I met with Dr Laurent Bozec, University of Toronto, last autumn to find out more about his research into collagen and how AFM is used in the fields of dentistry and oral care.

It was an inspiring session. Laurent is an enthusiast for his specialist subjects and his desire to see how each new question can help to push the development and use of AFM was much in evidence.

Laurent Bozec (Image credit Jeff Comber)

Background & Career Development

We started by talking about his background, career progression and his early successes. After a PhD at Lancaster University, which resulted in him writing the first thesis worldwide on AFM Infrared, he was eager to leave the world of instrumentation and focus on applying what he learnt to other research fields. “I am excited to see the current development of AFM-IR, an idea that I worked during my PhD with Professor Pollock at Lancaster university.”

His move, in 2002, to work with the late Professor Mike Horton at the London Centre for Nanoscience (LCN) was what Laurent describes as one of two ‘sliding door’ moments in his career.

The first of three postdocs recruited in the LCN to run the AFM facility, Laurent was working with a Professor (and Clinician) at the Bone and Mineral Centre in the UCL Faculty of Medicine. On the first day he said: “Laurent, you know about AFM, I know about collagen, I don’t mind what you do, I want to work on collagen, AFM and write papers.” Overnight Laurent went from being in a field of instrumentation and technical design to working with a medic who gave him carte blanche. “It was one of the biggest jumps in my career.”

“This was my first introduction to collagen and now for the past 18 years I have not stopped talking about collagen!”

His second ‘sliding door moment’ came on a staircase in the Rayne building at UCL, and led ultimately to Laurent’s transition from the purely biophysics of collagen towards to research in dentistry:

“I happened to pass the late Professor Robert Brown, extremely well known in the field of tissue engineering and he said to me, ‘Laurent, I know you work on collagen, but have you heard they are looking for a lecturer at Eastman to do research related to collagen? And I know they recently bought an AFM.’ I had two days to spruce up my CV and submit my research plan, and the rest was history! I was appointed the lecturer at the Eastman Dental Institute at UCL and began building my group.”

Early successes

In Laurent’s early career his biggest success was to be able to resolve the collagen molecule – the height of which, measured with an AFM, is 0.5nm (vs say the size of DNA which is 10nm across a long filament).

“The images I published back in 2005 were the first ever taken of collagen molecules by atomic force microscopy. It was really a great paper as the way that I imaged the collagen molecule has not changed for almost 20 years. It took roughly 6 months to get that image of the collagen single molecule!

“Seeing the work from Dr Alice Pyne who has resolved the triple helix in DNA, I would love to go back and resolve in greater details the collagen triple helix, which also happens to be one of the more conservative protein structure in our body. If we can image the double helix, then surely, we can image the triple helix!”

Collagen Molecule, credit Dr Laurent Bozec University of Toronto Dentistry

O’ Canada

In 2018, Laurent decided to move his lab to Canada at the University of Toronto. “The reasons for this move were many. First, with the uncertainty of the Brexit, I wanted to bring stability to my family and raise my children in a dual French-English country. I also wanted a new challenge. Having been at UCL since 2002 and being Head of Research at the Eastman, I had an urge to explore research outside of Bloomsbury.

“The University of Toronto is growing at a rapid pace and it felt natural to be joining the great research team at the Faculty of Dentistry. Here, I am surrounded by collagen experts and this enables my greater collaborations! The new AI centre is attracting much interest and considering my current research interests, all the stars seemed to align. However, I would not recommend this move to everyone. It was so challenging to uproot from the UK after 20 years and settle down as a newcomer in a new country! Let alone rebuilding your lab!”

One of the projects that Laurent was working on before he left for Canada, was to create an AFM imaging suite in a clinical setting (at the Royal Free Hospital London). As part of his move, he knew that his lab would be co-located with the new imaging suite, CAMiLoD.ca within the newly refurbished UofT Dentistry research labs. “This is an amazing facility, beyond what we were planning for in the London. Its creation was very timely indeed and enabled my group to be productive from day 1.”

Dentistry and oral biology

We turned to the subject of dentistry and AFM. Laurent explained traditionally AFM is used on the hard surface of the tooth (the enamel), to look at the topography of the surface and its hardness.

