Advances in Tissue Engineering

The following sections are included:

• Contents

• Contributors

• Foreword

• Introduction

In the last few decades tissue engineering has emerged as a technology, and this has now evolved into what is called regenerative medicine, including not only the replacement of tissues and organs, but also repair and regeneration. This field is an outgrowth of the biological revolution. Although research in this field goes back well into the 20th century, it was in the 1990s that research accelerated and that an industry began to emerge. In this same period the Tissue Engineering Society was formed, and this has evolved into what is now the Tissue Engineering and Regenerative Medicine International Society. Although the industry still may be characterized as being in a fledgling state, there are some positive signs appearing. Furthermore, the field has become a global activity, one that has the potential to alter the practice of medicine as we know it. Thus, building on the past, the future of tissue engineering and regenerative medicine remains extremely promising.

For any tissue engineering or regenerative medicine strategy its success is dependent largely on controlling the biology of the cells at the site of repair or regeneration, since it is the cells that constitute and co-ordinate the basic structure and function of tissues. The ideal situation would be to augment intrinsic self-repair mechanisms by stimulating the mobilisation, recruitment and activity of cells within the body. At present, however, this approach is quite limited, but as our knowledge of cell biology, cell environments, cell signalling and cell trafficking increases such activation of self-repair mechanism might become possible. The alternative is to supply cells exogenously and this raises a number of questions and challenges, such as what are the most appropriate sources and types of cell, how to control the growth and differentiation of the cells and how to deliver the cells to site of repair. This chapter provides a brief summary of some of the various cell types, including differentiated somatic adult cells, somatic stem cells, foetal cells and embryonic stem cells that are being and which might be used in promoting and understanding tissue repair and regeneration.

Although human embryonic stem cells may have enormous potential for the treatment of degenerative diseases, their origins and derivation have raised unprecedented controversy in many societies. Many organisations, on both international and national levels, have responded with regulatory systems that seek to manage and supervise such work. These systems vary from voluntary professional guidelines and international treaties to government agencies and binding national legislation. This chapter briefly summarises the main controversies surrounding human embryonic stem cell research and the existing international mechanisms that address its conduct. We then offer a more detailed overview of the relevant regulatory structures of five nations — the US, the UK, China, India and South Korea — where research with human embryonic stem cells is commonly practiced. A review of the responsible state and professional bodies in each country is included, along with a brief discussion of any relevant legislation. Regulations relevant to the conduct of international collaboration with national research groups are included where possible.

There has been continued substantial interest from both scientists and the public in the therapeutic and scientific potential of stem cells since the first isolation of human embryonic stem cells (hESC) in 1998.¹ Pluripotent hESCs derived from the inner cell mass of preimplantation embryos following fertilisation in vitro (IVF) have been well studied, and proposed not only as potentially useful in treating degenerative diseases, but invaluable clinically relevant alternatives to animal models for studying early development, and for identifying novel pharmaceuticals with high throughput drug screens in vitro.² In addition, due to ethical controversy surrounding the use of embryos in stem cell research, there has been a paradigm shift in some research groups who have reported alternative methods of obtaining embryonic stem-like cells without the use of embryos. Most recently there has been some enthusiasm for exploring the use of induced pluripotent stem cells (iPS) which may be able to be derived from somatic cells by manipulation of transcription factors.³ The derivation, culture and characterisation of hESC are currently a labour intensive and time consuming process. Emerging tissue engineering technology such as robotic control of culture will overcome such hurdles and facilitate the scale-up needed for clinical therapies.

Stem cells (both embryonic and somatic) have the capacity to self-renew and differentiate into specific lineage under permissive conditions. Although progress has been made on differentiation of pluripotent ES to a defined cell type, it is far from clear which are the signalling pathways controlling differentiation of a defined cell type and even less known about how to differentiate ES cells to be a functional cell type. Along with ES cells, foetal mesenchymal stem cells (MSC) are the only stem cells to be pluritpotent, expressing Oct-4 and Nanog, while adult MSC remain multipotent.

