Asking the Pro | Dr. Ludo van Haasterecht

22. Week


Hello Dr van Haasterecht,

As a starter, can you tell us about yourself please? Who are you, what are you currently working on, and/or can you work in that strange period? Did it affect your studies?

I am currently a 3rd year PhD student at the Vrije University in Amsterdam. My background is in medicine and I did both my bachelor and master’s in Brussels, Belgium. Then I became interested in reconstructive surgery and did an internship in Amsterdam. After that, at the end of my master’s period, I applied for this project, which was not medical at all! I had some doubts about starting at first, but I took it, nonetheless.

It basically about the rearrangement of the collagen matrix in the human skin in the context of scar formation, but also in the context of skin mobilization during surgery; you want to decrease the tension in wound to minimize the chances of getting hypertrophic scarring. That is simply the background of my project and then whole lot of other stuff basically centered around the microscopy technique that we will talk about later.

I have already finished some papers and working on finishing a few others right now. I was really looking forward to getting into the main portion of my lab work, but then pandemic hit…

So far for the last 3 months, I have been at home, which was tough for everybody, sadly. I was trying to get a clinical trial of my running experiments, just trying to do my lab work and all just fell short right now. So, I have tried to do the most for the preparation standpoint; writing the beginning of the papers and trying to wrap up other papers. But after 3 months, it gets tougher and tougher… I have been lucky to do lots of work from home, but it is getting harder. We basically got back to an almost quasi-normal situation here, but I am dependent on getting fresh human skin for my experiments. I get that from cosmetic surgeries and those are not happening yet. So that is my main problem for now.

Can we say wound healing is one of your main topics in you PhD?

Yes, I think wound healing would be one of the big general themes for my studies. Pathologic scarring is a more specific way to portray the topic. I guess wound healing is something vital to the survival of you as an individual when you get a wound. That wound has to heal and when we are talking about wounds in the context of skin, we are confronting with scarring first. One of the challenges for scarring is healing. That is neither a goal nor the theme of my PhD. My PhD is about pathological scarring; hypertrophic scarring in the context of burn wounds. Burn wounds are such a horrible type of scarring that you get terrible hypertrophic scars. That is basically wound healing of steroids. That is the main theme of my PhD.

You are a medical doctor working about biophotonics in the department of physics and astronomy. How did that transition happen?

In most cases you have a university medical center next to a university. At the VU, it is almost the same building, just separated by a street. We have a really nice small geographic distance between the medical center and the applied sciences department I have been working at. So as a geographical standpoint, they are very close. There are lots of nice collaborations going between medical doctors and the hardcore scientists, and one of those collaborations is between both my bosses. One of them is Prof. van Zuijlen, a surgeon, and one of them is Prof. Marloes Groot at physics department. Those two have been working together for a few years and they both saw the translational value of using this technique in skin/scar research. They both earn this grant together and that is how I got there. I think it is a really nice way of working. One of your supervisors is focused on the clinical aspects and that is what I tend to focus on as well. I have been supervised really in depth by my other boss, who is a master of hypothetical science. That is a really nice way of doing this translational research.

Dr Haasterecht Presentation

Was it a smooth transition for you?

The transition was not smooth though. I had my ups and downs, especially in the beginning. It was really tough to get into the stuff because it is really technical. I am a bit of a geek, so I am not too afraid of a challenge from a technical point of view, but it was hard to get into especially when your entire education has been focused on helping patients and working with people. And then, all of a sudden, you are in a lab 24/7. That was tough. Especially, in every PhD project, you really need to know what you are getting into and you really need to prepare for a type of work what you are going to do. If you just got out of a science oriented education, then that is what you have trained for; but if you trained to work with people, you find yourself in a lab all of a sudden. It can take some time to get used to.

Wound healing is one of the spectacular topics for tissue engineering studies, too. You have a considerable amount of experience in burn wound healing [1] and you are using some imaging techniques for follow-up studies. In fact, your recent article [2] was including one interesting microscopy technique, Second Harmonic Generation (SHG), to assess burn samples. Can you tell us about the basis of that imaging technique?