“We did some early work exploring the small changes that occur in the enamel when you drink your favourite soda. We showed how enamel became softer and how its topography changed. This early work was carried out by my first ever PhD student in 2007, Dr Laurance-Young who is now Assistant Professor at Plymouth University”.

In dentistry, Laurent describes the use of AFM as, “a direct use of the technology, it’s a very nice application but we don’t really push the technology very hard.”

Collagen Digestion, credit Dr T. Ahmed UCL

However, there are several other ways in which AFM is increasingly being used in dentistry and oral biology – in addition to this traditional approach of looking at the surface of the teeth, bone related research is conducted looking at the structure of the collagen inside the dentine.

“We have had some real success in exploring with clinicians how bacteria adhere to surfaces. We’ve been doing this using single cell force spectroscopy. Your mouth is full of bacteria and if we want to understand how caries (tooth decay) and plaque form, we need to understand how these individual bacterial cell binds to dental surfaces and to implants.”

“A key problem in dentistry is the contamination of implants for example. They are made of titanium and the surface of the titanium oxide is fairly rough to ensure osteoinduction. Oral bacteria love to attach themselves onto that roughened surface during surgery. This can lead to a secondary infection – and is unavoidable as it all happens in the moment between removing the implant from the sterile packet and placing it into the patient’s mouth.

“We are interested in understanding how oral bacteria are binding onto different surfaces of the mouth and especially by ‘early colonisers’ or the first invaders of the bacteria world. They are the bacteria that land first and colonise the surface, creating a welcoming environment for the second and third colonisers and so on.

“One of my PhD graduates, Dr Sebastian Aguayo (now Assistant Professor in Pontificia Universidad Católica de Chile) worked on the adhesion of first colonisers – Sebastian did a piece of work for an Oral Health Consumer Good company investigating the efficacy of mouth wash towards the killing of oral bacteria. Our research found that the active agent in the mouthwash did indeed kill all the first colonisers but dead or alive they still created a high level of adhesion which is what the secondary colonisers require. Thus, dead or alive the primary coloniser had done its job regardless. I am sure that alternative mouthwashes are now being developed accordingly!”

Single Bacterium on Collagen, Credit Dr Laurent Bozec University of Toronto Dentistry

Development and future of AFM

For Laurent the application of AFM into dentistry is there and has its uses. However, it is all about confirming pre-existing questions or challenges.

“We use AFM mostly as a routine tool which complements other types of measurement or assays. AFM is not really perceived as the innovative instrument that it used to be when I first started my career. When I interview students, they all know about AFM (sometimes in detail!) which is awesome.

“However, the future of AFM as a technology relies on its use in fields that require the daily use of imaging modalities, outside traditional University or company laboratories.

“I think what’s exciting at the moment with AFM is that we are really on the cusp of being able to use it for medical diagnostics of tissue sections.

“My group is working on oral cancer, skin ageing and connective tissue disorders. What makes this so fascinating is that the way we have had to approach the concept of AFM is completely differently to what we’re used to normally, in particular as regards the way we analyse the data.

“Our data set is in the order of several tens of thousands of force-distance curves, and several tens of thousands of images. The key here is how you segment and interpret your data. Having one image is no longer sufficient, you need to have a population of images. You need to convince the clinicians that what you are seeing in that image is real and represents your entire sample. This is no longer a single image of collagen molecules!

“We started this idea of AFM histology quite some time ago, together with Professor Mervyn Miles and Dr Loren Picco from Bristol University. At the time, we use the high-speed AFM to image a full histological section of skin. We generated a lot of images very quickly. However, at the time, the roughness of the section became problematic to clearly identify morphological features. Another way to go faster is to process the data very efficiently and not spend hours looking at images.

“For us the AFM has become a tool in our toolbox in the same way that we use other technology. We have reached a point that we feel confident that we can start to differentiate between different types of skin in the body, different anatomical sides, we can find markers for aging and diseases. We are so excited about the upcoming projects, but our test of time will be to see if we can convince the clinicians of our super-high-resolution approach (compared to traditional histology) for diagnosis or prognosis.”