The skeletal system of adult mammals fulfills many roles including providing structural support for the body musculature and functioning as a storehouse for calcium. Additionally, it also serves as a repository for the bone marrow, which represents a rich source of stem cells. The hematopoietic stem cell (HSC), which generates all lineages of the immune system and blood, was the first stem cell shown to reside within adult bone marrow and remains the best characterized with respect to phenotype and function. Soon after the discovery of the HSC a separate cell population in marrow was described with the capacity to form ectopic bone tissue in vivo. This stem cell population, referred to as mesenchymal stem cells or multipotent marrow stromal cells (MSCs) has since been shown to differentiate into various connective tissue cell types including adipocytes, chondrocytes, myoblasts, and osteoblasts. Most recently, adult bone marrow has also been shown to harbor endothelial progenitor cells (EPCs) that participate in postnatal vasculogenesis. Therefore, bone marrow appears unique with respect to the fact that it harbors three physically and functionally distinct adult stem cell populations. Herein I review the discovery of these stem cells and emphasize their characterization and function. Moreover, I describe recent findings that indicate these stem cells are derived from a common precursor during embryonic development. This fact may account for their phenotypic similarities and functional interdependency.

With approximately 130 million babies born worldwide every year umbilical cord blood represents perhaps the largest potential source of stem cells for regenerative medicine. Between 1972 and 2008, it is estimated that over 10,000 patients would have been treated by cord blood cells for over 80 different clinical conditions. Cord blood stem cells are used clinically mostly to support patients suffering from haematological and immunological diseases but they also provide emerging therapeutic solutions for limited cases of type 1 diabetes or infant cerebral injuries. Cord blood samples are collected after birth and bio-processed before cryopreservation in either public biobanks for unrelated allogenic use or private family biobanks for related allogenic and autologous uses. Regenerative medicine research demonstrated the existence of multipotent stem cells with embryonic characteristics in cord blood, which can produce over 20 tissue types including liver, neural or insulin-secreting cells. Cord blood stem cells not only offer therapeutic benefits at present but also show real potential for the advancement of regenerative medicine.

Adipose tissue is an abundant, accessible, and replenishable source of adult stem cells. Adipose-derived stem cells (ASCs) can be isolated from human lipoaspirates by collagenase digestion, differential centrifugation, and plastic adherence. The ASCs display a consistent immunophenotype that is similar, but not identical, to that of bone marrow-derived mesenchymal stem cells (BMSCs). Like BMSCs, ASCs inhibit mixed lymphocyte reactions in vitro, suggesting that it will be possible to perform allogeneic transplants in clinical settings. The expanded ASCs are multipotent and can differentiate into adipocytes, chondrocytes, endothelial cells, myocytes, neuronal-like cells, and osteoblasts, among other lineages. Furthermore, genetic engineering methods can be applied to ASCs, allowing their potential use as gene delivery vehicles in vivo. This chapter reviews the expanding literature base relating to ASC applications for regenerative medicine.

Stem cells are characterized by their dual ability to self-renew and differentiate, yielding essentially unlimited numbers of progeny that can replenish tissues with either high turnover such as blood and skin or contribute to the regeneration of organs with less frequent remodeling such as muscle. In contrast to their embryonic counterparts, adult stem cells can only preserve their unique functions if they are in intimate contact with an instructive microenvironment, termed niche. Stem cells integrate a complex array of niche signals that regulate their fate, keeping them in a relatively quiescent state during homeostasis, or controlling their numbers via symmetric or asymmetric divisions in response to the regenerative demands of a tissue. This chapter provides an overview of the current state of knowledge of structural and functional hallmarks of mammalian stem cell niches and offers a perspective on how bioengineering principles could be used to deconstruct the niche and providing novel insights into the role of its specific components in the regulation of stem cell fate. Such "artificial niches" constitute powerful tools for elucidating stem cell regulatory mechanisms that should fuel the development of novel therapeutic strategies for tissue regeneration.

The field of stem cell biology is fast approaching a degree of maturity that will allow novel therapies to be exploited both clinically and commercially. The first such treatments are likely to rely on the use of a patient's own (autologous) stem cells in order to avoid immune rejection. However if these approaches are to have any long-term viability then the use of donor (allogeneic) cells instead of individualised therapies will be essential in order to allow scale-up of production. There is some evidence that stem cells are hypoimmunogenic as they express only very low levels of major histocompatibility antigens and they are therefore likely to avoid immune rejection. In this chapter we review the evidence for hypoimmunogenicity of stem cells and highlight some differences between adult and embryonic stem cells in their immune stimulatory properties. We also review data demonstrating that the hypoimmunogenic properties of stem cells may sometimes be lost once they have differentiated or after they have been exposed to inflammatory cytokines.

