**Introductory Chapter**

### **Tissue Regeneration – A Clinical Science Whose Time Has Come**

Jamie Davies *University of Edinburgh UK*

#### **1. Introduction**

Tissue engineering is the application of knowledge gained in the study of basic developmental cell biology to the construction and repair of human bodies.

The surgically-focused side of the field has a long history, resting mainly on experience with wound healing and *ad-hoc* attempts to improve it. A well-known and long-standing example is modulation of bone healing by the application of physical force that gives rise to the image of a patient in traction, so common on humorous 'get well soon' cards.

The more cell biological side of the field is younger because its development had to await the gaining of significant amounts of basic knowledge in molecular cell biology, a field that is only a few decades old. The coming together of cell biology and experimental surgery to drive forward the development of tissue engineering is therefore a relatively recent phenomenon and only in this century has tissue engineering really taken off as a major area of research (Fig 1).

Fig. 1. Rapid growth of tissue engineering as a 21st century discipline. The graph shows the number of publications returned by a Pubmed search for ' *"tissue engineering" <*year*>'*.

Tissue Regeneration – A Clinical Science Whose Time Has Come 3

Fig. 3. Research in the young field of tissue engineering is a more global enterprise than research in synthetic biology, a field chosen for comparison because it is about the same age,

There are two broad methods for assisting endogenous healing; physical and biological. Physical forces are relevant to healing because many cell types sense and respond to mechanical forces using sensory systems in their cell-cell junctions, cell-matrix junctions and cytoskeleton. The sensing systems, though currently the subject of intense research, remain poorly understood (Katoh et al. 2008). Their detection of appropriate mechanical forces can nevertheless be critically important and the development of many cells and tissues, including heart muscles (Vandenburgh et al. 1996), heart valves (Jacot et al. 2010), and blood

Three chapters in this book illustrate how purely physical interventions can be used to assist healing. Monici and Cialdai (chapter 1) set the scene by reviewing in detail the ways in which living cells sense and respond to physical forces. These include mechanical and gravitational forces (which are, in terms of how they interact with living things, effectively mechanical) and also electric fields, which modulate various types of cell signalling and can be used to stimulate cell motility. Electrical forces are relatively easy to apply from outside the body and therefore offer exciting possibilities for safe, long-term treatment to encourage the regeneration of damaged nerves amongst other things (Hamid & Hayek 2008). Highfrequency electromagnetic radiation, in the form of microwaves, infra-red and light, has a long history of medical use (Kenkre et al. 1996, McCulley & Petroll 2008, Gravas et al. 2003, Owens et al. 2003, Simon et al. 2005, Goldberg & Sand 2000). Chapter 1 presents examples of laser light being used to promote regeneration of muscle and bone. In Chapter 2, Li illustrates how a very specific type of mechanical force, pressure waves from ultrasound, can be used to promote the repair of damaged nerves. Again, this technology holds

is taking off about as quickly and is also an application of molecular cell biological knowledge. As with figures 1 and 2, data plotted were obtained by conducting PubMed searches for the name of the field AND the name of the country, or a Boolean expression

vessels (Poelmann et al. 2008), is modulated or determined by mechanical force.

**2. Understanding and manipulating endogenous healing**

particular promise because it can be applied from outside the body.

combining several countries as appropriate.

Unlike many other young sciences, tissue engineering is growing very much as a global enterprise, perhaps because of the ubiquity of surgery and therefore the visibility of obvious need. It is noticeable, for example, that the contribution of China to research into tissue engineering is currently approximately equal to that of the European Union (judged by numbers of publications on a simple search: Figure 2).

Fig. 2. Growth of tissue engineering output by country. This graph was produced by searching PubMed for '<year> *"Tissue Engineering" xxxx'*, where <year> was '2000', '2005' or '2010' and 'xxxx' was the name of a country, or a Boolean expression combining, with a logical OR, a list of countries such as constituents of the European Union.

This global spread of research effort stands in marked contrast to the pattern seen in other new fields such as synthetic biology, which has grown more-or-less in parallel to that of tissue engineering and which is again very much of the twenty-first century. A comparison of pie charts of the national origins of papers in the two young sciences shows the difference immediately, about a third of research in tissue engineering coming from outside the USA and the European Union while only around fifteen percent does so in synthetic biology. The active engagement of so many countries and cultures in problems and applications of tissue regeneration ought to be a great strength for the field, encouraging the development of techniques suited to a wide range of problems and also to a wide range of health care economies.

Wherever it is done, research into tissue regeneration can be divided into three complementary sub-fields, and this book is organized around them. First, there is research that aims to understand and manipulate the endogenous healing processes in human tissues. This is the oldest part of the field. Second, there is the application of stem cell science to the regeneration of tissues (or to their *de novo* generation). Third, there is the construction of engineered scaffolds to guide normal healing processes and the behaviour of stem cells either in culture or *in vivo*. These different aspects of tissue regeneration link and overlap but, for convenience of organization, they will be considered in different sections of this book.

