Sarah Yim, Seoul International School
Abstract
The effect of fish collagen on the transmogrification of chicken skin tissue was investigated using LifeCare low molecular fish collagen which contains 50% low molecular fish collagen. Chicken tissue specimens obtained from a local supermarket were cut into multiple one-centimetre pieces for experimentation. The objectives of this experiment were to obtain data on the transmogrification of chicken adipose tissues from diverse solutions and assess the impacts of each solution. The experiment was conducted with three different groups — one negative control group where the chicken skin was submerged in formaldehyde solution and two experimental control groups where each of the cut chicken skins were submerged in distilled water and collagen-dissolved water, respectively. Colour, shape, and size among the three groups were then compared and evaluated. The results revealed that collagen strengthened chicken skin tissues over time and was the most optimal solution that decelerated decay rate compared to formalin or distilled water.
Introduction
Collagen is a complex macro protein that represents the main structural component of the extracellular matrix in connective tissues (skin, ligaments, bones) and interstitial tissues. It is also known to be used for structural support in biomedical devices and health applications. There are at least 16 types of collagen known today, but 80-90% of the collagen in the body consists of types I, II, and III collagen [1]. Type I collagen, the most abundant collagen in the body of humans and marine organisms, forms large eosinophilic fibres and improves skin elasticity and hydration. Type II collagen forms the cartilage and supports joint health. It is largely found in the intestines, muscles, and blood vessels, often paired with type I collagen to enhance skin elasticity and hydration [2]. Recently, researchers have considered marine organisms as a promising source of collagen as they have no limitations or records of transmissible diseases [3]. In particular, fish collagen, primarily made of type I collagen, holds several beneficial properties to the human body such as anti-aging properties, prevention of wrinkles and cellulite, and enhancement of skin hydration and firmness. Fish collagen is rich in two major amino acids: glycine and proline, which help with the production of DNA, RNA and more collagen [4].
To investigate and evaluate the effects of fish collagen on chicken tissue, we focused on one specific tissue of the chicken specimen known as the adipose tissue. Adipose tissues are specialized, connective tissues composed of lipid-rich cells called adipocytes. These are cells that store energy with large globules of fat known as lipid droplets [5]. Adipose tissues have been classified into two major types: white adipose tissues (WAT) and brown adipose tissues (BAT). These tissues are mainly distinguished through colour and physiological differences: white adipose tissues store energy and brown adipose tissues generate body heat [6]. White adipose tissues are the predominant type of fat in the body and are located under the skin around internal organs. Besides storing energy through large lipid droplets, they also insulate the body through homeostasis, support vital organs, and secrete useful hormones. Brown adipose tissues are primarily found in fetal life and infancy and are located above clavicles around vertebrae [5]. These tissues contain multiple small lipid droplets and express uncoupling protein 1 (UCP1), disrupting the respiratory chain to release heat [7].
Methods [8]
<Figure 1>
To begin the experiment, a fresh specimen was obtained. The fresh chicken tissue specimens utilized in this investigation were obtained from a local supermarket. It was important to be extremely cautious and meticulous when cutting the tissue specimen, for they could easily be damaged and unusable in the next experimental steps. The specimen was cut into three one-centimetre pieces, one piece for each of the three groups — the negative control group with formaldehyde solution and two experimental control groups in distilled water and collagen-dissolved water, respectively.
Each of the pieces was then placed in a liquid fixing agent for approximately 28 hours. The piece for the control group was placed in formaldehyde solution (formalin). This would be the most optimal solution to place a specimen piece into, for the formalin would slowly penetrate the tissue, hardening and preserving it. The piece for experimental group 1 was placed in distilled water. Lastly, the piece for experimental group 2 was placed in collagen-dissolved water.
The next step, dehydration, was necessary to proceed to wax infiltration. The specimen had to be water-free before infiltrated with paraffin wax because melted paraffin wax is hydrophobic. Therefore, each specimen piece was immersed into a series of ethanol solutions of increasing concentrations. Increasing concentrations were used to avoid sudden damage or distortion of the tissue. The specimens were dipped into 70% ethanol, 90% ethanol, and 100% ethanol for 15 minutes. Then, they were dipped into 100% alcohol for another 15 minutes, 30 minutes, and 45 minutes.
One more vital step before proceeding into wax infiltration was clearing. An intermediate solvent that is miscible to both paraffin wax and ethanol was used to displace the ethanol present in the tissue. This ‘clearing’ agent, xylene, also helped to remove fat from the tissue, which may have been barriers to the wax infiltration process. Three trials of xylene clearing were undergone to completely displace the paraffin wax on the specimens. The specimens were dipped into xylene for 20 minutes, 20 minutes, and 45 minutes.
<Figure 2>
For the wax infiltration process, paraffin wax was used, for it had particular physical properties such as water insolubility and a relatively low freezing point that allowed tissues infiltrated with the wax to be properly sectioned into thin ribbon-like sections through the microtome.