Stem cell cultures are complex and intricate processes. There are many contributing factors that affect and influence the outcome of the culture, including (1) factors affecting the physicochemical environment, (2) nutrients and metabolites, (3) growth factors, and (4) the various cell types that exist within the cell culture system, as shown in Fig. 1A.^1–8 Physicochemical factors, such as pH, temperature, dissolved oxygen and carbon dioxide levels, determine the cellular environment and affect cellular behaviour and functionality. Slight changes in the levels of these physicochemical parameters will result in large changes in the cellular output. For instance, the optimal pH for the process of megakaryopoiesis in haematopoietic cell cultures has been reported to be at pH 7.60 while for the process of granulopoiesis it has been reported to be at pH 7.21.^1,2 Such reports highlight the transient nature of cell bioprocesses and the existence of gradients occurring in the in vivo microenvironment. Specifically, the inherent heterogeneous and transient nature of cell bioprocesses, which includes cells at different maturation and differentiation stages (stem cells, progenitors, precursors, and mature cells), necessitate that culture conditions are dynamic during the culture time. Consequently, control of cell culture bioprocesses is difficult to achieve and culture conditions are generally not optimal.

The provision of central resources of human stem cell lines will be an important element in enabling progress in stem cell research and the development of safe and effective cell therapies. These resource centres, commonly referred to as "stem cell banks", could promote advances in the field of stem cell research, by providing access to well-characterised and quality controlled seed stocks of human stem cell lines that have been checked for appropriate ethical provenance. Such banks can also deliver benefits for the field through establishing international collaboration and standardisation between the developing national stem cell banking centres. This chapter will review the key issues that centres banking and distributing stem cells must address to support the research community in the development of exciting cell therapies for the future.

Cells in our tissues are exposed to complex arrays of biochemical and biophysical cues from their protein- and sugar-rich extracellular matrix (ECM). In concert with cell-intrinsic regulatory cascades, these temporally and spatially coordinated signals instruct cells to acquire specific fates, controlling, for example, cell division, differentiation, migration or apoptosis. Conversely, cells are constantly secreting signals that can trigger structural and biochemical microenvironmental changes, as is most evident during proteolytic remodeling of the ECM. The resulting reciprocal and dynamic cell-matrix interaction is crucial for tissue development, maintenance and regeneration and, if gone awry, it can be involved in disease progression such as tumor metastasis. Recent efforts in the development of synthetic biomaterials for tissue engineering aimed to mimic the cell-instructive and cell-responsive function of ECMs. This chapter focuses on the molecular design, function and application of such smart biomaterials as cell-responsive artificial ECMs that can for example actively participate in cascades of morphogenesis during tissue regeneration (see also other chapters in this book).

Synthetic bioactive and bioresorbable composite materials are becoming increasingly important as scaffolds for bone tissue engineering. Next generation biomaterials should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, stimulating the body to heal itself and leading to replacement of the scaffold by the regenerating tissue. In the present chapter composite materials based on smart combinations of biodegradable polymers and bioactive ceramics, including hydroxyapatite and bioactive glasses, are discussed as suitable materials for scaffold fabrication. These composites exhibit tailored physical, biological and mechanical properties as well as predictable degradation behaviour. The appropriate selection of a particular composite for a given application requires a detailed understanding of relevant cells and/or tissue response. Knowledge concerning interactions between cells and their immediate local environment in composite scaffolds has deeply improved in the last years. An overview of these findings is presented highlighting the influence of material processing methods, scaffold microstructure as well as the importance of the nature and amount of the bioactive ceramic particulate included in specific polymer matrices. The chapter also emphasises the response diversity according to the cell type used in vitro or the chosen in vivo models (species and location), suggesting the utility of standardisation in this field of biomaterials science. Bioactive composites discussed in this chapter, enhanced by microstructural optimisation and surface engineering, are suggested as the materials of choice for development of optimal bone tissue engineering scaffolds.

Aggregation of cells into clusters is a tissue engineering approach used for rapid and controlled formation of structures that have certain architectural and functional properties of native tissue. Some mammalian cells aggregate spontaneously in suspension, for others, methods have been devised to force them to aggregate. This chapter focuses on approaches based on biomaterials and bioreactors to induce or accelerate cell aggregation. It also describes a recently introduced cell surface engineering method used to induce aggregation of homotypic as well as heterotypic cell types.