Unlike many other young sciences, tissue engineering is growing very much as a global enterprise, perhaps because of the ubiquity of surgery and therefore the visibility of obvious need. It is noticeable, for example, that the contribution of China to research into tissue engineering is currently approximately equal to that of the European Union (judged by

Fig. 2. Growth of tissue engineering output by country. This graph was produced by searching PubMed for '<year> *"Tissue Engineering" xxxx'*, where <year> was '2000', '2005' or '2010' and 'xxxx' was the name of a country, or a Boolean expression combining, with a

This global spread of research effort stands in marked contrast to the pattern seen in other new fields such as synthetic biology, which has grown more-or-less in parallel to that of tissue engineering and which is again very much of the twenty-first century. A comparison of pie charts of the national origins of papers in the two young sciences shows the difference immediately, about a third of research in tissue engineering coming from outside the USA and the European Union while only around fifteen percent does so in synthetic biology. The active engagement of so many countries and cultures in problems and applications of tissue regeneration ought to be a great strength for the field, encouraging the development of techniques suited to a wide range of problems and also to a wide range of health care

Wherever it is done, research into tissue regeneration can be divided into three complementary sub-fields, and this book is organized around them. First, there is research that aims to understand and manipulate the endogenous healing processes in human tissues. This is the oldest part of the field. Second, there is the application of stem cell science to the regeneration of tissues (or to their *de novo* generation). Third, there is the construction of engineered scaffolds to guide normal healing processes and the behaviour of stem cells either in culture or *in vivo*. These different aspects of tissue regeneration link and overlap but, for

convenience of organization, they will be considered in different sections of this book.

logical OR, a list of countries such as constituents of the European Union.

economies.

numbers of publications on a simple search: Figure 2).

Fig. 3. Research in the young field of tissue engineering is a more global enterprise than research in synthetic biology, a field chosen for comparison because it is about the same age, is taking off about as quickly and is also an application of molecular cell biological knowledge. As with figures 1 and 2, data plotted were obtained by conducting PubMed searches for the name of the field AND the name of the country, or a Boolean expression combining several countries as appropriate.

#### **2. Understanding and manipulating endogenous healing**

There are two broad methods for assisting endogenous healing; physical and biological. Physical forces are relevant to healing because many cell types sense and respond to mechanical forces using sensory systems in their cell-cell junctions, cell-matrix junctions and cytoskeleton. The sensing systems, though currently the subject of intense research, remain poorly understood (Katoh et al. 2008). Their detection of appropriate mechanical forces can nevertheless be critically important and the development of many cells and tissues, including heart muscles (Vandenburgh et al. 1996), heart valves (Jacot et al. 2010), and blood vessels (Poelmann et al. 2008), is modulated or determined by mechanical force.

Three chapters in this book illustrate how purely physical interventions can be used to assist healing. Monici and Cialdai (chapter 1) set the scene by reviewing in detail the ways in which living cells sense and respond to physical forces. These include mechanical and gravitational forces (which are, in terms of how they interact with living things, effectively mechanical) and also electric fields, which modulate various types of cell signalling and can be used to stimulate cell motility. Electrical forces are relatively easy to apply from outside the body and therefore offer exciting possibilities for safe, long-term treatment to encourage the regeneration of damaged nerves amongst other things (Hamid & Hayek 2008). Highfrequency electromagnetic radiation, in the form of microwaves, infra-red and light, has a long history of medical use (Kenkre et al. 1996, McCulley & Petroll 2008, Gravas et al. 2003, Owens et al. 2003, Simon et al. 2005, Goldberg & Sand 2000). Chapter 1 presents examples of laser light being used to promote regeneration of muscle and bone. In Chapter 2, Li illustrates how a very specific type of mechanical force, pressure waves from ultrasound, can be used to promote the repair of damaged nerves. Again, this technology holds particular promise because it can be applied from outside the body.

Tissue Regeneration – A Clinical Science Whose Time Has Come 5

of the cells diverge so much between species, but the work described in the chapter narrows down the difference to a specific stage in the time-course of the response and should help researchers to discover exactly why the cells of the different organisms end up behaving so

Compared to the research in stem cell biology that is catching so much public attention, research on simple physical and biochemical methods to promote regeneration may strike some readers as pedestrian. From the point of view of the patient, though, the important requirement is not that a researcher keeps publishing in the trendiest stem cell journals, but rather that they invent something that actually works. With the honourable exception of bone marrow transplantation, most stem cell treatments are very new and most clinical trials have been small and have not been running long. It is therefore too early to say, in most cases, whether stem cells will prove as effective as is hoped (Trounson et al. 2011). If they are not, it may well be that, in the short-to-medium term at least, real patients will benefit more, and in larger numbers, from the development of 'conventional' physical and

Stem cells are cells that can maintain their own population and give rise to daughters that are committed to differentiate. Depending on where they come from, they can have an ability to differentiate into any cell type of the body (for example, embryonic stem cells) or only a restricted range of cell types (for example, haematopoietic stem cells). Because of their potential to make new cells, stem cells have for some years been seen as a very promising source of new tissue. Initial experiments were generally designed on the assumption that a useful stem cell therapy would work by the stem cells themselves making new tissue to replace that lost to injury. In recent years, though, the field has been made more complicated by the realization that some stem cell types, in particular mesenchymal stem cells, exert significant beneficial effects by secreting factors that modulate inflammation and healing by the host tissue, and that much new growth is from the cells that were already there rather than being from the stem cells. The dual mode of stem cell action, which places different emphasis on each mode according to the precise situation, makes analysis of stem cellmediated repair complicated. On the positive side, the realization that secreted factors may be of significant, or even primary, importance in the mechanism of this repair is exciting from a clinical point of view because it may be possible to apply the factors directly without any need for patients to undergo potentially dangerous stem cell transplantation. This is not yet possible, though, and at the moment attempts at this type of therapy requires the use of

Development of effective stem cell therapies can be divided into two sub-problems; that of finding the most suitable type or source of stem cells to use, and that of applying them to the injured body in the most effective way possible. For convenience, section 2 of this book has been divided along precisely these lines, chapters 8-11 covering sources of stem cells and

Arguably the most convenient source of stem cells for therapy is the patient himself. Bone marrow is a rich source of stem cells. Many attempts to use them have involved mechanical recovery of marrow followed by purification of stem cells, perhaps with additional steps of

differently from the same initial pathway.

chemical based therapies.

stem cells themselves.

chapters 12-16 covering their application.

