Building with biology: The engineering in tissue engineering

The engineering in tissue engineering

Genetic science has provided some of the most astounding research breakthroughs of recent years, often accompanied by high-profile debate of the ethical issues involved. Now, clinical success in the regeneration of bone, cartilage and skin is being achieved, with huge advances in the creation of even more complex structures. David Williams writes on the processes of creating human tissue from stem cells and chondrocytes and explains the role of the engineer in the ever-developing world of tissue engineering.

Hardly a day goes by without the publication of a news item that concerns some aspect of man’s interference in natural processes in the name of medical or scientific progress. Many of these news items appear to have equal capacity to alarm and excite us. Not long ago, on the same day, one group of scientists in the USA announced that they had successfully cloned human beings while another in London explained how gene doping was being used to alter human characteristics in order to enhance athletic prowess. To the lay person these were things we were promised would never happen. Just as we were promised that IVF, originally developed to facilitate reproduction to help infertile couples, would never lead to interference with the process of creation other than to assist eggs and sperm to meet under optimal conditions. ‘No designer babies’, we were told; then along came surrogate parents, the ability to determine the sex of children and the production of babies to assist in the treatment of siblings by the provision of their closely matched genetic material.

In spite of the best endeavours of many individuals and organisations, the inevitable has happened, and, even if that story of human cloning turned out to be false, the reproductive embryologists, geneticists and others now have the opportunity and technology to control and manipulate reproduction. Taking centre stage in this rapidly evolving scene are cloned animals, from the original Dolly the sheep to Cc the cat, ANDi the monkey and many more, and the embryonic stem cells that have the potential to revolutionise medicine.

Nuclear transplantation technology (cloning) involves transferring the complete genetic material from the nucleus of a cultured donor cell to a mature recipient egg whose own nucleus has been removed. The resulting offspring are genetically identical to the donor that supplied the nuclear material. Needles rather than nature control the process. The technique also offers the opportunity to manipulate genetically the cultured cells.

These are but two examples, albeit among the most controversial, of the rapid move towards the manipulation of life, where it is quite possible to see both the enormous potential in terms of the improvement in the quality of life that may be realised by the successful implementation of the technology and the enormous risks, both physical and psychological, that are being taken in the name of progress.

Terminology

What, one might ask, does this have to do with engineering? Intuitively we might consider that these are branches of science, within molecular biology and genetics, that are about as far away as you can get from the engineering sciences. But even here we can begin to see a relationship, since the practice of gene manipulation is referred to as genetic engineering, and the industry that is rapidly growing around these practices is encompassed by the expression ‘biotechnology’. Furthermore, at the heart of the therapeutic measures that may well arise from these types of techniques is the area that is now known as tissue engineering. It might seem as though the engineer was right in the middle of this debate. The discipline of tissue engineering is now beginning to provide the tools, techniques and strategies to enable the optimal regeneration of tissues, and here both genetic manipulation and the control of stem cell behaviour are having a substantial influence on this process.

With so much emphasis being placed on this subject at the moment, it is useful to step back and consider why this is so and what the ‘engineering’ in tissue engineering really means. If engineering usually means the application of scientific principles to the design, production and performance of technical devices, there does not appear to be any connection with tissues that, by definition, develop through a natural process, usually without any assistance. Certainly, tissue engineering does not mean manufacturing a piece of tissue in a workshop. On the other hand, if we take the original meaning of engineering as the art of creation, we can see the relevance since tissue engineering involves human intervention in the creation of new tissue.

Key factors

In order to better appreciate the story, a few words about the key factors and a few definitions might be helpful. Central to the process of tissue engineering is the human cell and the ability to persuade cells to perform in certain ways. As we shall see below, for the purposes of tissue regeneration, we can either use the fully differentiated cells of the tissue in question, i.e. bone cells for new bone, skin cells for new skin etc., or use stem cells. Stem cells are the precursors to the differentiated cells. In adults they are predominantly found in bone marrow, but also to a lesser extent in circulating blood, and these have the ability to transform, or differentiate, into specific cells as required. A very rich source of stem cells is the embryo, since, by definition, these embryonic stem cells are primed to differentiate into the various types of cell that are required for human growth. The manipulation of stem cells has become a vital factor in the control of tissue regeneration. In general, two different types of signal influence cells, including stem cells, one being of chemical origin, where agents such as growth factors provide molecular signals to the cells, the other being mechanical, where forces (such as tensile stress or fluid shear stress) provoke certain types of cell behaviour. A few definitions of key terms in tissue engineering are given in the accompanying box.

