Stuck in a Hurricane – Don’t Know When I’ll Be Back Again!

Hurricane Sandy will go down in history for what it did to New York and New Jersey. But it will also be a story I will tell my grandchildren.

 

It’s stressful enough to move out of an apartment.  You have to sell or give away all of your furniture. You have to throw out all the detritus of a private life that you would keep using if you were still there – but which everyone else already seems to possess and doesn’t want more of: stainless steel pots anyone?  You have to clean the apartment thoroughly if you expect any of your deposit back. And you have to pack your entire life into 2 check-in bags (I paid extra and brought 3), one computer bag, and one “personal item”.

 

It’s also stressful to finish your PhD experiments (see my previous post: “Experiments – Done!!”). To simply circle a date on a calendar and say, “I’m going to finish this day and what I’ve got is what I’ve got. It is, what it is, and I’m going to write up and be done.” Particularly stressful when it gets to 3 days before you leave and you’re starting an 18 hour experiment and still have immunohistochemistry to work out, and to make sure you have all your files off all the various computers in the department.

 

And then: Hurricane Sandy. Oh, how I love you Hurricane Sandy. I’ve seen stronger wind and rain growing up in Albuquerque; and yet, for some reason, Washington D.C. completely shut down. I had just gotten back from my birthday/going away party on Sunday night, when they announced that the metro was going to be shut all day Monday. I therefore had a choice: I could either be stuck in the apartment with no way to get into work, or I could be stuck at work, with no way to get back to my apartment.  The choice was simple, I had an experiment that had to run overnight Monday to Tuesday. Either that experiment was going to be in my thesis or it was not. Also, my immunohistochemistry wasn’t working yet. I had to go into lab. So, I grabbed a set of pajamas, and hoofed it into lab.

 

I spent two nights in the lab.

The experiments got done.

The files were obtained.

The immunohistochemistry finally worked.

I ate the cheese, salami, and cracker platter leftover from the Friday birthday party from the lab fridge. I ate leftover pie.

Did I mention I spent my birthday in the lab with a hurricane outside?

 

Then I went home.

I packed everything I could.

I threw out everything I couldn’t.

I scrubbed every appliance and floor in the apartment.

I slept one night on an air mattress (a step up from the chairs in the lab).

Then I went to the airport and flew to England.

Experiments – Done!! a.k.a. I Never Want to Do Immunohistochemistry Again

Those who have been following me the past month on Twitter have witnessed my countdown to leaving the NIH. For those reading my blog, apologies for not posting, but time was limited and there was a lot of work to be done. So I now post a recap:

 

I needed to make sure I had all the information necessary to create a coherent project. To this end, the main focus of the last month was making sure I had all the staining I needed from my transplants in order to correlate angiogenesis and good bone formation, so that I could justify my thesis.

 

To this end, a preliminary survey of all of the “good transplants” – ones that I had transplanted since my return to the states in 2010 was made. This meant that 180 slides were observed in H&E. It was found that a number of transplants were in fact mammary glands or plain brown fat. The explanation for this is that sometimes transplants are resorbed – one of the reasons, after all, why I focused on collagen-based scaffolds is because they are resorbed over time. However, the ideal would be for the scaffold to be resorbed but simultaneously replaced by bone. Since this does not always happen, I was occasionally hunting around in the mouse for anything that resembled a transplant. After the preliminary survey, samples that were not transplants, or which were transplants but for reasons unknown were very fragmented and deemed “impossible to section” were abandoned. This left about 130 samples.

 

Some of the remaining samples were re-stained, and all were scanned using an Aperio slide scanner. This is an amazing machine, which is the saving grace for someone like me – a graduate student who vehemently despises microscopy. Slides are scanned at 40x resolution and then the entire image can be viewed on a computer, zoomed, and images captured of any section desired – without having to constantly refer back to the actual slide. Also very handy for me because it meant all I had to bring to Oxford was my computer. Welcome to the digital age of science.

 

Finally, all of the 130 slides to be analyzed were graded for bone formation and hematopoiesis (marrow formation) on a scale of 1-4 by 3 “blinded” researchers. I use blinded in quotation marks here, since the identities of what the samples were clearly written on the slide, but I have a habit of using acronyms for anything, so no one knew what they were. As one of the “blinded” researchers myself, I simply didn’t look at the slides based on identities, but by their numbered position in the box – and, as they were not arranged in the most logical order, my gradings can also be seen as unbiased.

 

Some of the slides appeared to be either pre-osteoid (i.e. in the process of forming bone) or cartilage, so I stained with toluidine blue. However, none of these samples turned out to be cartilage. For grading purposes these samples were deemed unsuccessful.

 

Then, I came to what I had been dreading for months. Immunohistochemistry.

 

Often shortened to IHC, immunohistochemistry is simply the staining of sections of tissue for specific expression of markers through the use of antibodies. Sounds simple, but not all antibodies are created equal and it took me about 10 different antibodies to find one that was specific for blood vessels and worked on my sample. Adding to the complexity of this process is the need for heat retrieval of some markers – for which every company and scientist will tell you a different protocol. Yet another variable exists in how to show where you have specifically selected with your antibody (i.e. choice of a secondary antibody). Since most antibodies aren’t themselves fluorescent or colorful (chromogenic) this means that you have to create another layer of antibody by selecting for the first one with your secondary which is either fluorescent or colorful. Thus, I think it’s easy to see that what, in theory, could be a very simple process, is indeed a very complex one.

 

But, I got it to work. I finally got really good immunohistochemistry of blood vessels on 16 representative samples from one experiment. The rest of the samples I am going to have to attempt to count the blood vessels by eye based off the H&E staining scans that I have.

 

And then there was the hurricane. But that’s for my next post!