**3. Application of stem cells**

One of the major problems faced by surgeons and their patients is the replacement of bones that have to be removed because they harbour neoplasia. Amputation of a limb is clearly a major loss for a patient, so surgeons attempt to conserve whenever possible. If a large section of bone has to be removed, there is a problem finding something to replace it. The ideal replacement would be the excised bone itself, freed from all traces of the tumour. Crude attempts to kill tumour cells, by autoclaving or by severe chemical treatment, do result in a sterile bone but destroy so many of the normal guidance cues for cells that the bone is not properly integrated into the body and maintained when it is put back into the patient. In chapter 3, Diehl and colleagues describe an adaptation of a technique for sterilization that uses high hydrostatic pressures. This kills any cells in the bone but seems to leave intact the guidance cues necessary for the bone's efficient recolonization. It may therefore allow tumour-infested bones to be removed, sterilized safely and replaced, with the aim of leaving the patient with a normal limb.

Biochemical manipulation of cell behaviour is as ancient as medicine itself and the herbal remedies of the first physicians worked - when they worked at all - in this way. An improved understanding of the natural communications during healing and regeneration is allowing researchers to develop biochemical techniques for encouraging regeneration and discouraging damaging scar formation. An approach that is simple in principle even if not straightforward in practice, is to alter wound chemistry to make naturally-produced growth factors more effective. One way of doing this is to alter the extent to which wound matrix sequesters signalling molecules that would, if able to interact with cells, promote healing. In Chapter 4, van Neck and colleagues take this approach and discuss the use of synthetic, glycanase-resistant heparan sulphate mimics in mobilizing endogenous, regenerationpromoting signalling molecules. The chapter presents interesting data that suggest a beneficially-altered balance in favour of repair and against scar formation. Healthy tissue needs a good blood supply and, in chapter 5, de Mendonça describes how angiogenesis is controlled and draws attention to potential targets that would allow the process to be manipulated pharmacologically. Platelets are small cell fragments that travel in blood and that play a critical role in limiting the immediate consequences of wounding by formating a clot when a blood vessel is damaged. As well as performing this mechanical function, platelets are a rich source of growth factors and cytokines. In chapter 6, Ohkohichi and colleagues present data that argues that platelets can significantly promote the regeneration of damaged liver and possibly promote the survival of small grafts as well.

A well-known, and frustrating, fact about natural regeneration is that so-called 'lower' animals are a lot better at it than are humans. Have humans completely lost an ancient regenerative response still present in fish and amphibians, or is the difference a matter of degree, humans still retaining elements of the repair pathway but not activating it strongly enough? The difference is important, because the second of these hypotheses allows for the possibility of developing a clinical intervention that builds on an existing pathway but pushes it harder to give humans an amphibian-like power of rebuilding. Eleonora Grigoryan, in chapter 7, analyses the early cellular events that follow damage to the retina in amphibians and mammals. Although the final outcome of the type of damage is very different in these different organisms (amphibians making a new retina, mammals failing to do so), the early responses of the cells are the same but the conversion of non-neuronal cells to neurons and glia follows them only in amphibia. It is still not clear why the eventual fates of the cells diverge so much between species, but the work described in the chapter narrows down the difference to a specific stage in the time-course of the response and should help researchers to discover exactly why the cells of the different organisms end up behaving so differently from the same initial pathway.

Compared to the research in stem cell biology that is catching so much public attention, research on simple physical and biochemical methods to promote regeneration may strike some readers as pedestrian. From the point of view of the patient, though, the important requirement is not that a researcher keeps publishing in the trendiest stem cell journals, but rather that they invent something that actually works. With the honourable exception of bone marrow transplantation, most stem cell treatments are very new and most clinical trials have been small and have not been running long. It is therefore too early to say, in most cases, whether stem cells will prove as effective as is hoped (Trounson et al. 2011). If they are not, it may well be that, in the short-to-medium term at least, real patients will benefit more, and in larger numbers, from the development of 'conventional' physical and chemical based therapies.

#### **3. Application of stem cells**

4 Tissue Regeneration – From Basic Biology to Clinical Application

One of the major problems faced by surgeons and their patients is the replacement of bones that have to be removed because they harbour neoplasia. Amputation of a limb is clearly a major loss for a patient, so surgeons attempt to conserve whenever possible. If a large section of bone has to be removed, there is a problem finding something to replace it. The ideal replacement would be the excised bone itself, freed from all traces of the tumour. Crude attempts to kill tumour cells, by autoclaving or by severe chemical treatment, do result in a sterile bone but destroy so many of the normal guidance cues for cells that the bone is not properly integrated into the body and maintained when it is put back into the patient. In chapter 3, Diehl and colleagues describe an adaptation of a technique for sterilization that uses high hydrostatic pressures. This kills any cells in the bone but seems to leave intact the guidance cues necessary for the bone's efficient recolonization. It may therefore allow tumour-infested bones to be removed, sterilized safely and replaced, with