How tissue forms and is repaired

The formation of tissue, as inferred above, is a natural process. Tissue is formed by the sequence of events following conception, through foetal development and postnatal growth that involves cell multiplication and differentiation (i.e. becoming a specific type of tissue), to the production of extracellular matrix and the organisation of the resulting structure into recognisable functional tissues and organs. However, in contrast to certain lower-order animals, humans have a very limited ability to repair that tissue once it has been formed, and indeed an extremely limited ability to regenerate tissue at all once we have reached maturity. We do have the ability to repair some tissues, for example bone following fracture through the generation of new identical bone at the fracture site, and skin, although often the new ‘skin’ is usually more scar tissue than normal dermis and epidermis. With all other tissues, including muscle and nerve, this process of regeneration is difficult if not impossible.

As we age, we are subjected to many diseases and injuries and one of the biggest obstacles to maintaining quality of life is this ability to regenerate tissue once it has become damaged. Since our tissues originally possessed this capability, the question arises as to whether it is possible to switch this tissue formation process back on when required and, equally importantly, to then switch it back off again once we have produced the right amount and right quality of tissue. This switching off process is not trivial, since the unregulated growth of tissue may lead to cancer, and cancerous cells notoriously do not respond to signals that tell growth to stop.

Let us take the cartilage of human joints as an example. The role of articular cartilage is to bear load and to minimise wear between articulating joint surfaces. The extracellular matrix is the main structural component of this cartilage. It is formed by the cartilage-producing cells, the chondrocytes, which become embedded within this matrix. In the developing embryo, the long bones of the skeleton originate from the cartilage. As the skeleton grows and matures, the osteoblasts, cells that are responsible for bone formation, produce bone from this cartilage by a process of endochondral ossification, that is calcification of the soft cartilage. In order to act in this manner and produce the optimal form of tissue, the cells receive signals from a variety of molecules, and specifically from a multitude of growth factors. Growth factors are polypeptide hormones that regulate the activity, and especially the proliferation, of cells, and are therefore extremely important in controlling the nature and quality of the tissue that is produced, and the speed of production.

A variety of genes also controls the orchestration of these events and these determine the resulting structure, the long bones eventually consisting of tubular cortical bone along the length, a softer cancellous bone towards the ends and a layers of articular cartilage at the points where they articulate with the other bones within the joints. At maturity, the cartilage, which, incidentally, is a superb engineering structure in its own right, loses all of its hitherto excellent powers of tissue generation, and exists henceforth without any significant blood supply. From about the age of 16 onwards, our cartilage becomes very susceptible to damage, typically from sports injuries in youth (especially in the knee) and osteoarthritis in older age (especially in the hip). The main switches that are required to recover the ability to generate new cartilage are the growth factors and genes that controlled the cells in the first place, these providing the ‘molecular signals’ for tissue regeneration. In addition, chondrocytes require mechanical stimuli in order to function properly and produce the extracellular matrix, and so it is usually necessary to apply mechanical signals simultaneously with the molecular signals in order to achieve functional regeneration.

We therefore have the essential elements of tissue engineering. All we need are exactly the right cells for the tissue in question, and to provide them with an environment in which we can supply the optimal mechanical and molecular signals for an appropriate length of time, and the result is the creation of new tissue which can be used to replace the damaged or diseased structures. It is for this reason that my definition of tissue engineering is ‘the persuasion of the body to heal itself, through the delivery to the appropriate sites, of molecular signals, cells and/or supporting structures’. The engineering in tissue engineering is indeed one of the best examples of the concept of an engineering-inspired creation.

