Primer

Primer

 

Introduction

The goal of the introduction is merely to serve as a primer to the foreskin, circumcision, and restoration. In which we summarize the functions of the foreskin and consequences of circumcision. Following, it's common practice in biomedical science & engineering research articles to describe current therapies and their downsides, which is why we discuss foreskin restoration methods. The reason for all of this is because the primary audience of this journal (and other journals of this type) are not intactivists. As I mentioned before, they're engineers, doctors, and different kinds of researchers, many possibly based in the United States. Nothing in this section is likely all that new to many of you, however, without this context, it may be somewhat puzzling to the journal's primary audience as to why there is a need for this type of therapy.

 

Materials and methods

ETHICS STATEMENT

The foreskin tissue we obtained came from France. France is not a country that has routine circumcision, and it's not as ingrained into the culture, like in North America. This is important to consider, as the ethics committee that reviews the procurement of the foreskin tissue is more likely to be aware of its functions. The foreskin tissue was provided by adult donors who underwent medically-necessary circumcision, after providing written and informed consent. Sources are scarce, but from what I can find, the rate of medically-necessary circumcisions in France is about 7.5% [1].

Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation (Ministry of Higher Education, Research and Innovation) is a government institution in France, and one of their duties is to review the ethical procurement of human tissue for research purposes. The appropriate ethics committee within the ministry would have reviewed all materials associated with procurement of the donor tissue, ensuring it was done so in an ethical manner, and that all donors gave proper informed consent.

Additionally, the work itself at Emilia Romagna Regional Skin Bank in Italy needed to be approved by a separate ethics committee, as human tissue is being used. The Italian ethics committee that reviewed the proposed work was Comitato Etico IRST IRCCS-AVR (CEIIAV). On a side note, that ethics committee has since been dissolved, and Comitato Etico della Romagna (CEROM) now operates in its place. CEIIAV approved the use of human tissue in this work, evaluating that it was ethically procured and that the proposed work does not violate any other ethical rules or regulations and will be treated with respect.

 

PROCUREMENT OF HUMAN FORESKIN SAMPLES AND DECELLULARIZATION

There is a lot of technical language in this section, but I will do my best to make it as easy to follow as possible.

Following the surgical harvesting of the donated tissue, the tissue needed to be packaged appropriately for its transportation to Emilia Romagna Regional Skin Bank in Italy. Tissues are typically frozen at extremely low temperatures to prevent degradation during transport. To facilitate this, tissue samples were dipped in a freezing solution with antibiotics, and a cryoprotectant added. Cryoprotectants are used to avoid the formation of ice crystals during freezing, which can damage biological materials. After being dipped in this freezing solution the tissue samples were frozen at -80℃, and shipped to Emilia Romagna Regional Skin Bank.

When the tissue samples arrived, they underwent processing. The method of decellularization used is one that was developed and patented at Emilia Romagna Regional Skin Bank, with Dr. Elena Bondioli being one of the patent owners. This decellularization method uses trypsin, which is a digestive enzyme that hydrolyzes, or cuts apart, proteins. With the use of a trypsin solution, the dermal and epidermal layers of the foreskin samples were separated. The isolated dermal layers were sectioned into two pieces. One half was then decellularized, and the other half was left as is (fresh-frozen foreskin or FFF). The halves that were to be decellularized were placed into cell culture flasks and covered with a more concentrated trypsin solution and incubated at 37℃ for 24 hours, after which they were washed with a sterile saline solution. For storage, both decellularized and fresh-frozen samples were dipped in another freeing solution with antibiotics and frozen using liquid nitrogen vapor at -195℃.

 

MICROBIOLOGICAL ANALYSIS

Because we are dealing with biological materials and tissue samples, contamination by bacteria or fungi are always a concern. Because sterility of the experiment needed to be ensured, both the fresh-frozen and decellularized foreskin sections were placed in culture plates with a growth media, which was selective for bacteria or fungi. The growth media itself is a polymer gel, much like gelatin, but it is formulated to promote the growth of microorganisms for the purpose of detection.