Biochemical manipulation of cell behaviour is as ancient as medicine itself and the herbal remedies of the first physicians worked - when they worked at all - in this way. An improved understanding of the natural communications during healing and regeneration is allowing researchers to develop biochemical techniques for encouraging regeneration and discouraging damaging scar formation. An approach that is simple in principle even if not straightforward in practice, is to alter wound chemistry to make naturally-produced growth factors more effective. One way of doing this is to alter the extent to which wound matrix sequesters signalling molecules that would, if able to interact with cells, promote healing. In Chapter 4, van Neck and colleagues take this approach and discuss the use of synthetic, glycanase-resistant heparan sulphate mimics in mobilizing endogenous, regenerationpromoting signalling molecules. The chapter presents interesting data that suggest a beneficially-altered balance in favour of repair and against scar formation. Healthy tissue needs a good blood supply and, in chapter 5, de Mendonça describes how angiogenesis is controlled and draws attention to potential targets that would allow the process to be manipulated pharmacologically. Platelets are small cell fragments that travel in blood and that play a critical role in limiting the immediate consequences of wounding by formating a clot when a blood vessel is damaged. As well as performing this mechanical function, platelets are a rich source of growth factors and cytokines. In chapter 6, Ohkohichi and colleagues present data that argues that platelets can significantly promote the regeneration

of damaged liver and possibly promote the survival of small grafts as well.

A well-known, and frustrating, fact about natural regeneration is that so-called 'lower' animals are a lot better at it than are humans. Have humans completely lost an ancient regenerative response still present in fish and amphibians, or is the difference a matter of degree, humans still retaining elements of the repair pathway but not activating it strongly enough? The difference is important, because the second of these hypotheses allows for the possibility of developing a clinical intervention that builds on an existing pathway but pushes it harder to give humans an amphibian-like power of rebuilding. Eleonora Grigoryan, in chapter 7, analyses the early cellular events that follow damage to the retina in amphibians and mammals. Although the final outcome of the type of damage is very different in these different organisms (amphibians making a new retina, mammals failing to do so), the early responses of the cells are the same but the conversion of non-neuronal cells to neurons and glia follows them only in amphibia. It is still not clear why the eventual fates

the aim of leaving the patient with a normal limb.

Stem cells are cells that can maintain their own population and give rise to daughters that are committed to differentiate. Depending on where they come from, they can have an ability to differentiate into any cell type of the body (for example, embryonic stem cells) or only a restricted range of cell types (for example, haematopoietic stem cells). Because of their potential to make new cells, stem cells have for some years been seen as a very promising source of new tissue. Initial experiments were generally designed on the assumption that a useful stem cell therapy would work by the stem cells themselves making new tissue to replace that lost to injury. In recent years, though, the field has been made more complicated by the realization that some stem cell types, in particular mesenchymal stem cells, exert significant beneficial effects by secreting factors that modulate inflammation and healing by the host tissue, and that much new growth is from the cells that were already there rather than being from the stem cells. The dual mode of stem cell action, which places different emphasis on each mode according to the precise situation, makes analysis of stem cellmediated repair complicated. On the positive side, the realization that secreted factors may be of significant, or even primary, importance in the mechanism of this repair is exciting from a clinical point of view because it may be possible to apply the factors directly without any need for patients to undergo potentially dangerous stem cell transplantation. This is not yet possible, though, and at the moment attempts at this type of therapy requires the use of stem cells themselves.

Development of effective stem cell therapies can be divided into two sub-problems; that of finding the most suitable type or source of stem cells to use, and that of applying them to the injured body in the most effective way possible. For convenience, section 2 of this book has been divided along precisely these lines, chapters 8-11 covering sources of stem cells and chapters 12-16 covering their application.

Arguably the most convenient source of stem cells for therapy is the patient himself. Bone marrow is a rich source of stem cells. Many attempts to use them have involved mechanical recovery of marrow followed by purification of stem cells, perhaps with additional steps of

Tissue Regeneration – A Clinical Science Whose Time Has Come 7

One theme that emerges from a large number of these chapters is that studies of the effects of stem cell treatment in humans are few, and tend to be small. Perhaps because of their small size and consequent low statistical power, these studies frequently produce contradictory results. This is not helped by the lack of standardization in how experiments are performed and assessed, which makes meta-analyses problematic. Overall, it is clear that, in most areas of application, it is still far too early to decide whether stem cell treatments really are a means of effective cure and repair, or whether other approaches, such

Where there are large-scale defects in tissues, caused either by injury or by congenital abnormality, simple stem cell treatments – however well they can be made to work – are unlikely to be able to make a proper repair. In terms of directly producing new tissues, stem cells are expected to work by recapitulating the processes of natural development or tissue maintenance. Embryonic development tends to take place at small scale and tissues then grow; when an embryo first makes a trachea, for example, it is less than a millimetre long, not the many centimetres it is in adult life. Also, many embryonic events depend on signals from other embryonic tissues that move or disappear by birth. There are therefore good reasons that a stem cell, even in a state that corresponds perfectly to the cells that would make a tissue in an embryo, would not be able to make it in an adult. Similarly, the stem cells concerned with tissue maintenance are regulated by the environment of the tissue in which they are situated and there is no reason to assume that they can rebuilt tissues across a large gap or scar, in which this environment is missing. In the case of genetic abnormality, the cells may be incapable of making the body part normally anyway. For all of these reasons, there is a strong argument that large scale regeneration requires the construction of

Bone tissue, which in its mature form is mostly inorganic matrix, lends itself to a scaffoldbased approach. In chapter 17, Magdalena Cieślik and colleagues compare the ability of different matrices, based on the natural structure of bone with additional components such as bioglasses, to promote effective bone repair. On a related topic, Peter Emans and colleagues propose, in chapter 18, the use of scaffolds designed with the normal developmental process of endochondral ossification in mind. The chapter includes a critical review of clinical trials (which, as with much regenerative medicine, are less effective than

