Three years ago, Jan Křivánek compiled an atlas of mouse dental cells, categorised them, described how they work and discovered several previously unknown cell types. Nowadays, the associate professor and head of his own research group at the Institute of Histology and Embryology of the Faculty of Medicine of Masaryk University can talk not only about his passion, but also about his life project, through which he brings fundamental knowledge in the field of basic developmental biology. The idea that we will grow third teeth is premature, but in regenerative medicine, in testing new approaches to wound healing or in monitoring developmental defects of bones and teeth, it is possible to build on them today. The recent success of doc. Mgr. Jan Křivánek, Ph.D. and his colleagues in the form of a new universal method enabling the monitoring and quantification of bone and tooth growth dynamics across species has been recently reported by the prestigious scientific journal Science Advances.
When did you start researching dental stem cells?
During my Ph.D. studies in Brno. However, my real passion was awakened only during my stay in Sweden, at Karolinska Institutet, where I had the opportunity to study the structure and development of the tooth directly on living organisms. It was then that I also got a job offer to go to Vienna as a postdoc, where I started to research the tooth 100% and we started to use the new techniques of analyzing the structure and function of tissues at the level of individual cells to map all the cells in the teeth. Not just in mice, but in humans.
When did your focus turn to dental tissue regeneration? Did it stem from a fascination with the animal kingdom, where mice, for example, grow incisors, and the idea that why shouldn't humans have such "everlasting teeth"?
This is a good question, since the human capacity for regeneration is quite limited. At least in comparison to some other animals, be it newts, for example, which can grow a whole lost limb, or mice, for example, whose teeth are still growing. That's terribly interesting, because they have to have stem cells that are constantly active and continuously form the hard tooth tissues - the dentin and enamel. By studying them, we are trying to understand the general principles of organ and tissue regeneration, which may eventually lead to the discovery of new approaches in regenerative medicine.
What is the most important thing you have come up with in the time you have been working on this topic?
The Dental Cell Atlas project has produced a huge amount of data, of which we have focused on two main things. The first is stem cells, for which we have both discovered several completely new types and described to some extent how they work. And we have also described how these cells further differentiate. Stem cells are characterised by their high differentiation potential. Figuratively, you can think of it as the trunk of a tree that branches out to the terminal (differentiated) branches. Similarly, a stem, pluripotent, cell has the ability to transform into any cell in our body. However, the process of differentiation, i.e., specification into functional tissues, is very complex. We have been able to describe how stem cells in teeth can transform into functional cells that are involved in different roles within the tooth. That is, what molecular pathways are sequentially activated within them.
So this cataloguing of stem cells lays the foundation for your further research...
Yes. It's terribly important to understand how things work in vivo, in living organisms. Which we have done to some extent, and now we are trying to show that we can apply this knowledge.
“Not only mice can grow teeth, but when a mouse breaks off a tooth, the growth accelerates dramatically. We can now describe and quantify it.”
doc. Mgr. Jan Křivánek, Ph.D.
The BEE-ST method, developed by you, allows monitoring and quantification of the development and regeneration of calcified tissues in space and time. Can you summarize what this is about?
It allows us to monitor the development of hard tissues, i.e. bones and teeth, in all three dimensions and over time. We developed it because we wanted to see how fast a mouse tooth grows. What is interesting is not only the fact that it grows, but also that when a mouse breaks off a tooth, it has evolved a special principle that the tooth grows even faster. And dramatically so. We've now been able to describe and quantify on a micrometer scale how fast it grows, what the differences are between males and females or between young and old. In parallel, we have found that our method can be applied not only to incisors, which was our original focus, but also to other teeth and bones.
Have you found out what factors influence this growth?
For example, the composition of the diet plays an important role. For example, when a little mouse goes from a soft diet to a hard diet, or when it starts using those teeth, which then affects the length of the tooth root...
So if we're talking about people, what kind of lifestyle...
(smiles) Yes. I don't want to completely translate this into human medicine because we don't have those results, but theoretically our findings may suggest that the time at which a child switches from breastfeeding to solid food may affect the resulting length of their teeth. Theoretically!
Which would be supported by your earlier finding that some mouse tooth cells are identical to human ones, no?
