In 1997, the scientific world got their first glimpse of the ‘Vacanti mouse’ – an eerie, hairless mouse with
a human-sized ear growing out of its back. Despite the public controversy that the experiment initially
faced over animal ethics, the techniques used by the researchers to achieve this feat brought medical
research one step closer to an exciting goal: generating organs in the lab.

Tissue engineering is a recently new field in biology, and researchers have been using its potential to
synthesise ‘bioartificial’ tissue, which consists of both living and manufactured material. Bioartificial
tissue can be implanted into an organism, and the assimilation of this material within the organism’s
existing tissues can be a powerful tool to modify cellular functioning, division, and growth. With
healthcare systems consistently facing a severe shortage of suitable organ donors, building organs in the
lab could be a challenging but potent solution.

Extracting Chondrocyte Cells from Cows

In order to build the human-shaped ear and implant it into a mouse, a team of researchers led by
Joseph and Charles Vacanti first isolated cartilage fragments from cows under sterile conditions and
used the cells found within the cartilage to model the human-shaped ear. Cartilage is a type of
connective tissue that consists of cells known as chondrocytes, which are surrounded by an
extracellular matrix of water and proteins like collagen and proteoglycans. These bovine cartilage
fragments were treated with collagenase, an enzyme that digests collagen, and later filtered and
centrifuged. Centrifugation allowed the researchers to obtain suspensions containing the desired
chondrocytes within the pellet, which refers to the heavy components of the mixture that sink to the
bottom of the centrifuge tube (see figure 1).

Figure 1: A sketch illustrating how chondrocytes are obtained post-centrifugation

Building a Scaffold

However, without a guide, the chondrocytes cannot grow into a specific shape such as an ear. This is
where a scaffold comes in handy. A scaffold can be shaped into the form of a human ear, acting as a 3D
template for the cultured cells to grow in. The polymer biomaterial forming the scaffold can either be
derived from a natural source or produced synthetically, but researchers need to consider two
important factors before making their choice: the polymer’s biocompatibility and biodegradability.
Biocompatibility refers to how successfully the implanted tissue is able to assimilate into the new
biological system and interact with neighbouring tissues. This can vary based on its structural
properties, such as its surface chemistry or porosity. Researchers must consider the properties of the
target tissue type and use a compatible scaffold material to minimise toxic effects on the target tissue

Additionally, once the cultured cells have grown into their desired shape, the scaffold is no longer
required. Therefore, the material that makes up the scaffold needs to be biodegradable. This allows the
scaffold to degrade into waste products like carbon dioxide and water once it has fulfilled its purpose
within the body.

In the ‘Vacanti mouse’ experiment, the researchers created a plaster mould using a 3-year-old child’s ear
for reference. To create the polymer construct (see figure 2), they used a biodegradable polyester known
as polyglycolic acid and submerged it in an organic solution for a few seconds. Following this
procedure, the polymer construct was sculpted into an ear-shaped scaffold using the plaster mould.
The bovine chondrocytes were then planted into the polymer construct and placed into an incubator.


Figure 2: A sketch displaying the human ear-shaped scaffold

Implantation of the Tissue-Polymer Construct
Once cellular growth begins in the incubator, the tissue-polymer construct is ready for implantation
into the subdermal region of the mouse’s back. However, if researchers implant cells from a cow into a
healthy mouse, their experiment will unfortunately fail. This is because healthy immune systems are
trained to recognise unfamiliar cells and destroy them using antibodies, which means that the body of a
healthy mouse will reject an implant made of foreign bovine tissue.

To overcome this obstacle, the researchers decided to experiment on athymic nude mice, which lack
functioning immune systems and hair. An athymic mouse lacks a thymus gland and is therefore unable
to produce white blood cells called T lymphocytes, which act as soldiers in the fight against infection.
As a result, implanting bovine tissue into an immunodeficient mouse will not cause an immune
response, allowing the implanted tissue to continue growing in its new environment.

12 weeks after implantation, the researchers sacrificed the mice using an overdose of anaesthesia in
order to study the composition of the ears. Through careful examination using different stains like
eosin and haematoxylin, researchers found evidence of more cartilage growth in the implants.


Figure 3: An image of the lab mouse, post-implantation.

Applications of Tissue Engineering Techniques
Current tissue regeneration techniques can be used both therapeutically to replace damaged tissues or
cosmetically to change the appearance of certain body features. For instance, alloplastic implants,
which are similar to prosthetic devices and made of materials like silicone, can be used to reconstruct
the cartilage of the ear or the nose. However, these implants are usually temporary because they are
vulnerable to infection and may interact negatively with the patient’s immune system.

A more common material used to alter the shape of the external ear and the nose is autologous
cartilage, which is extracted from the patient itself, specifically from the costal cartilage in the ribs.
While this can be incredibly effective, the consistency of the surgical outcomes may vary depending on
the surgeon’s skillset, and extracting costal cartilage from the patient can sometimes lead to donor site
morbidity or complications like scars.

The potential benefits of using autologous cartilage within a scaffold could not only include a
consistent, predesigned shape for tissue implants but also reduce the operative time taken during
current surgeries. Furthermore, using the patient’s own tissue is usually more practical than finding a
suitable organ donor, since autologous tissue will not be rejected by the patient’s immune system and
the possibility of infection is also minimised.
A current limitation in tissue engineering is that the tissue being prepared for implantation often lacks
vascularisation or a blood supply, and relies mostly on diffusive processes to gain nutrients from the
culture medium. This limits the resulting size of the tissue, which is a major challenge for generating
larger organs in the lab.

However, there have been incredible recent advancements in tissue engineering such as the use of stem
cells (undifferentiated cells that can divide and form specialised cells) to build miniature organs in the
lab called organoids. It may even be possible to integrate blood vessels into organoids, which could
increase their lifespan. Alternatively, seeding stem cells into a scaffold could be a potential method for
building organs with numerous types of tissues. Significant scientific progress has also been made using
3D bioprinters which have been able to create liver tissue containing an embedded network of blood
vessels. The addition of vascularised systems to these tissues is a major accomplishment that could
allow scientists to synthesise larger organs in the future, revolutionising the field of bioengineering.

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