Future advances in tissue engineering are likely to require new tools and materials that provide novel capabilities for the construction of engineered tissue as well as the analysis and monitoring of such products. Nanotechnology research is yielding many new materials with unprecedented control over structure and function, often with highly unique properties. An example of nanotechnology in tissue engineering are nanoparticles, including quantum dots, where control over the size of semiconductor nanocrystals determines the emission spectra from these highly stable fluorescent probes, carbon nanotubes, which offer the potential to make very strong nanocomposites, and polymeric dendrimers, where the highly branched nature of the material offer opportunity to generate multi-functional particles. Nanotechnology also has advanced nanoscale control over material assembly. In this chapter, we will explore some of the ways that these technologies are being used to improve tissue engineering scaffolds, enhance cellular imaging capabilities, and enable monitoring of biological environments and processes.

Despite the enormous advances in tissue engineering, several challenges still prevent the widespread clinical application of tissue engineering products, such as how to acquire adequate number of cells, and how to engineer complex vascularized tissues that mimic the complexity and function of native tissues. The merger of bio-materials and microscale technologies offer new opportunities to overcome the challenges in tissue engineering to fabricate scaffolds and direct stem cell differentiation. In this chapter, various applications of microscale technologies have been illustrated in controlling stem cell fate and building complex artificial tissues. It is envisioned that with the rapid growth of this burgeoning research field, microscale technologies will transform the conventional tissue engineering approaches and greatly contribute to the therapeutic potential of tissue engineering.

Increasingly biological sciences are being built on a quantitative foundation, based upon our ability to accurately determine the temporal and spatial variations in the concentration of key molecules. It is this quantitative analytical data that provides the testable basis for building hypotheses about biological systems. Although many, diverse analytical techniques have been used to collect quantitative data these are often destructive of the system being analysed. Biosensors by contrast promise the ability to measure selected molecules continuously and in real-time with good spatial resolution. They use specific molecular recognition at the surface of a transducer such that an electrical signal is generated in proportion to the concentration of the target analyte. Many different molecular recognition reagents have been exploited in this respect and include enzymes, binding proteins and nucleic acids, whilst electrochemical, optical and mass sensitive signal transduction devices have been used to generate the electrical signal (typically a voltage or current).

Using biosensors in vivo presents additional challenges over and above simply relating signal to concentration. Biocompatibility is an ever-present issue and includes both the effect of the biological matrix on the sensor as well as sensor components on the biology. In this chapter a review of the different sensing modes is presented and their potential applicability to the monitoring of cells, tissue and tissue constructs is presented.

Monitoring of cell status and tissue formation within a three-dimensional cultured tissue is important to the success of engineered tissue development. Here we describe a method using microdialysis probes for the continuous monitoring of local extracellular environment, cell metabolic activity, cell functions and possible tissue formation. Using 3D culture of chondrocytes and culture of intervertebral disc explant as examples, the effectiveness of this method is demonstrated. Challenges and practical issues in using this method are discussed as well.

In regenerative medicine it is important to be able to understand how cells are behaving in response to stimuli. The stimuli can be biological signals or materials. Raman spectroscopy allows the non-invasive real time monitoring of live cells in vitro by interpretation of spectra. Materials are being developed to use as templates (scaffolds) for tissue regeneration. The morphology of the pore structure is critical if tissue is to populate the scaffold. X-ray microcomputed tomography is the only method that can obtain 3D images of pore networks. Novel image analysis has been developed that can quantify pore networks. There is potential for this technique to be used to image tissue growth into scaffolds ex vivo. The next challenge is to adapt these two promising techniques to monitor the response of cells to porous scaffolds, including that of cells within the porous network.

Stem cell research is undergoing a critical transition from being a discipline of the basic sciences to being recognised as a potential component of medical practice. Cell transplants to replace cells lost due to injury or degenerative diseases, for which there are currently no cures, are being pursued in a wide range of experimental models.

The monitoring of cellular grafts, non-invasively, is an important aspect of the ongoing efficiency and safety assessment of cell-based therapies. Magnetic resonance imaging (MRI) methods are potentially well suited for such an application as they produce non-invasive "images" of opaque tissues. For transplanted stem cells to be visualised and tracked by MRI, they need to be tagged so that they are "MR visible". We are developing and implementing a programme of molecular imaging in pre-clinical models that is directed towards improving our understanding of stem cell migration in the context of the whole organism.

In order to achieve these goals we are engineering novel MRI contrast agents and developing specific tagging molecules to deliver efficient amounts of contrast agents into stem cells. The intracellular contrast agents are based on either paramagnetic nanoparticles, such as dextran-coated iron oxide, or other MR contrast agents. Methods for monitoring implanted stem cells non-invasively in vivo will greatly facilitate the clinical realisation and optimisation of the opportunities of stem cell-based therapies.