Tissue engineering of soft tissues involves different considerations, such tissues generally being much more flexible and much more cellular in terms of the ratio of cells to surrounding matrix. Chao Feng and Yue-min Xu illustrate this in chapter 19, where they explore techniques for reconstructing the lower urinary tract. They compare different scaffold materials, such as fleeces, sponges and advanced materials that include signalling molecules, and consider techniques for populating them with cells before their use. The chapter includes a review of clinical data on reconstruction of human bladder and urethra,

In the last chapter in this section, chapter 20, Abir El-Sadik connects the rapidly developing field of tissue engineering with another 'hot topic': nanotechnology. Nanotechnology is

as those described in section 3 below, will actually prove more useful.

scaffolds to bridge gaps and to control cell behaviour.

original experiments gave hope to believe).

with an encouraging rate of success.

**4. Construction of scaffolds**

proliferation and reprogramming, followed by injection into systemic blood or directly into a site of damage. In chapter 8, Christian Drapeau and colleagues discuss an alternative approach that involves much less invasive manipulation Their strategy is to use the fact that endogenous bone marrow stem cells can be mobilized by cytokines, and they describe experiments that involve injecting pure cytokines into animals that have suffered cardiac infarction, in an effort to encourage mobilization of the animals' own stem cells to effect a repair. The authors also describe preliminary studies of this approach in humans, to treat stroke and kidney failure.

A novel source of pluripotent stem cells, capable of making any body cell, is the testis. Spermatogonial cells are the stem cells that naturally maintain production of sperm. Within the testis, they are constrained by their environment to have only the simple choice between self-renewal and spermatogenesis. Taken outside that environment, though, the cells can differentiate into a large range of cell types, making them effectively pluripotent. Liz Simon and colleagues analyze the abilities of these stem cells in chapter 9, and evaluate their potential for therapeutic use compared to the potential of other stem cell types. While spermatogonial cells can be obtained only from men, pregnant women can be a source of fetal membrane-derived mesenchymal stem cells. Shin Ishikane and colleagues describe the isolation and properties of these cells in chapter 10, and summarize their ability to modulate immune activity and to become a useful tool in regenerative medicine. The theme of mesenchymal stem cells is explored further by Arshak Alexanian in chapter 11, with a special emphasis on their ability to improve central nervous system repair.

Wherever and however they are obtained, stem cells have to be applied in a way that optimizes their ability to effect repair. Five chapters in this book focus on the application of stem cells to different clinical problems in the circulatory and the musculoskeletal systems. José Lamas and colleagues (chapter 12) address the potential of mesenchymal stem cells to treat osteoarthritis, a common and debilitating disease of joints, and outline current knowledge and future prospects for this important field. Namath Hussain and colleagues address another common and important chronic problem in chapter 13; that of debilitating lower back pain caused by damage to intervertebral discs. They explain the basic pathology of the disease and then review the results from the (very small) studies that have so far been conducted into the efficacy of stem cell treatment for disc damage in humans. Marianna Karagianni and colleagues consider a different problem in orthopaedics; the healing of defective bone. In chapter 14, they also examine the regulatory frameworks that govern the use of 'advanced therapy medicinal products' and consider how these frameworks shape research and development. Regeneration of bone is also addressed by Dilawhare Khan, Arnaldo Santos and their colleagues (chapters 15 and 16) . Khan *et al.* use an unusual source of stem cells – teeth – and having demonstrated that these show promise, they make the suggestion that milk tooth stem cells could perhaps be banked for a patient's later use. Santos *et al.* provide a wide-ranging review of different approaches to the regeneration of bone including, but not restricted to, the use of stem cells. Their chapter could have appeared in almost any section of this book: it was included as a final chapter of the stem cells section to highlight the need to compare stem cell approaches with the best of other techniques, because there is arguably a tendency, at the moment, to place undue emphasis on some ways of effecting repair perhaps to the detriment of developing others that may even show more promise in existing clinical trials.

One theme that emerges from a large number of these chapters is that studies of the effects of stem cell treatment in humans are few, and tend to be small. Perhaps because of their small size and consequent low statistical power, these studies frequently produce contradictory results. This is not helped by the lack of standardization in how experiments are performed and assessed, which makes meta-analyses problematic. Overall, it is clear that, in most areas of application, it is still far too early to decide whether stem cell treatments really are a means of effective cure and repair, or whether other approaches, such as those described in section 3 below, will actually prove more useful.

#### **4. Construction of scaffolds**

6 Tissue Regeneration – From Basic Biology to Clinical Application

proliferation and reprogramming, followed by injection into systemic blood or directly into a site of damage. In chapter 8, Christian Drapeau and colleagues discuss an alternative approach that involves much less invasive manipulation Their strategy is to use the fact that endogenous bone marrow stem cells can be mobilized by cytokines, and they describe experiments that involve injecting pure cytokines into animals that have suffered cardiac infarction, in an effort to encourage mobilization of the animals' own stem cells to effect a repair. The authors also describe preliminary studies of this approach in humans, to treat

