Please could you introduce yourself and your institution?
My name is Ioannis Papantoniou, a chemical engineer by training that has been always fascinated by living systems. Since August, I have been a principal investigator at the Institute of Chemical Engineering Sciences at the Foundation of Research & Technology(Hellas, Greece) and a visiting professor at Prometheus– the division of skeletal tissue engineering of KU Leuven (Belgium).
You’ve just had a paper published in Advanced Science, can you tell us a little about the research?
This is a collaborative paper, and I would like to start by thanking all the co-authors, and especially Gabriella Nilsson Hall (KU Leuven) for her commitment, persistence and high quality, as well as the co-senior author of this work, Prof. Frank Luyten (KU Leuven), for his insights in developmental biology and medical applications.
As an engineer working in a regenerative medicine context, I tried to bring forward strategies that would enable scalable manufacturing and also robust performance. This meant harnessing potent biological mechanisms in order to engineer living implants. By utilizing this approach, we hoped to treat large long bone defects, a major clinical challenge and a significant unmet medical need.
...these microtissues can be used as ‘raw materials’ to build larger tissue modules in the future...
Long bone defects heal through a process that is reminiscent of developmental events that take place during embryonic limb formation. Hence, a robust biologic foundation already existed based on the transition of a cartilaginous template to bone, termed endochondral ossification. We translated this developmental paradigm into an in vitro process, resulting in microtissue structures that we termed as “callus” organoids. These in vitro engineered organoids were able to turn consistently into bone once implanted, irrespective of scale, resulting in whole bone organ regeneration.
What is currently available for bone repair at the minute and what are the drawbacks associated with it?
The current gold standard for treating large bone defects is the use of healthy bone taken from the patient itself (autologous graft) or allografts. Both approaches have serious limitations and adverse side effects. Therefore, alternative treatments, with predictable clinical outcomes, that allow healing of critical sized defects are urgently needed.
Additional solutions for bone defect regeneration include the use and delivery of bone morphogenetic proteins through injection of a collagen carrier. This is a potent solution; however, it has been seen to affect signaling pathways in other organs, resulting in heterotopic bone formation and increased inflammation at the site of implantation. Calcium phosphate-based biomaterials have also been used in recovery and do show some relevance for smaller defects, but in order to deal with larger defects, these biomaterials have to be used in conjunction with stem/progenitor cells. Unfortunately, in large defects, the severely anoxic environment, due to the lack blood vessels, renders even this treatment challenging.
What was the most surprising result during your research?
We were all stunned when we first saw that our organoids, consisting of 200-300 cells each, where capable of turning into humanized bone mini-organs containing bone marrow compartments when implanted on their own. This was a challenging experiment but that was instrumental in showing that the essential mini-unit/building block was functional also at that small scale.
You refer to bottom-up tissue formation in your article, could you elaborate on the concept?
Bottom-up tissue formation aims to utilize small tissue modules that possess the necessary biologic information for them to undergo autonomous differentiation events, leading to the regeneration of larger defects. The small size allows for the containment of adverse environmental gradients within these modules and allows a standardization, or synchronization, in phenotype across a population. Once this has been achieved, these microtissues can be used as ‘raw materials’ to build larger tissue modules in the future by constituting a living bioink for the automated manufacturing of living implants, this can be coupled also to bioprinting technologies that can operate at these length scales.
These in vitro engineered organoids were able to turn consistently into bone once implanted, irrespective of scale...
What were some of the challenges you faced during your research?
The implants we generated were obtained through the self-assembly of the callus organoids. The resulting tissue structure was scaffold-free and, although it could maintain its structure during handling and implantation, it needed precise handling. This was a high-risk step where the work of many weeks was at stake.
This research is still in the early stages, but where do you intend to go with it next?
Based on the rapid rate of regenerated bone and the high survival rate of the implanted cartilage-intermediate implants (chondrocytes are known to be resistant to hypoxia) we are optimistic. The incorporation of 3D printed scaffolds, providing structural support, is an obvious next step. Obviously, this work needs to be validated for larger animal models and hence larger defect sizes, prior to considering clinical application.
The major challenge, however, is the development of the infrastructure to manufacture a high number of organoids/tissues, which can be used for the production of the final implant. We have recently obtained EU H2020 funding, called Jointpromise, that will try to tackle such challenges and, hopefully, we can make progress on the automation and manufacturing fronts implementing a Quality by Design approach for this type of advanced therapy medicinal product.
One of the drawbacks of many regenerative medicine approaches is the cost of scaling, could this research help overcome this?
Scaling up this process will most likely require the bespoke design of processing units for the dynamic culture of organoids, employing bioreactor and biofabrication technologies, although for some process steps solutions that exist already can cover the need. Our research will help manufacturing by implementing a Quality by Design approach. A defined and measurable mechanism of action, such as the endochondral ossification program evidenced during our work, can act as a cornerstone for ‘reverse identifying’ the critical attributes of the end product, to then forecast its potency. In my opinion, in the long term this will have implications in cost-cutting by further personalizing the production of these implants and by predicting optimal times for implantation.
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