 

HISTOLOGICAL PROCESSING AND ANALYSIS

Histology is the study of the microscopic structures of tissues. Histological examination is widely used in biomedical sciences and is frequently employed in Tissue Engineering research. Using sections of the samples, they're mounted on microscope slides and stained using a particular staining protocol and are then viewed (in this case with a light microscope). Stains are incorporated as they add contrast to the otherwise colorless samples. This contrast allows for the differentiation of tissue components. Here we stained both the fresh-frozen foreskin and the decellularized ECMs with three different staining protocols: Hematoxylin & Eosin (H&E), Weigert's elastic stain, and Masson's Trichrome. I don't want to get into the staining protocol or how they work on a biochemical level. Instead, I'm going to go through what each stain is used for, and how to interpret the histology images (although this takes practice).

H&E staining was used to qualitatively evaluate the removal of cells and cellular components in the decellularized foreskin tissue. Ideally, with a decellularized matrix, only collagen would be observable with this type of staining. With H&E staining, many different components of cells and tissues can be identified (although this takes a lot of practice):

  • Cell nuclei: Blue/Purple

  • Basophils: Purple/Red

  • Cell cytoplasm: Red

  • Erythrocytes: Cherry Red

  • Collagen: Pale Pink

Weigert's elastic stain was used in the identification of elastic fibers of the samples, in which the fibers can be differentiated by their dark blue/purple color, and collagen again by pale pink. These elastic fibers contribute to the elastic recoil of tissue; if you pinch the skin on the back of your hand and gently tug on it and release, you can observe this phenomenon. The principal role of elastic fibers in skin serve to facilitate movement or maintain the position of the skin covering, although human skin has been found to have an atypical origin and arrangement of the elastic fibers, suggesting there may be a more prominent role in stretch and recoil and maintenance of skin integrity [2].

The last staining method we used was Masson’s Trichrome, which allowed us to evaluate the maintenance of the collagen fibers (in blue) and the overall structure of the extracellular matrix after decellularization. For context, the extracellular matrix is a complex mesh network composed primarily of collagen fibers. I plan to write a future blog post that will go into more detail about the extracellular matrix, but for the time being if you’d like to know more, Khan Academy has an excellent introductory article here.

It should come to no surprise, that when you’re attempting to engineer a tissue via this approach, the scaffolding (the extracellular matrix in our case) should be as close to the original native tissue as possible. Masson’s Trichrome allows us to assess if the architecture and overall structure of the matrix is preserved following decellularization.

 

IMAGE ANALYSIS

To build on the work of the previous section, we employed image processing techniques to obtain a quantitative fraction of collagen and elastic fibers of the samples. This was done to determine if the collagen and elastic fiber content are preserved after decellularization. To do this, we employed an incredibly clever image processing technique known as Color Deconvolution. What this method basically does is utilize the colors of the stained histology images. As I mentioned previously, histological stains are used to add contrast and allow one to differentiate the cellular and tissue components.

Color Deconvolution separates the colors of a stained histological image into three isolated channels. Because each color corresponds to some specific tissue component, this method effectively isolates those tissue components into their own channel. From here, the pixel count and intensity can be measured. Measurement of pixel intensity is particularly useful because higher intensities correspond to higher amounts of stain, which in turn corresponds to a higher concentration of the tissue component in question, i.e., more collagen or elastic fibers. By utilizing these measurements, we can calculate the tissue component content, which we express as a "quantitative fraction" (which can be kind of thought of as the amount of a tissue component per analyzed histological area).

Although the workings of the algorithm and its mathematics are outside the scope of this post, if any of you are interested in reading more about the development and inner workings of Color Deconvolution, you can read the original paper by the creators Ruifrok & Johnston.

CELL VIABILITY ANALYSIS (MTT ASSAY)

One method of ensuring that the viable cells have been removed from the tissue by the decellularization process is by MTT assay (an assay is essentially a laboratory experiment or test), which measures the metabolic activity of cells. It can be easy to get hung up on the details with assays like this due to the overwhelming amount of underlying science involved. The important takeaway is that with this technique, a sample with viable cells (fresh-frozen foreskin tissue) will exhibit metabolic activity. Conversely, a sample without viable cells (decellularized samples) will not show metabolic activity. Because we are able to measure the metabolic activity, MTT assay enables us to indirectly measure and assess how effective the decellularization protocol was at removing cells. I'll do my best to explain how this all works briefly, but if you're not interested in reading about it, please feel free to skip ahead.