A novel source of pluripotent stem cells, capable of making any body cell, is the testis. Spermatogonial cells are the stem cells that naturally maintain production of sperm. Within the testis, they are constrained by their environment to have only the simple choice between self-renewal and spermatogenesis. Taken outside that environment, though, the cells can differentiate into a large range of cell types, making them effectively pluripotent. Liz Simon and colleagues analyze the abilities of these stem cells in chapter 9, and evaluate their potential for therapeutic use compared to the potential of other stem cell types. While spermatogonial cells can be obtained only from men, pregnant women can be a source of fetal membrane-derived mesenchymal stem cells. Shin Ishikane and colleagues describe the isolation and properties of these cells in chapter 10, and summarize their ability to modulate immune activity and to become a useful tool in regenerative medicine. The theme of mesenchymal stem cells is explored further by Arshak Alexanian in chapter 11, with a

Wherever and however they are obtained, stem cells have to be applied in a way that optimizes their ability to effect repair. Five chapters in this book focus on the application of stem cells to different clinical problems in the circulatory and the musculoskeletal systems. José Lamas and colleagues (chapter 12) address the potential of mesenchymal stem cells to treat osteoarthritis, a common and debilitating disease of joints, and outline current knowledge and future prospects for this important field. Namath Hussain and colleagues address another common and important chronic problem in chapter 13; that of debilitating lower back pain caused by damage to intervertebral discs. They explain the basic pathology of the disease and then review the results from the (very small) studies that have so far been conducted into the efficacy of stem cell treatment for disc damage in humans. Marianna Karagianni and colleagues consider a different problem in orthopaedics; the healing of defective bone. In chapter 14, they also examine the regulatory frameworks that govern the use of 'advanced therapy medicinal products' and consider how these frameworks shape research and development. Regeneration of bone is also addressed by Dilawhare Khan, Arnaldo Santos and their colleagues (chapters 15 and 16) . Khan *et al.* use an unusual source of stem cells – teeth – and having demonstrated that these show promise, they make the suggestion that milk tooth stem cells could perhaps be banked for a patient's later use. Santos *et al.* provide a wide-ranging review of different approaches to the regeneration of bone including, but not restricted to, the use of stem cells. Their chapter could have appeared in almost any section of this book: it was included as a final chapter of the stem cells section to highlight the need to compare stem cell approaches with the best of other techniques, because there is arguably a tendency, at the moment, to place undue emphasis on some ways of effecting repair perhaps to the detriment of developing others that may

special emphasis on their ability to improve central nervous system repair.

even show more promise in existing clinical trials.

stroke and kidney failure.

Where there are large-scale defects in tissues, caused either by injury or by congenital abnormality, simple stem cell treatments – however well they can be made to work – are unlikely to be able to make a proper repair. In terms of directly producing new tissues, stem cells are expected to work by recapitulating the processes of natural development or tissue maintenance. Embryonic development tends to take place at small scale and tissues then grow; when an embryo first makes a trachea, for example, it is less than a millimetre long, not the many centimetres it is in adult life. Also, many embryonic events depend on signals from other embryonic tissues that move or disappear by birth. There are therefore good reasons that a stem cell, even in a state that corresponds perfectly to the cells that would make a tissue in an embryo, would not be able to make it in an adult. Similarly, the stem cells concerned with tissue maintenance are regulated by the environment of the tissue in which they are situated and there is no reason to assume that they can rebuilt tissues across a large gap or scar, in which this environment is missing. In the case of genetic abnormality, the cells may be incapable of making the body part normally anyway. For all of these reasons, there is a strong argument that large scale regeneration requires the construction of scaffolds to bridge gaps and to control cell behaviour.

Bone tissue, which in its mature form is mostly inorganic matrix, lends itself to a scaffoldbased approach. In chapter 17, Magdalena Cieślik and colleagues compare the ability of different matrices, based on the natural structure of bone with additional components such as bioglasses, to promote effective bone repair. On a related topic, Peter Emans and colleagues propose, in chapter 18, the use of scaffolds designed with the normal developmental process of endochondral ossification in mind. The chapter includes a critical review of clinical trials (which, as with much regenerative medicine, are less effective than original experiments gave hope to believe).

Tissue engineering of soft tissues involves different considerations, such tissues generally being much more flexible and much more cellular in terms of the ratio of cells to surrounding matrix. Chao Feng and Yue-min Xu illustrate this in chapter 19, where they explore techniques for reconstructing the lower urinary tract. They compare different scaffold materials, such as fleeces, sponges and advanced materials that include signalling molecules, and consider techniques for populating them with cells before their use. The chapter includes a review of clinical data on reconstruction of human bladder and urethra, with an encouraging rate of success.

In the last chapter in this section, chapter 20, Abir El-Sadik connects the rapidly developing field of tissue engineering with another 'hot topic': nanotechnology. Nanotechnology is

Tissue Regeneration – A Clinical Science Whose Time Has Come 9

authors of other chapters. I hope that a sensible compromise has been struck, but am aware that there has had to be some repetition of some introductory material from chapter to chapter. In a book that could only be obtained as a whole, this repetition would have been removed. The editor and authors trust that readers will understand why some has had to

The chapters in this book were written only a short time before publication and represent a very up-to-date overview of the field. Regenerative medicine is moving so quickly, however, that the authors expect some details of their material – especially reviews of human trials – to become out of date in only a few years. Depressing as a work's rapid obsolescence might seem to be for an author, really we rejoice in the fact, for it is the mark of a vibrant and fast-moving field that promises to have a significant impact on twenty-first century medicine. It is our hope that this book may inspire some of the new work that will

Goldberg, R.P. & Sand, P.K., 2000. Electromagnetic pelvic floor stimulation: applications for the gynecologist. *Obstetrical & Gynecological Survey*, 55(11), pp.715-720. Gravas, S., Laguna, M.P. & De La Rosette, J.J.M.C.H., 2003. Application of external

