Tissue Engineering of Oral Mocosa

Scientists at the Centre for Biomaterials and Tissue Engineering have developed an extremely valuable multi-layered epithelial model of the human oral mucosa. This is currently being used in a range of clinical treatment evaluations, as well as research into the biological mechanisms of disease.
Evaluation of the biocompatibility of dental treatments
The use of the oral mucosal model, in place of animal or patient testing, enables us to carry out very much more extensive and exhaustive test programmes. This is ideal for evaluating the biocompatibility of new dental treatments, for example, where we can test complete formulations, plus the components individually and in any combination.
The effect of any treatment can be precisely measured. Commonly we monitor cell histology, plus release of cytokines and inflammatory markers. More in-depth information can be obtained using affinity arrays to characterise gene expression in the model epithelium under control and test conditions. These tests allow us to quickly identify any irritant or toxic components on the basis of the observed changes.
Disease models
The epithelial model is being used with some success to develop an understanding of oral candidiasis ('Thrush'). We now know that an invasive, hyphal form of the fungus Candida spp. causes this. We are currently trying to evaluate what factors trigger the conversion from the relatively benign 'yeast' form that is present in most people's mouths, to the active 'disease' form.
Right: Micrographs of two sections through the oral mucosal model. The oral mucosal model at the bottom has been treated with an invasive strain of Candida spp.
Tissue engineered skin and oral mucosa are also being used to develop models of other diseases that affect these tissues. These models allow us to study disease processes and evaluate new treatments without the need for animal experiments.
Cancer models
One of the models we are developing is a model of early invasive skin and oral cancer. This project, in its early stages, is following the addition of cancerous cells to tissue engineered skin or oral mucosa. This results in an in vitro model that we hope will open a way to the development of effective treatments and better early detection of these early cancers.
Later stage tumours, and metastases, form a solid ball of cells that steadily grows in size. We can also model these in vitro by growing tumour spheroids. As the ball of cells grows in size, the center becomes starved of oxygen and nutrients. This causes tumours to release factors that encourage the growth of new blood vessels and also makes them resistant to radio- and chemo-therapy. Using tumour spheroids we can study the interaction of tumours with their surrounding tissues and the immune system as well as developing better methods for targeting treatment at these more advanced tumours.
Right: Movie clip showing the first twenty five days in the life of a breast cancer tumour. After day 6 a dark centre can be seen in the tumour ball. This is where tissue is becomming necrotic due to lack of oxygen and nutrients in the core of the tumour.
Targeted Drug Delivery
We are also using these tissue and disease models to test and develop smart drug delivery systems. By combining our knowledge of tissue specific markers and smart drug encapsulation systems we are developing the means to selectively deliver payloads of genes, radioisotopes and drugs into target cells, such as head and neck cancer cells whilst avoiding other tissues in the body. Using our model systems we can rigorously test the selectivity and effectiveness of these smart drug delivery systems before moving to clinical trials.
Researchers active in this area include:Tissue engineering of oral mucosa, use in evaluation of dental materials,

Biomaterials

There is a range of materials being developed for use in medicine, prosthetic surgery and tissue engineering. Traditionally the tough 'biomaterials' developed for prosthetics uses have been quite different from the cell culture plastics used for tissue growth 'in vitro'. Increasingly, however, surface engineering is allowing us to adjust the properties of the materials used, so that tough biomaterials and delicate culture support media can express the same surface chemistry - if desired!
Above right: A surface patterned with two different chemical groups - methyl and carboxyl - visualised by lateral force microscopy (imaging technique based on atomic force microscopy).
Working with the Sheffield Polymer Centre gives access to a wide range of new synthetic materials whose properties can be tailored to carry out specific tasks within the body or in vitro. The range of materials includes biocompatible block copolymers, dendrimers and hyper-branched polymers.
Increasingly these 'biopolymers' are being designed to be 'smart', responding to changes in their environment. Currently scientists at Sheffield can synthesise materials that change their properties in response to changes in pH, temperature, salt, ions and light. Frequently polymer structures can be tuned to undergo sharp changes in properties under conditions close to those found in the body.
Current research programmes include work to develop new materials for:
tissue engineering - as scaffolds and surfaces
controlled or targeted drug release
materials for high efficiency protein separation
DNA transfection agents
anti-fouling coatings
Many of the materials being developed are 'hydrogels', polymers that are swollen with water and can resemble living tissues in their physical properties. The water content, water structure, phase morphology, polymer structure, biofunctionality and surface science ALL affect the biological performance of these materials in natural systems - which includes cells and the proteins and other materials they secrete.