Tissue engineering can be broadly defined as the combination of biology and engineering to repair or replace lost tissue function. From an industry perspective, the field encompasses implanted biomaterials, cell and tissue transplants and therapies, and even extracorporeal cellular devices. To achieve its goals, tissue engineering must effectively utilize not only multiple aspects of engineering but also several aspects of biology that govern mechanisms of organ development, repair and regeneration. The field has always had a strong focus on application yet the challenge of integrating biological science, engineering and medicine has kept many past efforts from reaching their therapeutic and commercial potential. This chapter covers the evolution of tissue engineering, looking at the change in emphasis from bioengineering to stem cell biology and the potential impact of this shift in focus from an industrial perspective. In addition, we have analyzed four major commercial thrusts from past to present: vascular tissue engineering, cartilage repair, liver-assist devices and skin constructs, paying particular attention to how the biomedical disciplines must be integrated to achieve commercial feasibility and therapeutic success. Each example yields one or more important and practical lessons learnt that could be instructive for most future medical and commercial efforts in tissue engineering.

The routine delivery of therapeutics to patients requires a translational infrastructure that includes a substantial level of financial support that is typically provided by the pharmaceutical industry and venture capitalists. However, there is little evidence that the stem cell arena has attracted any significant levels of translational support despite the anticipated revolutionary impact of stem cell-based therapeutics. The pharmaceutical industry has adopted a "watch and wait" stance for stem cell-related products and this attitude together with a complex and evolving patent landscape have to date deterred any major commitment by private investors to support the stem cell sector. However, timing is key and 2007 has been notable for a series of events that will positively influence the stem cell sector. The European Union accepted a harmonised regulatory system for advanced therapy medicinal products that include stem cells; major players within the pharmaceutical industry publicly announced that they would work with human embryonic stem cells and furthermore, stem cell therapy companies were able to successfully attract corporate investors. These events and their impact on the business of stem cells are the subject of discussion within this chapter.

Regenerative medicine is reaching maturity. There are numerous examples of translation of research efforts into initial clinical practices. Stem cells have played a pivotal role, as exemplified by the 2007 Nobel Prize accolade to one of the discoverers of embryonic stem cells (ESCs). In spite of this, numerous challenges lay ahead if the conversion from a few clinical successes to treatment to a vast number of people with incurable illnesses is to succeed.

Within these challenges the ability to obtain large and uniform number of specific cell lineages is of paramount importance. This short chapter reviews the continuous efforts that are being made towards this goal, by learning how to grow the cells in a more natural 3D environment to sophisticated and automated ways of stem cell bioprocessing.

The replacement of the "flat biology" of the Petri dish with three-dimensional (3D) cell cultures has shown to narrow the gap between cell behaviours and function in vitro and at the physiological settings. A fundamental challenge to realise the potential of the 3D cell culture is the design and application of "smart" bioreactor systems. These systems should provide homogenous mass transport into the internal volume of the cultured cell constructs as well as to efficiently propagate physical and mechanical stimuli. Herein, we describe the design principles of various bioreactors, starting with the conventional spinner flasks, the rotary wall vessels and up to the latest perfusion vessels. In particular, the key role of perfusion bioreactors in regenerating the dynamic 3D cell microenvironment is demonstrated by providing a few successful examples of engineering thick functional tissues, such as the cardiac muscle tissue. In closing this chapter, we envision future innovations in bioreactors.

The United Kingdom regulatory landscape as it applies to cell-based therapies is rapidly evolving and constantly produces new information for researchers. This chapter brings together the plethora of information in the form of a process map of the key stages in the life cycle of a cell-based product, from cell/tissue procurement, processing and manufacture, through pre-clinical trials, clinical trials and on to commercialisation and post-launch activities. The critical components of each stage are described, and key issues which are pertinent to the UK researcher are discussed, for example, use of pre-clinical models, documentation requirements for clinical trials. The text goes on to identify which regulations, codes of practice and standards are already available for use in the UK and links them to the life cycle stages. The most recent regulation to be agreed in 2007 in Europe is also discussed. EC 1394/2007 is an amendment of EU Directive 2001/83/EC, and describes overarching regulations of advanced therapy medicinal products (which encompasses cell-based therapeutics).