<Figure 3>
Each specimen infiltrated with paraffin wax was cut into smaller blocks for the next step, microtomy. The smaller blocks of each specimen were placed on the centre of the moulds filled with molten paraffin wax. Then, a cassette was placed on top of the mould and topped with more molten paraffin wax to eliminate all bubbles and to make sure a sufficient amount of wax had infiltrated the specimen on the bottom side of the cassette. After each of these blocks was placed onto ice plates to solidify quickly, the attached cassette was removed and excess wax on the sides was cut and discarded.
<Figure 4>
Each of the completed blocks was clamped onto a microtome and sectioned into ribbon-like sequences to be placed on a warm water bath. From the water bath, sections were transferred onto labelled microscopic slides.
Finally, tissue staining with haematoxylin and eosin was conducted for each of the specimens on the microscopic slides. Staining would help easily identify and contrast the specimen’s nucleus (purplish-blue by haematoxylin) and cytoplasm (pink by eosin). The microscopic slides were dipped into xylene, 100% alcohol, 95% alcohol, and 80% alcohol for three minutes. Next, they were transferred to haematoxylin for ten minutes and rinsed with water to wash off the haematoxylin they were previously subdued in. The same rinsing procedure was followed after a dip into the ammonia solution. The slides were then stained with 80% alcohol, followed by three dips into eosin, one dip into 95% alcohol, and one dip into 100% alcohol. The slides were placed in xylene until the coverslips were applied after two drops of mounting medium were located on each slide.
Results
<Figure 5> Microscopic view of chicken tissue in control group (formalin)
<Figure 6> Microscopic view of chicken tissue in experimental group 1 (DW)
<Figure 7> Microscopic view of chicken tissue in experimental group 2 (collagen + DW)
Firstly, by observing the colours from the H&E staining, it was revealed that the skin tissue in the control group (formalin) displayed a colour closest to light purple resembling pink, whereas experimental group 1 (distilled water) and experimental group 2 (distilled water and collagen) displayed darker shades of purple. Experimental group 2 exhibited relatively darker colour shades of purple, whereas experimental group 1 showed an intermediate colour of the other two groups. In other words, the group exposed to collagen displayed the warmest tone of colours in the cross-sections surrounding the adipose tissues, demonstrating a relatively fresher state of the tissue compared to the other two groups. The group exposed to distilled water and formalin, on the other hand, displayed a faster decaying rate, as shown by the lighter, subdued purple colour shades. The nucleus and cytoplasm in tissues that had decayed faster had lost the responsiveness to haematoxylin and eosin more quickly.
The size of adipose tissues shown by each figure also varied depending on the solution each tissue was exposed to. The size of the adipose tissues increased in order of experimental group 1 with distilled water, control group with formalin, and experimental group 2 with collagen. The tissues exposed to collagen resulted in the adipose tissues with the largest size and firmest adipose tissues, exhibiting collagen’s ability to strengthen and enhance the elasticity of skin tissues. The tissues exposed to distilled water, however, resulted in adipose tissues with the smallest size, for they did not receive optimal treatment to prevent or decrease decay of the tissue.
Conclusion & Discussion
Overall, results showed that collagen was the best solution to preserve the freshness of the exposed chicken skin tissues. On the other hand, distilled water was the least effective solution, for it displayed the fastest decaying rate with subdued, unclear colouring from the staining. Formalin exhibited an intermediate performance that fell between the performances of distilled water and collagen. This investigation revealed collagen’s abilities to enhance skin elasticity, strengthen skin tissues, and serve as structural support for connective tissues. Because this investigation had only focused on the adipose tissues of chicken specimens, other tissues must also be studied for a more accurate and reliable interpretation of the effect of collagen on the transmogrification of chicken tissues. Specimens from different organisms could also be tested to analyse similarities and differences of transmogrification.
References
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2. Collagen: The Important Differences Between Types 1, 2, and 3. (n.d.). Retrieved from https://naturalforce.com/blogs/nutrition/collagen-differences-types-1-2-3
3. Coppola, D., Oliviero, M., Vitale, G. A., Lauritano, C., D’Ambra, I., Iannace, S., & De Pascale, D. (2020, April 15). Marine Collagen from Alternative and Sustainable Sources: Extraction, Processing and Applications. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230273/
4. Further Food. (2021, January 29). What Is Fish Collagen? Everything You Need To Know Explained. Retrieved from https://www.furtherfood.com/fish-collagen/
5. Hernández, Anna. (n.d.) Adipose Tissue: What Is It, Location, Function, and More. Retrieved from https://www.osmosis.org/answers/adipose-tissue/
6. Adipocytes. (2021, June 15). Retrieved from https://www.umassmed.edu/guertinlab/research/adipocytes/
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8. Geoffrey Rolls, B. (2019, April 15). An Introduction to Specimen Processing. Retrieved from https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/