Not just some, but the vast majority look very similar! So even though mouse and human teeth look different on the surface, they are similar at the molecular and cellular level. That's why we can use the mouse as our research model and apply these results to the human dentition. But again, we have not measured the results in humans, so this is just an assumption.
Is it reasonable to think that if humans gnawed more than chewed, they would also evolve this regenerative mechanism?
In the very long term, I think, there is no doubt. Teeth are hugely variable formations across organisms; from mouse rodents, to elephant tusks, to, say, shark's ever-changing generations of teeth. That evolution is going on all the time even now in our own species, if we look at the gradual disappearance of human wisdom teeth, for example...
Maybe I'm asking off topic, but humans are growing nails, or derivatives of skin, in addition to hair and hair. Can these findings be applied to your research?
This is not at all off-topic, because the development of some skin derivatives takes place at the cellular level in a very similar way to teeth... In mice, we even observe, exceptionally, that their teeth grow hairs. I don't want to go into detail, but molecularly, something just slightly wrong happens in them, and the cells that are supposed to form the tooth cells form a hair, which then grows out of the same base as the tooth. Which is due to the fact that evolutionarily they have a similar history.
An essential part of the BEE-ST method is finding the optimal combination of chemical stains with which to monitor tooth growth, right?
Our method relies on a combination of several approaches, some of which have been known about for a long time. The dyes that we incorporate into the emerging calcified tissues have been known for many years. However, there are dozens of them that we have tested, and ultimately selected two - alizarin and calcein - that work best. Moreover, in collaboration with the Faculty of Science (the team of Associate Professor Marcela Buchtová and Dr Jakub Harnoš), we were able to apply them in various animals - besides mice, also in fish, reptiles, birds and amphibians - and we found that they work universally. To do this, we have written precise procedures on how to apply them and devised new approaches for their visualisation and subsequent quantification. We also found that our method can be used to monitor wound healing.
Testing on more animals increases the versatility of the method and ultimately the prestige of the results themselves, am I right?
Exactly. We have a long-standing and happy collaboration with the Faculty of Science. But we mainly wanted to show that our method can be used wherever calcium-based hard tissues are formed.
Your article is not only about the development of a new imaging method, but also about the results you have achieved with it...
Yes, which is, for example, the description of the detailed growth dynamics of mouse incisors or molars. That was never known before. We could have divided our findings into several smaller papers, but we were aiming more at having one large comprehensive paper that we could send to a prestigious scientific journal.
The text is primarily methodological, but you mention that in the future your method could be used in areas such as developmental biology, tissue or dental engineering...
Yes, because we can use it to test new approaches in wound healing, bone healing, regenerative dentistry or to monitor various defects such as congenital developmental defects of bones and teeth in animal models. For example, we can observe exactly when the development of bones or teeth stops... Today, it is possible to create a mouse in laboratory conditions that will model a disease, and then we can use these models to invent new diagnostic approaches, new drugs, or to investigate new interventions to find out how to improve the quality of life in humans. Our method will thus allow us to monitor, for example, the effectiveness of treatments in these animals. If we have two mice with a genetic bone or tooth disorder, one of which we treat and one of which we don't, we give them both these dyes and by observing them over time we can see not only whether the treatment was effective, but exactly when and how it worked. With that information we can then work out more precisely how to treat the organism.
What is the next phase of your dental tissue research?
We are currently focusing intensively on the ability to repair teeth and how stem cells are involved in the repair or growth rate of teeth. As I mentioned, no one has yet figured out what it is that makes a mouse grow a broken incisor so quickly. No one knows how it is detected, how stem cells affect it, and no one has been able to quantify the growth rate accurately. We can do that now, and we can model the tooth and its regrowth. In doing so, we are discovering the mechanisms behind this and uncovering new properties of the stem cells that regenerate the tooth. The BEE-ST method intersects all of this because it allows us to know exactly when a tooth will grow back, what needs to happen for it to start growing, or when it will slow down again.
Publication of the research in the Science Advances 2023/08 journal
How fast teeth grow, what influences their growth dynamics and how dental tissues regenerate. All this can be monitored in detail by a new monitoring method developed by the research group of Assoc. Mgr. Jan Křivánek, Ph.D.