The Tissue Engineering Paradigm

For my PhD I’m working with a general tissue engineering setup that has been worked on by countless scientists over the years. The system consists of three components which can be used in combination or separately: a load-bearing scaffold, cells, and growth factors. This concept can be applied to any tissue a scientist wished to try and “tissue engineer”. The scaffold is used to give structure to the system. It can be used to fill a gap, e.g. a hole in bone, or to completely replace something missing, e.g. an aortic valve. In some cases it is weight bearing (often the case in bone), though not always (skin). Many different materials have been tested for the engineering of various tissues, some of which you will have heard of, and others of which you almost certainly have not. In addition to providing structural support, the scaffold can be used to deliver cells and growth factors to a region. Which cell type a scientist chooses to use is very dependent on the organ desired, and the “stem-ness” of the cell varies from study to study. Growth factors can also be added and these can stimulate cells the scientist has put on the scaffold (through a process known as “seeding”) and also tissues and cells surrounding the transplant once it has been placed in a living animal. They can change whether the cells live or die, whether they move around or stay still, and even whether they turn into heart, bone, or other tissues

 

Tissue engineers really have a lot of optimizing to do for all sorts of different tissues using this general paradigm. To support research into this system there has been a lot of basic materials science and biochemistry done that really allows each scientist to make informed decisions about the capabilities of different types of scaffolds, cells, and growth factors.

The trick is to find the correct set of conditions. It’s that simple.

The 3D Challenge

The biggest challenge in tissue engineering is the 3D nature of the enterprise. There is only one tissue which is essentially 2D, skin, which exists in a sheet-like form; and tissue-engineered skin is already commercially-available and approved for use. So why is 3D such a problem? 3D is a problem because it makes growth of the tissue ex vivo, that is, outside the body, much more complicated. If you have a 2D structure everything is exposed, whereas by creating a 3D structure you automatically create the distinction between what is “inside” vs. “outside”.

Studies have shown that simply placing cells on a 3D structure changes which genes are expressed versus just plating them on a 2D plane. Additionally, in order for cells to survive they need to constantly receive nutrients from the outside and get rid of waste products. This is the purpose of blood vessels (vasculature) in the body. In culture in a dish in the lab, or in a newly transplanted synthetic material, however, there are no blood vessels. In these instances nutrient exchange occurs by diffusion. Diffusion is very easy from a 2D surface, but much more complex from a 3D structure. In a 3D structure, cells on the outer surface behave as if they were on a 2D plane, and are uniformly exposed to the outside, whereas cells on the inside rapidly deteriorate in terms of viability and functional capacity the further away from the surface they are. Once blood vessels enter the transplant, however, nutrient exchange becomes uniform throughout, and the distinction between inside and outside increases.

Think, for example, of a loaf of bread. Loaves can be very hard and crusty on the outside, where they are in direct contact with hot air, but the inside of the loaf can still be airy and light because it has been sheltered by the outer crust. The result is a loaf of bread with very different outside versus inside, although all the dough was uniform prior to baking. Similar concepts apply to tissue engineering of bone, where often, in the literature, one can find the production of a hard bony exterior to the scaffold, while the inside is airy and empty, due to different conditions experience by the transplant even though the original components were the same throughout. Unlike bread, however, we want our transplant to be uniformly boney, instead of an empty center. The empty center occurs when cells on the inside don’t have blood vessels and hence they die through lack of nutrient exchange. The goal of my PhD is to increase the rate of blood vessel formation and ingrowth into the scaffold to make a transplant that is uniformly boney. A loaf full of crust.

Beginnings

Tissue engineering is a new field of research that wasn’t a distinctive field prior to the 1980s. Science has been moving forwards at an incredible rate in all fields for the last century. Just think about it – In terms of biochemistry, the DNA helix structure was published in 1953 and opened up a whole world of opportunity, with the Human Genome Project being only a stepping stone in the grand scheme of things.

There are two main thrusts of research, that which seeks to increase our knowledge of how biology works, and that which seeks to use this knowledge to create new things (drugs, therapies, other products). Most scientists fall into either one camp or the other. I knew very early on in my days as an undergrad studying biochemistry that I was a creator, not a discoverer. I found my home in tissue engineering.

The field of tissue engineering encompasses research that tries to combine different sciences to create living tissue therapies. To be able to perform tissue engineering you need to understand materials, medicine, and a whole bunch of biochemistry.  I received a Masters in Biochemistry from the University of Oxford in 2008. My knowledge of materials science came from working as a research intern at Sandia National Laboratories in Albuquerque, New Mexico during the summer and winter vacations of my first few years in undergrad. It was actually while working at Sandia that I worked with a postdoc who had done his PhD in tissue engineering, which in turn inspired me to do the same. Following my masters research project in Oxford, I was anxious to do graduate work that would allow me to continue on a similar project. That was how I discovered the Oxford-NIH fellowship which allowed me to keep my mentors at Oxford, while adding in the NIH experience and funding. I also received an NSF GRFP award which paid for the first years of my project.

Am I happy? Yes. Am I stressed? Very, very, much.

Welcome!

The goal: to submit my thesis by February 2013.

The traveler: one intrepid 26-year old American female doing a joint PhD between the University of Oxford and the National Institutes of Health in Bethesda, MD.

Welcome to my blog! As a graduate student heading (very quickly) towards completion, or, at least, the goal of completion, I plan to keep this blog to show the wider community what a PhD in tissue engineering entails. You will see what goes into thesis writing, scientific research, and related topics that a graduate student encounters in the final months. I promise that nothing will be incomprehensible to anyone who has had basic high school science. As for myself, I hope that this blog will somehow keep me from complete panic as the final months turn into weeks, days, and hours.