This information is taken from a Publicly Available Specification (No. 83) which was written by the authors of this chapter and published by the British Standards Institution (BSI) in 2006. It is intended that this PAS acts as a quick reference source to increase clarity for users on the requirements needed for exploitation of a cell-based therapy in the UK, rather than an in-depth examination of the supporting literature.

The observations that mice exposed to otherwise lethal irradiation could survive if their spleens or marrows were shielded, or if they received an infusion of bone marrow, led to the first attemps of bone marrow transplantation in humans in the mid-1950s by E. D. Thomas and J. Ferrebee. Thanks to Thomas' persistence despite criticism and initial clinical failures, and thanks to the development of a canine model of bone marrow transplantation by Thomas and Storb, the role of allogeneic hematopoietic cell transplantation (HCT) changed during the last 50 years from a desperate therapeutic maneouver plagued by apparently insurmountable complications to a curative treatment modality for thousands of patients with hematologic diseases. Further, it was recognized that allogeneic immunocompetent cells contained in the graft mediated therapeutic antitumor effects independent of the action of the high-dose therapy. These were termed graft-versus-tumor (GVT) effects. This prompted the recent development of non-myeloablative conditioning regimens for allogeneic HCT that have allowed offering this treatment modality in elderly patients and those with comorbid conditions. While hematopoietic stem cells were identified in the early 1960s, identification of other types of stem cells such as mesenchymal stem cells or embryonic stem cells might pave the way for stem cell therapy in regenerative medicine in the future.

Tissue engineered skin was the first out of the stable of tissues that could be made in the laboratory from biopsies of patients skin expanded and then delivered back to them. As patients have been benefiting from cultured skin cells since 1981,¹ at 25 years old this is far from being a new area. In this article the question of to what extent tissue engineered skin has finally come of age will be reviewed.

There are currently three clinical areas where it can benefit man — for the treatment of patients with extensive skin loss due to burns injuries, to accelerate or initiate healing in patients with chronic non-healing ulcers and for reconstructive surgery purposes (an area which is still in its infancy but can encompass the treatment of pigmentation defects and diseases such as vitiligo and scarring and hopefully blistering diseases). There are also many in vitro applications where having a physiologically relevant model of skin can teach us more about normal and pathological skin biology than working with monolayers of skin cells.

This chapter looks at why tissue engineered skin was initially developed for burns injuries and how it is now also used for chronic wounds and beginning to be used for reconstructive surgery. The challenges that remain are then considered followed by a summary of some of the in vitro applications for tissue engineered skin.

Stem cell therapy is currently one of the most exciting areas of biomedical research with hopes of providing therapeutic treatments for a myriad of diseases, including liver diseases. Several liver diseases fall under this category, including fibrosis of the liver, and hepatitis B and C viral infection. At the cirrhotic stage, liver disease is considered irreversible and the only alternative is orthotopic liver transplantation. While orthotopic liver transplantation cures chronic liver disease and a variety of metabolic and genetic deficiency disorders, the increased shortage of donor organs restricts liver transplantation. Therefore novel therapeutic options are in demand.

Adult stem cells with their multilineage differentiation potential and self-renewal are possible candidate cells and it is believed that novel cellular therapeutics can perform better than any medical device, recombinant proteins or therapeutic agents. In this chapter we have presented our own experience using adult stem cells for therapeutics for liver disease as well as reviewed the latest literature on this topic.

Teeth are clinically important, easily accessible, non-essential organs whose development, cell biology and physiology are well-understood. Missing or damaged teeth are currently replaced by non-cellular structures such as metal implants. Replacement of teeth with cell-based implants that will form whole teeth or specific tooth structures is a realistic goal of regenerative dentistry. Putative stem cell populations have been identified in several different tooth tissues and the ease with which these cells can be obtained, for example from naturally lost deciduous (milk) teeth makes them an attractive source of mesenchymal stem cells.

Congenital abnormalities, trauma, infection, and cancer can all necessitate reconstructive surgery in the genitourinary tract. Currently, such surgeries may be performed with native non-urologic tissues, homologous tissues from a donor, heterologous tissues or substances, or artificial materials. However, these materials often lead to complications after reconstruction, including rejection of the implanted tissue. The field of tissue engineering may soon allow replacement of lost or deficient urologic tissues with anatomically and functionally equivalent ones that are derived from a small sample of the patient's own tissue. This would improve the outcome of reconstructive surgery in the genitourinary system and lead to new methods of treating these disorders.