That interesting microscopy technique is Second Harmonic Generation (SHG) microscopy, which is basically the main technique that I use for my PhD. It is actually a really cool technology. It has been used in skin related research for a few decades now. Because it is so specific to Type I and II collagen, which of course present vast amounts of in skin, but also in cartilage and a lot of other tissues, it is the most prominent protein in the human body. I think it is a really specific tool to look at collagen without doing anything harmful to your samples; you do not have to stain or cut them. At the result, you get this really nice images that gives you subcellular resolution of collagen fibers in 3D.

Some SHG images of collagen (red) in between chondrocytes (green). Please click on the images for bigger versions.

For me, I look at pieces of dead skin. We did a project together where we looked at the production of collagen by chondrocytes (pictures above). We used the technique in a whole different context than I do for my PhD studies. The paper you cited in your question ([2]) is basically about the disappearance of collagen. We looked at the thermal distraction of collagen Type I related SHG signal disappearance. We can use it to look at the conformational changes at the rearrangement of the collagen at the supramolecular level. At the other end of the spectrum, we are looking at the extra production of collagen by different cell types. This single microscopy technique really encompasses a range of collagen related research, which is great!

Annotation 2020-07-17 210606
SHG signal disappearance due to thermal degradation

Since SHG works based on collagen presence, can we say it may be useful for tissue engineering applications, too? Can we use it with any other polymers?

There are second harmonic dyes that work in the same way. The technique is not specific to collagen because of the chemical formula, but because of the molecular structure. Collagen fibers have a non-centrosymmetric structure because they do not have internal symmetry, which makes them emits second harmonic signals. There are other materials as well that do this, actin for instance. Actin is a SHG emitter but compared to collagen, there is so little of it in samples, thus you hardly see it usually.

So, from a biological standpoint, collagen is the most prominent protein in tissues and that is why it is the most usable one for this technique.

I was at the latest biofabrication conference last year at Würzburg, and I was really expected this technique to be presented in there. Most of the biofabrication projects are about collagenous tissues, so why is this not used in this field? I cannot understand that…

Additional Info:

There is also third harmonic generation microscopy, but it works on a completely different basis. It is a lot less specific. Basically, you just see the changes in refractive index. For instance, you can see lipid layers, but it is not really specific. You see a lot of stuff, but you cannot really be sure about what you see. You are lot more dependent on image processing. For a case of SHG, when you look at collagenous tissue sample, you know it belongs to collagen.

18-10-15
Multiphoton-microscopy image of ex vivo human dermal skin tissue. Second-harmonic generation (red) images collagen fibers; third-harmonic generation (green) shows the fiber interfaces; and two-photon excited autofluorescence (blue) captures the elastin fibers – Link

I am also wondering why they do not use that in a wider way. Can that be because of the expenses? Or because of the experience need to use it?

Well, it is not the same as using a regular light microscope. We are not talking about hundreds of euros, also. It is a relatively expensive setup if you do not have any microscopy material in your lab, but not every biofabrication lab has to have one for themselves! You have this microscopy departments to do this kind of stuff for you. In addition to that, when you look at the new high-end 3D bioprinters, I think those are a lot more expensive than what we are talking about here.

Well, I believe it is not the only microscopy technique that is being used for polymer detection, and I know a perfect example in your carrier for that topic. One of your articles was about an imaging technique, Stimulated Raman Scattering (SRS), for the detection of silicone breast implant leakage within the healthy tissues [3]. What do you think about the impact of your study on your field?

