, Zexing Deng3,¥, Ilyas M. Khan5, Baolin Guo3,*, Jing Yang1,2* 1School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom. 2. Biodiscovery Institute, University of Nottingham, Nottingham, NG7 2RD, United Kingdom. 3 Frontier Institute of Science and Technology, and Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, 10054, China 4School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand. 5. Centre of Nanohealth, Swansea University Medical School, Singleton Park, Swansea, SA2 8PP, Wales, UK.
Start Year 2019
 
Description Engineering collagen 
Organisation Procol
Country United Kingdom 
Sector Private 
PI Contribution Using the extensive knowledge of collagen dynamics we have gained from our research under this grant we have approached a local company, ProCol Ltd who isolate bovine dermal collagen using proprietary methods, and are working with them to engineer the collagen the extract so that it has added value.
Collaborator Contribution ProCol Ltd have supplied the group with soluble collagen that we are using as the base substrate to develop new biomaterials using genetically engineered cells.
Impact work is still ongoing.
Start Year 2020
 
Description Human articular cartilage derived stem cells for cartilage repair 
Organisation Keele University
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided data and knowledge from our experience in isolation and characterisation of articular cartilage derived progenitors to this collaboration which aims to deliver manufacturing grade human articular cartilage progenitor cells to the NHS for cartilage repair.
Collaborator Contribution The partners Professor Martyn Snow (Birmingham University) and Prof Sally Roberts and Dr Karina Wright (Keele University) are generating preliminary data that shows that progenitors from human joints can be expanded using the Quantum Bioreactor to generate millions of cells required for allogeneic transplantation. In addition the Khan group is assisting in trying to add value to these processes through techniques acquired during the UKRMP funding in terms of maintenance of phenotype, biomarkers for progenitors and developing optimal methodologies for chondrogenesis.
Impact As a collaborative group we have applied for UKRI funding, through a special scheme linked to the Advanced Therapy Treatment Centres, led by University Hospitals Birmingham NHS Foundation Trust and with the following partners; CELL THERAPY CATAPULT LIMITED CRYOGATT SYSTEMS LIMITED TRAKCEL LIMITED Velindre NHS Trust ROYAL ORTHOPAEDIC HOSPITAL Swansea University Keele University The University of Manchester ORTEQ SPORTS MEDICINE LIMITED
Start Year 2018
 
Description Human articular cartilage derived stem cells for cartilage repair 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided data and knowledge from our experience in isolation and characterisation of articular cartilage derived progenitors to this collaboration which aims to deliver manufacturing grade human articular cartilage progenitor cells to the NHS for cartilage repair.
Collaborator Contribution The partners Professor Martyn Snow (Birmingham University) and Prof Sally Roberts and Dr Karina Wright (Keele University) are generating preliminary data that shows that progenitors from human joints can be expanded using the Quantum Bioreactor to generate millions of cells required for allogeneic transplantation. In addition the Khan group is assisting in trying to add value to these processes through techniques acquired during the UKRMP funding in terms of maintenance of phenotype, biomarkers for progenitors and developing optimal methodologies for chondrogenesis.
Impact As a collaborative group we have applied for UKRI funding, through a special scheme linked to the Advanced Therapy Treatment Centres, led by University Hospitals Birmingham NHS Foundation Trust and with the following partners; CELL THERAPY CATAPULT LIMITED CRYOGATT SYSTEMS LIMITED TRAKCEL LIMITED Velindre NHS Trust ROYAL ORTHOPAEDIC HOSPITAL Swansea University Keele University The University of Manchester ORTEQ SPORTS MEDICINE LIMITED
Start Year 2018
 
Description Human cartilage implants 
Organisation The Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution We have tried to work together to develop the systems to manufacture articular cartilage implants for tissue therapy. We have provided the details of implant manufacture, including the material and methods required to make large >2cm diameter cartilage implants.
Collaborator Contribution The Robert Jones and Agnes Hunt Orthopaedic Hospital has unique facilities in the UK to perform cell therapies for cartilage repair. They have access to human tissue and the ability to initiate clinical trials for medicinal products. They have provided their expertise in these areas to help facilitate development of human cartilage implants.
Impact 01 March 2017 - the two parties jointly submitted an outline application for a pilot human trial of tissue engineered human cartilage to Arthritis research UK, the title of the application was, 'Biofabrication of tissue engineered allografts for the repair of isolated osteoarthritic lesions in man: a pilot study.'
Start Year 2017
 
