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

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.

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.

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.