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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. 

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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).

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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. 

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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 type.

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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. 

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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.

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

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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.

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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. 

 

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. 

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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. 

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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.

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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.

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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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