 

Small biopsies of the samples are placed into a solution with a dyeing compound, known as “MTT,” which is the abbreviation of its IUPAC name: 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide. When viable cells are placed into the dyeing solution, enzymes produced by the cells react with the MTT, which results in the solution turning purple. A larger number of viable cells will, of course, produce more enzyme, resulting in a solution with a deeper shade of purple [3]. 

 

Because the number of viable cells is proportional to the color of the solution, we are able to quantify the number of cells indirectly by measuring the absorbance or optical density of the solution. Without going into too much depth, and not being too rigorous with my description, absorbance is a property that tells you, more-or-less, how “effective” a material is at absorbing light. The property is described by the Beer-Lambert Law, which states that the absorbance is proportional to both the concentration of the attenuating species, as well as the thickness of the material sample [4].

 

Absorbance can be measured experimentally using a spectrophotometer, which is a machine that essentially shines a light through the sample. A detector on the opposite side of the device measures the intensity of the light after it has passed through the sample, which allows for the calculation of absorbance. Because the shade of the solution corresponds to the number of viable cells, measuring absorbance thus provides for the determination of cell viability and ultimately how effective the decellularization method is at removing cells from the foreskin ECM.

 

This material, in particular, can be a little tough to understand at first, and in my opinion, there is still a lot that can be discussed, but in the interest of keeping this as brief and straightforward as I can, I'm going to end this section here. However, if all I did was confuse you, and you are entirely lost, or instead you’re just interested in a more thorough explanation of absorbance, I highly recommend the two spectrophotometry videos on Khan Academy, which can be found here

MEASUREMENT OF BASIC FIBROBLAST GROWTH FACTOR VIA EXTRACT

As we mention in the original article, we are interested in measuring the content of basic fibroblast growth factor (FGFb) of the samples. In the future, I plan to write a more detailed blog post about the purpose of growth factors, as well as other regulatory stimuli. Until then, the important thing is to understand that they play an essential role in stimulating cell and tissue growth, wound healing, and cellular proliferation and differentiation, particularly in the tissue types relevant to Foregen's work. If you'd like to read more on FGFb in particular, an excellent open-access review article can be found here.

 

A method known as enzyme-linked immunosorbent assay (ELISA) was employed to measure the FGFb content of the samples. FGFb was extracted from the samples through incubation in a specific culture medium. The extracts were then submitted to ELISA to measure FGFb content. There are a variety of types of ELISA assays, and they all work a little bit different. To keep this brief, I'm going to make a gross oversimplification to avoid having to get into protein biochemistry, because the fundamental idea is basically the same. ELISA takes advantage of enzyme's affinity for binding to specific substrates. Generally speaking, enzymes will only bind to one type of substrate (molecule). While it's not necessarily 100% correct, an easy way to conceptualize this is through the "lock-and-key" model: you need a specific key to open a particular lock. Enzymes work similarly. For a more detailed explanation, Khan Academy has an excellent article on enzymes and their active sites.

 

A commercial ELISA kit more-or-less provides you with a container, with specific enzymes bound to the inside walls of it. For our purposes, an enzyme that binds to FGFb would be present. When the extract as mentioned earlier is put into contact with these specific enzymes they bind. We can measure the quantity of the enzyme-substrate complex using the same spectrophotometric methods as we used in the cell viability analysis section [5].

 

MECHANICAL PROPERTIES

Those of you with backgrounds in chemistry, biology, or other life sciences were likely familiar with the assays previously mentioned. The next section may be foreign to many of you, but I will do my best to keep it as brief as I can. Mechanical properties are physical properties that a material exhibits upon the application of mechanical forces [6]. In tissue engineering, mechanical properties of a material (in our case, the scaffold) should match, as closely possible, the properties of the tissue to be replaced [7].