The lack of appropriate regenerative capacity of the heart has been the spur to early clinical trials using autologous stem cells (bone marrow-derived and skeletal myoblast) for cardiac repair. Associated in vivo and in vitro laboratory experiments have been essential in advancing understanding of mechanism of benefit or harm from these cells, and in suggesting other sources of stem cells for cardiac application. Skeletal myoblasts have dangers, in that lack of integration with myocardium produces an arrhythmic substrate. Bone marrow-derived stem cells, while safer, do not generate significant new cardiac muscle as part of their beneficial actions, suggesting an angiogenic mechanism or paracrine protection of existing cardiomyocytes. Other adult stem cells, primarily from heart but also from organs such as testis, are currently being characterised as source of new cardiac tissue. Embryonic stem cells reliably produce contracting cardiac muscle, and are a likely candidate for future repair when problems of teratoma formation and immune response have been solved. They are also more suited to tissue engineering application, because of their ready availability and potential for expansion. Study of the biology of stem cells is also generating new paradigms for understanding the intrinsic regenerative capacity of the heart.

Cardiovascular disease is a major problem worldwide, and remains the leading cause of death within Europe. Clinical trials of stem cells have been conducted in the setting of acute myocardial infarction and chronic heart disease. At the time of writing there have been a number of different cell types, preparation and doses delivered by a number of different delivery routes to a variety of patients in small, mostly uncontrolled trials, generally with positive outcomes. Larger placebo-controlled trials are ongoing and we eagerly await their results. The challenges currently facing the field are to define the optimal target patient population, cell source, preparation, dose, delivery and retention of cells within the myocardium and the appropriate assays to detect meaningful clinical changes. Clearly not all of these questions can be answered in the clinical trial setting and it is imperative that basic science and translational research are conducted simultaneously to help guide clinical research. At present there appears to be no clear steer in answering these questions but we attempt to discuss the evidence to date and the future of stem cells in the management of cardiovascular disease.

The use of left ventricular assist devices (LVADs) has been increasing in patients with end-stage heart failure in order to bridge them to transplantation and these devices are now also being used as "destination therapy". Some of these patients have demonstrated signs of recovery of the native heart and this recovery has been sufficient in some cases to allow explantation of the device and hence a new concept of bridge-to-recovery has been described. The rate of successful recovery leading to device explantation, however, has been only 5%–24% until recently when we showed it to be possible to increase the frequency and durability of myocardial recovery using drug therapy combined with LVAD unloading. This chapter summarises different types of LVADs and describes the clinical implications of myocardial recovery following device implantation, monitoring of myocardial recovery and gives the up-to-date findings of the histological and molecular changes in recovery.

In vivo animal models are currently the gold standard for testing the capacity of stem/progenitor cells, smart biomaterials and novel growth factors for successful tissue engineering. In vitro models ultimately fail to provide the appropriate physiologically relevant microenvironment and hence animal models are an essential pre-requisite in the translation of any new therapy to the clinic. The aim of this chapter is to consider the available animal models commonly in use for tissue engineering, with a particular focus upon bone and cartilage research. Factors driving the choice of a given animal model are reviewed, according to the requirements of experimental design, hypothesis and the specific parameters to be tested. A number of animal models, together with their respective advantages and limitations are described, ranging from relatively simple experimental designs such as the subcutaneous implant and muscle pouch models, through to the diffusion chamber model and chorioallantoic membrane assay, to the more complex in vivo bioreactors and (arguably the most clinically relevant) bone and cartilage defect models. The need to consider the ethical issues of using animal models and the principles of reduction, replacement and refinement are emphasised in selecting the final experimental model of choice.

The use of animal models is essential in bridging in vitro studies with clinical outcomes for tissue repair and regeneration. However, in vitro cell culture systems and animal studies have significant limitations that impede translation of the research findings to successful clinical outcomes. This limitation also applies to the study and treatment of human osteochondral tissues and associated diseases. An alternative approach, based on a combination of tissue engineering and knowledge of developmental biology, can be employed to establish relevant in vitro 3D human disease model systems for osteochondral needs. To address this goal, tissue-specific microenvironments to simulate hydrodynamic fluid flow, biomechanical functions and cell biology have been established in vitro. As a result, more physiologically relevant human tissue engineered disease model systems can be established to provide better input into a variety of osteochondral diseases, as well as to prescreen treatment options.