Hamid, S. & Hayek, R., 2008. Role of electrical stimulation for rehabilitation and

*European Section of the Cervical Spine Research Society*, 17(9), pp.1256-1269. Jacot, J.G. et al., 2010. Cardiac myocyte force development during differentiation and maturation. *Annals of the New York Academy of Sciences*, 1188, pp.121-127. Katoh, K., Kano, Y. & Ookawara, S., 2008. Role of stress fibers and focal adhesions as a

Kenkre, J.E. et al., 1996. A randomized controlled trial of electromagnetic therapy in the

McCulley, J.P. & Petroll, W.M., 2008. Quantitative assessment of corneal wound healing

Owens, B.D., Stickles, B.J. & Busconi, B.D., 2003. Radiofrequency energy: applications and

Poelmann, R.E., Gittenberger-de Groot, A.C. & Hierck, B.P., 2008. The development of the

Simon, C.J., Dupuy, D.E. & Mayo-Smith, W.W., 2005. Microwave ablation: principles and

Trounson, A. et al., 2011. Clinical trials for stem cell therapies. *BMC Medicine*, 9, p.52.

*American Ophthalmological Society*, 106, pp.84-90; discussion 90-92.

*Endourology / Endourological Society*, 17(8), pp.659-666.

*Health and Risk Management*, 4(6), pp.1273-1282.

microwave thermotherapy in urology: past, present, and future. *Journal of*

regeneration after spinal cord injury: an overview. *European Spine Journal: Official Publication of the European Spine Society, the European Spinal Deformity Society, and the*

mediator for mechano-signal transduction in endothelial cells in situ. *Vascular*

primary care management of venous leg ulceration. *Family Practice*, 13(3), pp.236-

following IntraLASIK using in vivo confocal microscopy. *Transactions of the*

basic science. *American Journal of Orthopedics (Belle Mead, N.J.)*, 32(3), pp.117-120;

heart and microcirculation: role of shear stress. *Medical & Biological Engineering &*

applications. *Radiographics: A Review Publication of the Radiological Society of North*

remain, given the necessity for each chapter to stand alone.

one day make it obsolete.

**7. References**

241.

discussion 120-121.

*Computing*, 46(5), pp.479-484.

*America, Inc*, 25 Suppl 1, pp.S69-83.

young and still raises significant safety concerns so there is little clinical data, but the chapter explains the ways in which, in principle, nano-materials can modulate the function of stem cells and other tissue cells. It also illustrates, using nerve regeneration as an example, how scaffolds that incorporate nano-materials can promote useful neuronal growth and decrease glial scarring in experimental systems.

#### **5. Assessing tissue regeneration**

Having ideas about how to improve tissue regeneration is all very well, but it is essential both to the process of research that evaluates these ideas, and also to the proper care of a patient undergoing regeneration, that progress can be monitored, evaluated and perhaps even predicted. The two chapters in the last section of this book address this specific issue.

Magnetic resonance imaging is an excellent non-invasive technique that can provide highresolution images of any part of the body. In chapter 21, Miyata illustrates how the ability of advanced MRI systems to provide quantitative information, particularly on the state of water and whether it is a free liquid or mainly bound to glycosaminoglycans, can be used to monitor the state of cartilage. This offers both researchers and clinicians an opportunity to monitor the progress of regenerative treatments over a long time-course, optimizing care and leading the way to patient-specific treatment regimes. It is possible that this approach will be extended in future to monitoring events in soft-tissue repair.

In the final chapter, Vermolen and van Rijn take an approach very different to most other authors in this book: they describe mathematical models of the processes involved in the 'healing' (by scar formation) of accidental wounds, particularly burns. The interest in the biophysics of scar formation is not simply academic and 'basic science' because, as the authors point out, creating tools that can *predict* the natural outcome of a patient's specific wound could be very useful to the design of a patient-specific programme of treatment designed to resolve that wound with as little aesthetic impact as possible. In particular, it can help physicians apply the right boost to regeneration (by whatever method) for each part of the wound, navigating successfully between the Scylla of overgrowth and the Charybdis of under-regeneration and consequent scarring, neither of which would be aesthetically satisfactory.

### **6. Using the chapters of this book**

The whole philosophy of this multi-author book, and of its publisher, has placed unusual demands on both the authors and the editor. Unlike a conventional volume, which can only be bought or borrowed in its entirety, this book can be viewed two ways. The first is the traditional one – purchase of a single bound volume containing everything in order. The second is downloading of individual chapters from the Internet, using the Open Access model. There is much to be said for this, not least by clinicians and researchers who do not work in rich institutions that are blessed with a large library.

The knowledge that the authors are writing both for a traditional, whole-book-owning readership and for readers who may view just one chapter has, however, presented the Editor with an unusual problem: that of judging how much introductory material is necessary for a chapter to be readable in its own right, and how much can be left to the authors of other chapters. I hope that a sensible compromise has been struck, but am aware that there has had to be some repetition of some introductory material from chapter to chapter. In a book that could only be obtained as a whole, this repetition would have been removed. The editor and authors trust that readers will understand why some has had to remain, given the necessity for each chapter to stand alone.

The chapters in this book were written only a short time before publication and represent a very up-to-date overview of the field. Regenerative medicine is moving so quickly, however, that the authors expect some details of their material – especially reviews of human trials – to become out of date in only a few years. Depressing as a work's rapid obsolescence might seem to be for an author, really we rejoice in the fact, for it is the mark of a vibrant and fast-moving field that promises to have a significant impact on twenty-first century medicine. It is our hope that this book may inspire some of the new work that will one day make it obsolete.