This research did not come out of nowhere. You must have read all this in news lately about women having issues with their breast implants. There are a lot of discussions about that, and there is a lot of concerns among women with the silicone breast implants. We know for a fact that these old implants are mechanically or structurally unstable, and they tend to leak a lot. In the last few decades, there has been a lot of improvements on the structural integrity of these implants, but there are still lots of women having complaints and we are not really sure about where the problems rooting. Is this an issue of the direct interaction of the implant shell with the surrounding tissue, or the shedding of implant material that then migrates further into the tissue and lymph nodes?

This was also a really nice example of how these collaborations start. I was working at the plastic surgery department, and I knew that there was a lot of research going on about the breast implants at the physics department. A colleague of mine in the physics department was working on microplastic detection in biological samples, and we started together. One and one was two, so he started to helping me with these mussel samples and all of a sudden, we have this really nice paper.

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Microplastic detection (green in images) with SRS in biological samples. For detailed information, please go to the paper cited in this question

Currently, we are working on a second paper, which we actually apply this technique to a lot of samples from women that have complaints, and also women that do not have complaints. It is coming up soon! I think the impact of this study will be higher because we have the actual data now. I hope that this technique will be used for this issues. It is still a relatively new technique and it takes time to getting used to. But in the end, I hope it will be added value for pathologists. I do not expect hospitals to buy such a machine or make one themselves. I think this chemically specific imaging can be really an added value to people working at the pathology department. You cannot always be sure about what you see when you look at the histology slides, and chemically specific imaging can be a solution for that.

Speaking of SRS, there is poly-dimethylsiloxane (PDMS) example as a common point with tissue engineering applications. It is not only one of the most used polymeric material for breast tissue implants, but also has a wide application in tissue engineering studies, such as lab-on-chip applications or its use as a bioink, too. Do you think SRS can have a potential use in tissue engineering?

I guess it depends on what your application will be. These techniques are relatively slow, but that should not be too much of a hurdle. What we did in histology slides takes about an hour to process for an entire slide. SRS in our paper was beneficial because we use slides that we do not want to destroy by processing. If you want to have chemically specific information and information on its localization in the surrounding tissue, then it is a great technique! If you want to do relatively standardized toxicology studies, then I guess chemical techniques are better in this sense. SRS has a good resolution, but the detection limit is not that small as well. So, if you are looking at basic toxicology information, then chemical side will be more beneficial. SRS imaging was beneficial because we wanted to have information about the localization of the chemically specific signal in the histology slides.

If you are looking at the lab context for tissue engineering, you do have a lot of time on your hands, so you could think of as an application.

What general aspects can be improved in microscopy techniques to be used more widely in tissue engineering? Can you give us any advice about how can we merge these two fields?

I think there are a lot of options to be used in tissue engineering and the most obvious one is SHG microscopy. I really do not understand why it is not being used right now. I think it is because of the lack of collaboration between the fields of tissue engineering and physics. A better collaboration would be really beneficial. There is still a lot to be done, but I think both fields can learn from each other and that would be the ideal way. If anybody would like to collaborate, people in the physics department also do not have a clue of what tissue engineers working on! So, be patient with each other, and try to explain what you want to do. Understand the limitations and the advantages of both your projects. Just knock on the door at physics department and see what they can do for you.

And the younger generations?

For younger generations, they need to be curious and be motivated. These are the most important advices for an enthusiastic student!

Thank you again for your time and interest! It was a great pleasure for me to talk with you. Wishing you the best in your studies!

Thank you! I was really enjoyed too!

 


References:

[1] Jaspers, Mariëlle EH, et al. “A systematic review on the quality of measurement techniques for the assessment of burn wound depth or healing potential.” Burns 45.2 (2019): 261-281

[2] Kelly, A. A., et al. “Effective Enzymatic Debridement of Burn Wounds Depends on the Denaturation Status of Collagen.” Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[3] van Haasterecht, Ludo, et al. “Label‐free stimulated Raman scattering imaging reveals silicone breast implant material in tissue.” Journal of biophotonics 13.5 (2020): e201960197

Multiphoton imaging: https://www.osa-opn.org/home/gallery/image_of_the_week/?page=10

 

 

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