Challenges and barriers

Obviously, the reality is far more complex than this simplistic scenario suggests and it is necessary to flesh out some of the details, so to speak. There are, or will be, many different situations in which this process can occur, but the following paradigm gives a good idea of the steps that might be involved. It will become obvious that not only will there be many technical challenges, but also a number of infrastructural barriers to progress. This paradigm has the following stages:

• the derivation of appropriate cells

• the identification of the appropriate recipe of biomolecules for the signalling process

• the identification of a structural support for the tissue while it is growing

• the provision of the optimal sterile environment in which molecular and mechanical signalling can occur

• and the incorporation of the resulting structure into the patient.

Obviously there is insufficient space here to discuss all of these aspects, but a few can be highlighted as being of special interest.

The starting point is the right cell, and, as noted earlier, we have several generic choices. There are three types of sources of cells for use in tissue engineering. First, they may be derived from the patient to be treated, in which case they are referred to as autologous cells. In cartilage tissue engineering, this implies that we could take chondrocytes from the patient, or alternatively take stem cells from that patient, for example from their bone marrow or peripheral blood, and cause these to be differentiated into chondrocytes. Secondly, they could be derived from a human donor, in which case they are referred to as allogeneic cells, which again could involve the direct use of donor chondrocytes or allogeneic derived stem cells, which would normally imply embryonic stem cells. Thirdly, it is possible for the cells to be derived from animal sources. This choice has largely been taken out of our hands by regulations that essentially ban the use of xenotransplants, that is live tissues or cells transplanted from animals to humans, largely because of the unknown risks of disease transmission.

Autologous cells make a great deal of sense biologically, since there will be no question of immunological rejection when the patient’s own tissue is placed back in them. Logistically, clinically and economically, however, there are quite serious limitations to this use and much has to be said for the allogeneic approach if the rejection process can be modulated. This is a matter of serious scientific research and debate at the present time. For example, the autologous route may require a dedicated facility, with appropriate sterility and security over several weeks, that is essentially customised to the patient and, therefore, very expensive. The allogeneic route is far more consistent, with an off-the-shelf, manufactured product, with economy of scale and greater possibilities for quality control.

So too is the possibility of using stem cells, alluded to above. A stem cell is one that has yet to decide which specific cell it will turn into. Bone marrow contains a rich supply of stem cells and if the patient is willing to undergo the procedure of bone marrow biopsy, the autologous tissue engineering process can start right there. Alternatively, circulating blood contains some stem cells, but these are few in number and major process work on the blood, involving separation and multiplication techniques have to be used. The alternatives are embryonic stem cells, that is stem cells derived from aborted foetuses. This clearly has major ethical dimensions, and governments worldwide have taken this seriously with respect to the control of the use of such stem cells, in many cases actually banning their use, not just for therapeutic purposes but also for any experimental work.

The scaffold

Once an appropriate number of the right cells has been derived and manipulated, they can be seeded onto a material substrate that will form the matrix for tissue formation. It is necessary to design this substrate very carefully, since many cells change their structure and nature when grown under the wrong conditions and, indeed, some stem cells having being differentiated down the right pathway can then de-differentiate (i.e. reverse the process) under inappropriate conditions. This substrate is usually referred to as a scaffold. It is usually designed to be biodegradable since it acts as a temporary porous template for tissue formation. Several synthetic biodegradable polymers, such as certain aliphatic polyesters, are in widespread use as such templates. It is obviously important that they degrade with the right kinetics and without generating any toxic or proinflammatory degradation products. There are certain drawbacks with these synthetic polymers with respect to the responses they generate in the host, and attention is rapidly turning towards natural biopolymers, such as proteins (e.g. collagen and elastin) or polysaccharides such as chitosan, alginates or hyaluronic acid. It is necessary to produce these scaffolds with a high degree of control over three-dimensional architecture, and there are some very interesting developments in advanced manufacturing techniques such as soft lithography, three dimensional printing and solid free form fabrication.

The bioreaction

The critical point of the whole process is now the creation of the right environment where, under sterile conditions and meeting all of the usual requirements for cell nutrition and metabolism, the cells and scaffolds are brought together and the molecular and mechanical signals applied. This could involve the use of a cocktail of growth factors delivered in a time-dependant manner, the insertion of genes into the cells, and the application of mechanical stresses, either via structural stresses imparted by the scaffold or via fluid stresses through the culture medium. The equipment in which this takes place is usually called a bioreactor.