Description ITN Partnership 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution The two partners in our UKRMP collaboration, UMC Utrecht and Swansea University Medical School led the drafting and submission of an Innovative Training Networks (ITN) (Call: H2020-MSCA-ITN-2016) proposal named 'TraiNFAB'. The aim of the submission is to train up the next generation of cartilage tissue engineers in close collaboration with industrial partners and imbuing a sense of translational imperative in the trainees. Other collaborators joining us in this proposal are Profector (Ireland), LifeTec Group (Netherlands), Tecnologica Research Institute SRL (Italy), KMWE Precisie Eindhoven B.V. (Netherlands), University Hospital Würzburg (Germany), Heriott Watt University (UK) and Nottingham University (UK).
Collaborator Contribution Our work at SUMS especially our discoveries in the past year has put us in the forefront of the collaboration to develop a training partnership programme for cartilage tissue engineering encompassing clinical, biological, materials and translational aspects in one scheme.
Impact The ITN proposal was submitted in January 2016, and SUMS and UMC see this as a natural extension of our UKRMP collaboration. The decision is expected in June 2016.
Start Year 2014
 
Description Osteochondral implants 
Organisation NHS Blood and Transplant (NHSBT)
Country United Kingdom 
Sector Public 
PI Contribution We have provided cells and expertise in tissue engineering cartilage to NHS B&T in an attempt to develop the techniques to produce osteochondral implants.
Collaborator Contribution NHS B&T Research & Development have provided us demiineralised bone, made using their proprietary methods in order for us to develop new applications for their intended product.
Impact We have produced first proof-of-principle data showing that our techniques of producing cartilage can be used to produce an osteochondral implant using demineralised bone as a anchor for implantation into patients.
Start Year 2017
 
Description Invitation to present research to the 10TH OSWESTRY CARTILAGE SYMPOSIUM "THE QUALITY OF REPAIR CARTILAGE" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presented research to clinical practitioners and peers on a National level. The activity did have a measurable impact, I was asked to join the Orthopaedic Department in making a outline grant application to the Arthritis Research UK, submitted 01 March 2017. Furthermore, our work has helped to significantly advance several important areas of the field of cartilage tissue engineering, most prominently in highlighting the usefulness of tissue specific stem cells and new methods of differentiating these cells.
Year(s) Of Engagement Activity 2016
URL http://www.sonhset.com/uploads/6/1/4/3/61436309/cartilage_flyer.pdf
 
Description Invited Oral Presentation TERMIS 2017 DAVOS: 0225 BMP9 is a potent chondrogenic factor for articular cartilage-derived chondroprogenitors Ben Morgan, Guillermo Bauza, Charles Archer, R Steven Conlan, Lewis Francis, Ilyas Khan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of an important breakthrough in the protocol for inducing chondrogenesis for cartilage tissue engineering to an International audience of the largest society of Regenerative Medicine & Tissue Engineering.
Year(s) Of Engagement Activity 2017
 
Description Invited to speak to to the Applied Digital Technologies in Head and Neck Reconstruction Conference 17-18th February 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invitation to talk about the potential to interface soft (biological) technologies with the much more advanced hard reconstruction technologies that are based upon 3D digital imaging methods.
Year(s) Of Engagement Activity 2017
URL https://www.lifescienceshubwales.com/events/adt-uk-conference-2017/
 
Description Pint of Science (Public Engagement) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Pint of Science seeks to inform (and entertain) the general public about the types of scientific research that is happening in their local universities.
Year(s) Of Engagement Activity 2020
URL https://pintofscience.co.uk/
 
Description Welsh Government Promotional FIlm 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact I was asked to appear in a Welsh Government sponsored film to promote Science in Wales, specifically my area Regenerative Medicine and Cartilage Tissue Engineering. They asked me to to talk about various initiatives the Welsh Government had setup in order to bring academia and business together. I represented the academic strand of the promotional film and the CEO of Reneuron Ltd, who are relocating to Pencoed in Wales represented the commercial sector.
Year(s) Of Engagement Activity 2015
 
Description Workshop on Innovative/biological therapies in elite sports injury 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact A Workshop was convened at the Welcome Institute, London on 28th March 2017 entitled 'Innovative/biological therapies in elite sports injury'. It was co-organised by Dr Ilyas Khan Swansea University and the BioSport consortium. It is likely that many of the innovative therapies for orthopaedics will be 'trialled' by elite athletes who are attempting to recover from injuries sustained as a consequence of taking part in their sports. Examples of these types of therapies include microfracture, where many of the advances in the technique and post-surgical rehabilitation have come through treatment of elite athletes. This workshop brought together virtually all the major stakeholders involved in sports orthopaedics including patients (elite athletes), physiotherapists, team dcotors (Premier League and Championship clubs represented), sporting organisations (FIFA and Team Sky representatives present), Insurance companies, Biotechnology companies, Ethicists and Biological researchers. The outcome of the meeting will be published in a report through the ESRC funded Biosport consortium.
Year(s) Of Engagement Activity 2017