 

Numerous mechanical properties can be measured, but for our purposes, Maximum load, Tensile strength, Young’s modulus of elasticity, and Stiffness were measured using tensile stress tests. Samples were placed in a specialized tensile machine, which stressed the samples until the point of fracture (material separation). Data for each sample is plotted as stress versus strain. Stress-Strain Curves provide a great deal of insight into mechanical properties of a material. I'll be able to devote a lot more time to explain these properties in a future blog post, but for a solid introduction into Stress-Strain Curves you can check out the following article.

 

STATISTICAL ANALYSIS

So you have the results of your experiment. Statistical analysis can be used to determine if the results are meaningful, or statistically significant. To resolve this, you put your experimental data through a hypothesis test. There many different types of hypothesis tests one can employ, depending on the scenario, however, we chose the commonly used Student’s t-test, which compares the means (averages) of two groups and if any differences are meaningful. You can read more about Student's t-test here.

 

Our data itself is presented as the arithmetic mean ± standard deviation. I'm going to provide both their mathematical definitions and a less rigorous explanation of what they more-or-less are. Arithmetic mean (AM or X̄) is what we commonly refer to as an average, which is the sum of all observations, divided by the number of observations (n):

 

Standard deviation (SD) is a little bit more complicated, mathematically. It's more-or-less the spread of observations; how much deviation is there across samples. The definition changes depending if you're doing statistics across a sample or a population, so to that end I'll stick with the definition for a sample:

 

Results & Discussion

The results of the work are quite promising for Foregen’s aims. To summarize, the decellularization method we employed removed virtually all viable cells in the extracellular matrices. Additionally, the architecture of the matrices was left intact. There was also no difference in the mechanical properties of the decellularized matrices and native foreskin tissue. The decellularized matrices also exhibited a drastic increase in FGFb content, which we believe is a consequence of the decellularization process. This increase in FGFb content indicates high bioactivity, which effectively translates to meaning the scaffolds have high regenerative potential.

 

There are always more experiments that can be done to characterize your material further. Don’t get me wrong; these additional experiments would no doubt be beneficial. We could have done more mechanical studies on the anisotropic characteristics of the foreskin tissue and the decellularized matrices. We could have quantified many matrix biomolecules. There are always more things that one can do. It’s regretful that more experimentation couldn’t be done, as our supply of foreskin tissue limited us. That said, for our purposes, what was done was adequate for us to feel comfortable enough to move forward to the next phases of research. We are looking into securing more sources of foreskin tissue to decrease that limiting factor as much as possible. In the future, we do intend to pursue 3D bioprinting methods, but that is a discussion for another post. In conclusion, this was a significant first step for Foregen.

 

I hope this was helpful to everyone who had difficulties making sense of the technical language of our publication. We have a lot of great things coming up, so stay tuned!

 

References

[1] Circumcision in France, Circlist. (n.d.). http://www.circlist.com/rites/france.html.

[2] B. Starcher, R.L. Aycock, C.H. Hill, Multiple roles for elastic fibers in the skin, J. Histochem. Cytochem. 53 (2005) 431–443. doi:10.1369/jhc.4A6484.2005.

[3] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1983) 55–63. https://www.ncbi.nlm.nih.gov/pubmed/6606682.

[4] J.D. Ingle, S.R. Crouch, Spectrochemical Analysis, Prentice Hall, New Jersey, 1988.

[5] G. Weiland, [The enzyme-linked immunosorbent assay (ELISA)--a new serodiagnostic method for the detection of parasitic infections (author’s transl)], MMW Munch. Med. Wochenschr. 120 (1978) 1457–1460. https://www.ncbi.nlm.nih.gov/pubmed/100702.

[6] Mechanical properties - Latest research and news | Nature, (2019). https://www.nature.com/subjects/mechanical-properties.

[7] J.S. Temenoff, A.G. Mikos, Biomaterials: The Intersection of Biology and Materials Science, Pearson Prentice Hall, 2008. https://books.google.com/books/about/Biomaterials.html?hl=&id=Y81NJQAACAAJ.

Please check your e-mail for a link to activate your account.