The process could take place over several weeks before a suitable volume of tissue is generated. This has to be placed in the patient where it should become properly incorporated into the host. This is not a trivial point. Apart from the control over any immunological recognition process should allogeneic cells have been used, it is essential that the tissue has an appropriate blood supply, this critical vascularisation process being known as angiogenesis, and that the incorporation takes place without stimulation of inflammation and with cessation of all regenerative activity in order to avoid any undesirable cell proliferation, which, as noted before, could lead to the formation of tumours.

The impact of tissue engineering

This short summary should give some hint that there is real practical engineering in the process of tissue engineering, with respect to bioreactors and scaffolds, for example, as well as the conceptual affinity with engineering creativity. In many respects, tissue engineering is in its infancy and many of the processes discussed in this article are still in the experimental or pre-clinical testing stage. From the clinical and commercial perspective, there is a strong customer led demand for progress, but also a strong resistance to leading the way because of the uncertainties. There are several ethical issues to face with respect to both stem cells and gene transfer. There are huge regulatory hurdles, since regulatory bodies around the world are struggling to cope with the new science. The health economic aspects are also very challenging: tissue engineering is undoubtedly expensive at the point of delivery and successful business models for companies have yet to be resolved, primarily because it is often so difficult to identify what the product is.

At the moment there have been some clinical successes in the tissue engineering of skin, in the treatment of burns and chronic ulcers, in the treatment of small injuries to cartilage and in the regeneration of bone. There has been a great deal of progress experimentally with more complex tissue such as nerve and muscle, and in the regeneration of more complex architectures, for example within the urogenital system and the cardiovascular system. Some blood vessels and heart valves have been successfully created and used in large animal models. The increased complexity means that the bioreactor may have to cope with more than one cell type at a time, and the very critical functionality of some of these systems, as in the arteries and heart valves mentioned above, will pose some significant challenges where it is not possible for the final product to undergo performance testing before implantation. Nevertheless it is envisaged that once the technical and logistics challenges have been met, tissue engineering will have a huge impact on the treatment of diseases and injuries that affect many parts of the human body, thereby improving the quality of life for the very many patients for whom there is no current effective therapy. Indeed, the concept of creating new tissue to alleviate suffering, especially if it can be done without serious ethical confrontations and unacceptable levels of interference with nature, is a powerful reminder of the contribution that engineering makes to mankind.

Definitions of key terms used in tissue engineering

These definitions are taken from The Williams Dictionary of Biomaterials, D.F.Williams, Liverpool University Press, 1999.

Allogeneic
Derived from individuals of the same species, or cell lines, that are not genetically identical.

Autologous
Relating to a product used in the treatment of a patient that is wholly derived from the tissues or fluids of that patient.

Bioreactor
Any device which is designed to contain structures, both cellular and molecular, that are capable of taking part in a specific biological process and from which the products of that process can be harvested or extracted.

Chondrocyte
Mature cartilage cell embedded in a lacuna within the cartilage matrix.

Clone
Genetically identical progeny produced by the natural or artificial asexual reproduction of a single organism, cell or gene.

Differentiation
The expression of cell- or tissuespecific genes which results in the formation of a specific cell type.

Extracellular matrix
The non-cellular matrix of proteins and glycoproteins surrounding cells in certain tissues.

Growth factor
Any of a group of polypeptide hormones which regulate the division of cells.

Receptor
A molecule on the surface or within a cell that recognises and binds with specific molecules, producing a specific effect in the cell.

Scaffold
The porous structure which serves as a substrate and guide for tissue regeneration.

Stem cell
A multipotential cell from which differentiated cells derive.

Xenogeneic
Relating to a product used in the treatment of a patient that is wholly or partly derived from the tissues or fluids of a different species to that patient.

Professor David Williams

University of Liverpool

David Williams is Professor of Tissue Engineering at the University of Liverpool and Director of the joint Liverpool–Manchester UK Centre for Tissue Engineering. He was trained as a materials scientist and has worked in the medical applications of materials for 35 years. He was elected as a Fellow of The Royal Academy of Engineering in 1999. He is Editor-in-Chief of the leading journal Biomaterials and scientific advisor to the European Commission on public health aspects of medical devices and pharmaceutical products.