Sean Murray
Homework 1
1. Cao paper (Cao et al., Plastic and Reconstructive Surgery, 1997, 100, 297302)
What was the engineering objective of the study?
The engineering objective of this study is to use a biodegradable polymer that is molded to the
shape of a human ear and to analyze the feasibility of seeding chondrocytes onto the mold to test
the potential feasibility of engineering tissue growth. The purpose of this objective is to
determine the feasibility of complete external ear reconstruction.
This paper discusses alloplastic implants and sculpted autogenous cartilage grafts. In your
own words, describe what these two techniques are and describe advantages and
disadvantages of these techniques.
Alloplastic implants are a device molded from a synthetic material such as silicon or
polypropylene. These implants can be specifically molded to represent a desired structure and
perform designated function. Advantages of this technique include a distinct predetermined
shape as well as the means of easier construction, while disadvantages include an increased risk
of infections that occurs when any foreign substance is associated with the body.
Meanwhile, sculpted autogenous cartilage grafts use autogenous tissue from the individual being
operated upon to reconstruct desired features. A distinct advantage is that there is an immense
decrease in the risk of infection due to the use of the individual’s tissue as well as higher
durability and increased longevity. However, this procedure could be more invasive, based on
where the tissue needs to be harvested from and could lead to a longer procedure.
This paper uses a number of histological stains such as hematoxylin and eosin, Fuschian
aldehyde-Alcian blue, and Masson’s trichrome. For each of these three techniques, describe
what the stains label and how you would be able to assess cartilage formation with these
stains (e.g., does a certain color correspond to a specific cartilage component?).
Hematoxylin & Eosin is a stain commonly used during bright field microscopy techniques. This
stain consists of Hematoxylin which is a basic dye that highlights acidic cellular structure and
stains them blue as a result. Whereas the Eosin component is an acidic dye that interacts with
basic structures highlighting them red. Using this allows differentiation between cells. Therefore,
in terms of cartilage growth there would be an increased number of cells which could be
observed using Hematoxylin & Eosin.
Fuschian aldehyde-Alcian blue is another stain that’s use will benefit the assessment of cartilage
formation. The blue component of the stain binds heavily to glycosaminoglycans (GAGs) and as
a result stains them blue. When there is cartilage growth the number of GAGs increases and as a
result more of the sample will be stained blue when using this histological stain.
Sean Murray
Homework 1
Masson’s trichrome is another form of histological stain that is also used in bright field
microscopy techniques. This stain is valuable in differentiating collagen fibers, muscles, and
cells. The stain works by staining nuclei black or a dark brown, muscles are stained red,
cytoplasm results in a light red or pink stain, and collagen is stained blue or green. Therefore,
implementing this stain into the examination of cartilage growth is integral because
differentiating between the distinct difference between collagen and muscle cells observed over
time can show the desired cartilage growth.
Why do you think they implanted the cell-seeded polymer ear mold into the backs of mice?
What would be an alternative to implanting it in the back of mice? Compare and contrast
some advantages and disadvantages of the different techniques.
The cell-seeded polymer ear mold was implanted into the backs of mice because the backs
offered adequate surface area and the soft tissue necessary when trying to replicate the size of a
human ear. Additionally, for ethical reasons relating to this study the mice were sacrificed at the
conclusion of the study, using other animals would present greater ethical concerns relating to
the study. However, there could be potential alternatives to backs of mice. One alternative could
be using a patients arm or leg as the host for polymer ear mold. This method was a performed in
2018 using a patient’s cartilage and a 3D printed mold and attaching the mold under a flap of
skin on the patient’s forearm. This represents a viable alternative to using the backs of mice. An
advantage of this approach compared to using the backs of mice is that using the patient’s own
blood, and cells will lower the possibility of infection as well as ethical advantages of not using
other animal parts within the human body. Meanwhile disadvantages of this approach include a
patient losses cartilage in certain areas and has an ear growing on another part of them for a
lengthy period. Also, there is increased operating time and time under anesthesia which is not as
ideal for a patient.