Oral Cancer

In his work on oral cancer, at the University of Toronto, Faculty of Dentistry, Laurent works with histologists from Mount Sinai Hospital (Toronto) on a weekly basis. Sitting around computers they look at the histological sections from patients who are going through surgery for diagnostic and prognostic purposes. Where histologists have an area of a section, they are not sure about, through Laurent’s use of AFM they are able to focus down and expand the area of interest using the AFM to look at the details in the ECM .

“The techniques that we have developed are called Quantitative Nano-Histology and can be used to distinguish between dermal layers for example.

“But this is for me where a transition in AFM metrology could occur and where we would bring technology to support effectively histopathology and histology, using machine driven analysis rather than relying on the human eye.”

Users can now be trained to run an AFM within half a day (Laurent has trained over 250 individuals on AFM and in 2008 he created the AFM training program now dispensed at the LCN -London UK). “We just need to democratise its access just as it happened for SEM or confocal microscopes.”

Laurent, as ever, has an eye to how the technology can be developed still further. “I think there is a potential avenue to try and create a histology probe. If that technology takes off, I can see an AFM being in every histological lab in the world.”

Latest update from the Bozec-lab

Checking in with Laurent this month to see what’s new we hear the Bozec-lab, is now fully running with 3 PhD students, 3 Masters and 1 Research Associate and that Laurent will also be looking for new Post-doctoral Research Associate (PDRA) with expertise in AFM/Collagen soon.

They have recently been awarded research grants to work on connective tissue disorders, Ehlers Danlos and are part of large team to tackle Fibrosis repair. A lot of work on collagen to come! They will be hosting some students from Cardiff, UK and Brazil to work on single cell mechanics and adhesion. Finally, they are in the process of writing up a huge story on collagen ageing detected by AFM!

Dentistry related with AFM

Ferreira S., Norman M., Jowett G., Bozec L.*, and Gentleman E.* (2021) Measuring the elastic modulus of soft culture surfaces and 3D hydrogels using atomic force microscopy, Nature Protocols, Accepted
Marshall H., Aguayo S., Kilian M., Petersen F., Bozec L.* and Brown J.*, (2019) In Vivo Relationship between the Nano-Biomechanical Properties of Streptococcal Polysaccharide Capsules and Virulence Phenotype. ACS Nano, https://doi.org/10.1021/acsnano.9b08631 – Joint Senior
Pattem, J., Davrandi, M., Aguayo, S., Allan, E., Spratt, D., & Bozec, L. (2018). A Multi-scale Biophysical Approach to Develop Structure-Property Relationships in Oral Biofilms. Scientific Reports, 8 (1), 5691. doi:10.1038/s41598-018-23798-1
Ibrahim S, Strange AP, Aguayo S, Shinawi A, Harith N, Mohamed-Ibrahim N, Siddiqui S, Parekh S and Bozec L, (2019) Phenotypic properties of collagen in Dentinogenesis Imperfecta associated with Osteogenesis Imperfecta, International Journal of Nanomedicine, 14, 9423
Aguayo, S.; Marshall, H.; Pratten, J.; Bradshaw, D.; Brown, J.S.; Porter, S.; Spratt, D. and Bozec, L. (2017) Early Adhesion of Candida albicans onto Dental Acrylic Surfaces. Journal of Dental Research, 96 (8), 917-923

Early paper with mentionned images:

Bozec, L., & Horton, M. A. (2005). Topography and mechanical properties of single molecules of type I collagen using atomic force microscopy. Biophysical Journal, 88(6), 4223-4231. doi:10.1529/biophysj.104.055228
Fenwick, O., Bozec, L., Credgington, D., Hammiche, A., Lazzerini, G. M., Silberberg, Y. R., & Cacialli, F. (2009). Thermochemical nanopatterning of organic semiconductors. Nature Nanotechnology, 4(10), 664-668. doi:10.1038/nnano.2009.254

Most cited

Wenger, M. P. E., Bozec, L., Horton, M. A., & Mesquida, P. (2007). Mechanical properties of collagen fibrils. Biophysical Journal, 93(4), 1255-1263. doi:10.1529/biophysj.106.103192