Tissue Engineering of Blood Vessel

Endothelial cells line blood vessels and regulate the passage of cells from the circulation into the tissues. This is an important 'gate keeper' function allows them to regulate many physiological and disease processes. However, this is a very dynamic process with circulating cells only interacting with endothelial under conditions of flow. Most studies of how cells interact with the endothelial lining of blood vessels are only performed under static conditions but this does not replicate what happens in life. Scientists at the Centre for Biomaterials and Tissue Engineering have therefore developed systems for studying how cells interact with the endothelial cells under conditions of flow and are using these to study a number of important disease processes.
Cancer cell binding to endothelium
Many cancers metastasise to other sites in the body. To do this, after entering the circulation, tumour cells must bind to the endothelial lining of blood vessels in order to leave the circulation and form a metastatic deposit in the tissues. This is an active process that involves the interaction of ligands on the cancer cells with adhesion molecules on the endothelium. Using flow systems we can study how different tumour cells bind to the endothelial lining of blood vessels and leave the circulation to form new cancer deposits. These studies enable us to identify markers for cancers that are more likely to metastasise and treatments that could prevent metastasis.
Right: Two looped movie clips showing fluorescently labelled cancer cells flowing over the endothelial lining of a blood vessels just as they would in the circulation. The cells in the top-most movie are from a cancer that metastasises and express a ligand that enables them to adhere to the endothelial cells. Whilst the cancer cells in the bottom movie do not express this ligand and are unable to adhere or metastasise.
Candida adhesion to endothelium
Circulating microorganisms, including bacteria and fungi, also have to bind to the endothelial lining of blood vessels in order to leave the circulation and infect the tissues. Candida albicans, is a fungal organism that is a major cause of death in patients who are immunocompromised or being treated in intensive care units. These patients are susceptible to systemic infection with this organism and once it leaves the circulation, it can cause massive tissue destruction. The mechanisms by which the organism adheres to the endothelial lining of blood vessels and invades the tissues is not known and we are using flow systems to study how Candida interacts with endothelial cells under conditions of flow.

Tissue Engineering of the Cornea

Two cornea projects are underway - addressing issues of making a biocompatible non-biodegradable replacement cornea, and of developing a carrier surface for delivering cultured corneal epithelial cells to the damaged cornea.
In the first project the challenge is to make a non-degradable, biocompatible polymeric cornea substitute, which will take the place of organ donor corneal transplants in the future. This material needs to be mechanically and optically appropriate but more exacting than this, to have a surface that promotes the attachment and migration of corneal epithelial cells. Cell attachment is a major challenge that is being tackled in a BBSRC project between Steve Rimmer in Chemistry and Sheila MacNeil in Engineering Materials. This project also involves collaboration with Dr Nigel Fullwood, University of Lancaster, who has some 10 years expertise of working on corneal epithelial cell culture.
In a second project, the University of Sheffield spin-out company, CellTran, has achieved a small business research initiative (SBRI) award from BBSRC to develop a contact lens as a carrier vehicle to deliver cultured cells from the laboratory to the patient.
Above right: Human corneal epithelial cell line cultured on (a and b) acrylic acid coated contact lenses, (c) collagen, fibronectin and BSA (positive control substrate) and (d) uncoated contact lens (negative control substrate)
Damage to the cornea can be treated by the grafting of autologous cells cultured from the contra lateral undamaged eye (if this is available) or by the use of donor corneal epithelial cells (which then requires immunosuppression). One of the challenges is how to get the cells from the laboratory to the eye and sutured onto the eye in such a way that they provide immediate cover and good take.
Currently the most commonly used method is to use human amniotic membrane. The amniotic membrane, with cells attached, is sutured onto the damaged cornea that has been denuded of cells. Clinical results with this approach are promising, but the membrane is taken from donors, and there are issues of screening and also of supply.
In the CellTran project the approach is to develop an alternative strategy, one that uses a chemically modified contact lens to transfer the corneal epithelial cells to the eye. The development of this treatment brings together two very different fields of expertise:
The development of an organ culture model (by Miss Pallavi Deshpande, 2nd Year PhD student in Professor MacNeil's group), allowing us to test the process.
Surface chemistry expertise, allowing us to treat standard contact lenses with an acrylic acid coating deposited by plasma polymerisation (by Dr Nial Bullett, Celltran), to provide a suitable transfer surface.
Bringing these together, we have recently been able to demonstrate in our model system that corneal epithelial cells can be plated onto the modified contact lens and successfully transferred from the contact lens to the eye.