This is a preliminary study, and the Zhou paper (Zhou et al., EBiomEdicine, 2018, 28, 287302) describes human trials. Name 3-4 improvements and/or experiments that had to be
performed before this research could be used in humans
To improve upon this preliminary study there needs to be immense improvements to the resulting
shape and structure of the ear grown. As experienced in the mouse study, there was varying
results of the shape that the final ear grows out to be, and little testing was done to determine the
long-term durability. Both need to be improved before implementation into humans.
Additionally, the cells used to grow the cartilage being source from calf’s and mice present risk
to rejection or infection by the receiving patient. Further tests need to be conducted to determine
how viable the ear will be once attached and whether there is a risk of infection. Building off this
improvement, it is important to determine whether the ear will have similar function to those of a
normal ear and whether it will contribute to an increased ability to hear. Therefore, functionality
tests of the ear must be conducted before it is used in humans.
Sean Murray
Homework 1
2. Villa-Diaz et al., Nature Biotechnology, 2010,6, 581-583
a. What was the engineering objective of the study?
The engineering objective of this study is to develop a culture matric for hES that is
standardized, controllable and sustainable by engineering a defined synthetic polymer coating.
Through the combination of the polymer coating and the hES there is progress made in the
optimization of biomedical applications.
b. At the time of this paper, what were some of the limitations for industrial scale-up and
use of human embryonic stem cells in clinical applications?
Limitations for industrial scale-up procedures is based on the intricacies of using polymers or
Matrigel’s in cell cultures. The preparation necessary to use polymer coated dishes is extreme
and takes significant time. This limits the scale-up ability due to the precise requirements
necessary to ensure a viable culture is reached. Meanwhile, limitations for the use of human
embryonic stem cells is that they still require recombinant extracellular matrix protein. Due to
this reliance there is a case which could lead to inconsistencies with the cell cultures or could
lead to the contamination of a batch. As a result there is significant variability within hES at the
time and limits the use their use in clinical applications.
c. What is Matrigel? What are the advantages and disadvantages of using it?
Matrigel is polymer membrane mixture that supports adhesion to other molecules and assists in
hES colony formation. Advantages of using it compared to other similar polymer mixtures
include its flexibility that mimics tissue structures in 3D cultures better. Meanwhile using
Matrigel for 3D cultures takes longer periods of time and is more cumbersome to use.
d. Describe what you consider to have been done well in this paper and what some limits to
the study were.
In this paper the researches compared the effects of Matrigel and PMEDSAH well. Controlling
these two mixtures and comparing the growth at different stages presents a good picture of the
effectiveness of culturing hES. Meanwhile, a limit to their study was what they defined as
matured cells, they compared the PMEDSAH cultured cells to the Matrigel cultured ones.
However, a defined control would be helpful to fully determine the effectiveness of the cell
culture.
e. What additional hurdles need to be overcome to translate hESC expansion use in
industry? Do a search of the literature to find at least one recent advance in this area.
Briefly describe the recent advance(s) and include relevant citations.
Additional hurdles that need to be overcome in order to translate hESC to industry is the ability
to mass produce this technique. Currently the procedure is very specific and translating this
procedure to industry could cause certain challenges. As expressed by Deniz Bartsch et al. in
mRNA Translations specialization by RBPMS presets the competence for cardiac commitment in
hESCs recent advances in and for continual advances to happen regarding the use of hESC and
Sean Murray
Homework 1
their translational capabilities it is integral to recognize the advancement of Wingless/Integrated
(WNT) signaling. This advance assists hESC and their ability to control the translation essential
factors required.
Deniz Bartsch et al., mRNA translational specialization by RBPMS presets the competence for cardiac
commitment in hESCs.Sci. Adv.9,eade1792(2023).DOI:10.1126/sciadv.ade1792