The potential applications of tissue engineering for craniofacial tissue reconstruction are numerous. However, much remains to be accomplished if translation of research findings is to change the way craniofacial surgeons manage tissue defects presented in the clinical realm. Currently, research in craniofacial tissue engineering aims to address concerns stemming from several shortcomings of strategies that intend to replace or repair tissue defects rather than regenerate them. The majority of this research is conducted in three areas employing various animal models. First, an increased understanding of the mechanisms underlying craniosynostosis/cranial suture biology (i.e. suture fusion or patency) and tissue interactions that may exist between dura mater and the overlying suture complex may impart insight into the molecular mechanisms underlying bone formation. Signaling cascades that have received significant attention in this area include BMPs, TGF-β and FGFs. Second, distraction osteogenesis, a form of endogenous bone tissue engineering, has served to further highlight not only the cellular and molecular components needed for successful bone formation, but also the interaction of mechanical forces with the microenvironment of osseous regeneration. Finally, cell-based therapies have received widespread attention, as multipotent mesenchymal cells have been shown to differentiate towards a multitude of lineages and cell types in a number of experimental designs and applications both in vitro and in vivo. As such, they may be suitable for application in craniofacial tissue engineering and reconstruction. These three models of tissue induction and de novo tissue formation form the basis of regenerative medicine for craniofacial reconstruction, and promise to reveal factors in recapitulating nature, which may be used in applications of tissue engineering to craniofacial reconstruction.

Osteoarticular repair involves replacing damaged cartilage and subchondral bone in an articulating, load bearing joint. The human and financial impact of osteoarticular defects is immense. In current clinical practice the most common non-surgical methods for treatment of joint degradation are symptomatic only. For patients who fail conservative treatment, surgical intervention is required. Standard surgical interventions addressing both bone and cartilage damage include osteotomies, mosaicplasty and joint replacements. Cell based treatments of cartilage defects have emerged as promising strategies, but their FDA approved use is limited. Clinical trials that employ tissue-engineering methods have so far focused predominantly on the regeneration of cartilage in damaged knees. Emerging strategies use autologous cells in combination with bioresorbable delivery scaffolds to provide initial mechanical support, homogenous three-dimensional cell distribution, improved tissue differentiation, and suitable handling properties for delivery into patients. The invasive surgical nature of osteochondral repair, the challenges of "blinding" in scaffold implantation procedures, and ethical considerations presents considerable difficulties in implementing large prospective clinical studies to evaluate tissue engineered constructs. Despite these obstacles, the convergence of technological advances in the fields of cell culture, biomaterials, biologics, and surgical techniques makes this an exciting and highly competitive field.

Recent advances in stem cell biology provide the conceptual framework for the development of cell-based therapies for life-threatening diseases affecting many organs, including the lung. Because of its complexity and structure, cell-based therapy for the lung faces significant technical challenges. Therapeutic goals span a spectra of expectations that might include: (1) regeneration of functional lung tissue, (2) replacement of specific cells affected by inherited or acquired diseases with genetically altered progenitor cells, (3) provision of cells capable of enhancing repair or influencing oncogenesis directly or indirectly, and (4) introduction of cells capable of expressing therapeutic molecules for local or systemic delivery. The technical hurdles required for accomplishing each of these goals are distinct and of various heights. None are trivial. Knowledge of the cellular and molecular basis for specification and differentiation of stem/progenitor cells will be required for the successful application of cell-based therapies for the lung. This chapter reviews concepts derived from study of lung morphogenesis and repair as well as stem cell biology that will be relevant to the development of novel therapies for pulmonary diseases in the future.

Membrane ventilators are medical devices that provide extrapulmonary gas exchange, thus enabling novel treatment protocols for lung failure. These protocols prioritize lung protection over pulmonary gas exchange. In the intensive care setting, these devices are applied from several days up to a month to bridge patients to lung healing in acute respiratory failure, exacerbated COPD, and weaning failure. In these clinical settings the durability of current membrane ventilators is sufficient. However, in long-term applications periodical exchange of the membrane ventilator is required due to biofilm formation and clotting. This failure mode can possibly be avoided by development of long term artificial lungs. In the near future, vascular access, safety and control features for membrane ventilators will be a priority. In the mid and long term artificial lungs will combine artificial and biological components, to mimic biological surfaces, thus improving long-term function. In the far future, organoid structures, either as capillary microfluidic systems or three-dimensional structures, generated from stem cells, could serve as bioartificial lungs.

The following sections are included:

• Index