#### **7. References**

8 Tissue Regeneration – From Basic Biology to Clinical Application

young and still raises significant safety concerns so there is little clinical data, but the chapter explains the ways in which, in principle, nano-materials can modulate the function of stem cells and other tissue cells. It also illustrates, using nerve regeneration as an example, how scaffolds that incorporate nano-materials can promote useful neuronal

Having ideas about how to improve tissue regeneration is all very well, but it is essential both to the process of research that evaluates these ideas, and also to the proper care of a patient undergoing regeneration, that progress can be monitored, evaluated and perhaps even predicted. The two chapters in the last section of this book address this specific issue. Magnetic resonance imaging is an excellent non-invasive technique that can provide highresolution images of any part of the body. In chapter 21, Miyata illustrates how the ability of advanced MRI systems to provide quantitative information, particularly on the state of water and whether it is a free liquid or mainly bound to glycosaminoglycans, can be used to monitor the state of cartilage. This offers both researchers and clinicians an opportunity to monitor the progress of regenerative treatments over a long time-course, optimizing care and leading the way to patient-specific treatment regimes. It is possible that this approach

In the final chapter, Vermolen and van Rijn take an approach very different to most other authors in this book: they describe mathematical models of the processes involved in the 'healing' (by scar formation) of accidental wounds, particularly burns. The interest in the biophysics of scar formation is not simply academic and 'basic science' because, as the authors point out, creating tools that can *predict* the natural outcome of a patient's specific wound could be very useful to the design of a patient-specific programme of treatment designed to resolve that wound with as little aesthetic impact as possible. In particular, it can help physicians apply the right boost to regeneration (by whatever method) for each part of the wound, navigating successfully between the Scylla of overgrowth and the Charybdis of under-regeneration and consequent scarring, neither of which would be

The whole philosophy of this multi-author book, and of its publisher, has placed unusual demands on both the authors and the editor. Unlike a conventional volume, which can only be bought or borrowed in its entirety, this book can be viewed two ways. The first is the traditional one – purchase of a single bound volume containing everything in order. The second is downloading of individual chapters from the Internet, using the Open Access model. There is much to be said for this, not least by clinicians and researchers who do not

The knowledge that the authors are writing both for a traditional, whole-book-owning readership and for readers who may view just one chapter has, however, presented the Editor with an unusual problem: that of judging how much introductory material is necessary for a chapter to be readable in its own right, and how much can be left to the

growth and decrease glial scarring in experimental systems.

will be extended in future to monitoring events in soft-tissue repair.

**5. Assessing tissue regeneration**

aesthetically satisfactory.

**6. Using the chapters of this book**

work in rich institutions that are blessed with a large library.

**Understanding and Manipulating**

**Endogeneous Healing of Tissues**

Vandenburgh, H.H. et al., 1996. Mechanical stimulation of organogenic cardiomyocyte growth in vitro. *The American Journal of Physiology*, 270(5 Pt 1), pp.C1284-1292 **Part 1**


**4. Aromatase inhibitors (AIs)-induced bone loss**

The majority of adverse reactions to aromatase inhibitors (AIs) affect the musculoskeletal system and can be divided into three groups [21]:

1.Metabolic bone disease, which increases the risk of fractures;

All of these adverse outcomes begin to manifest after varying amounts of time have passed since the start of treatment with AIs. Although the precise pathophysiology is not fully understood, the pathogenetic pathways endorsed to explain these

disorders are primarily related to the estrogen deficiency caused by a prolonged AIs treatment [21].


**5. Concluding remarks and future trends**

In this chapter, the main topics concerning the IJP manufacturing of printed electronics have been discussed. Being an additive manufacturing technology, some of its advantages concern lower associated costs, material economization, and design freedom. Since its establishment as an alternative for electronics manufacturing, different variations of this technology have emerged, nonetheless, piezoelectric IJP is the most widespread method. Many functional inks have already been optimized for IJP and are commercially available. Despite this, several factors linked to the printing process can still negatively influence the printing output in terms of both printing quality and electrical conductivity and demand optimization.

As for IJP application in the current PE market, there is an undeniable growing tendency. However, it is still early to anticipate the potential of IJP technology in the long run because of all the other options currently being developed. Because of the drawbacks associated with piezoelectric IJP, namely the tendency for nozzles to clog, the limited ink viscosity range (and consequently limited electrical conductivity), and the elevated number of factors that influence the printing process, it is possible that IJP gets to be used in specific niche applications, whereas needle-based and EHD-IJP might ultimately transcend piezoelectric IJP in terms of applicability.


**Author details**

Eleni Mousena1 \* and Nikolaos Raptis2

1 University of West Attica, Athens, Greece

2 University of the Aegean, Rhodes, Greece

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Beyond Teaching: School Climate and Communication in the Educational Context DOI: http://dx.doi.org/10.5772/intechopen.93575*


*4.1.2 Cultivation temperature*

Cultivation temperature is another factor that may influence the somatic embryogenesis response. Leaf explants of cultivar Catuaí and two hybrids showed higher formation of somatic embryos at 30°C compared to 25°C [90].


**2.2 Specificity of anti-8-nitroguanine antibody**

Specificity of the purified antibody was examined by a dot immunobinding assay and absorption test (Pinlaor et al., 2004a). Purified antibody gave a strong immunostaining only on the spot of 8-nitroguanine conjugate (Fig. 2A). The immunoreactivity disappeared only when the antibody was pre-incubated with 8-nitroguanine. In contrast, immunoreactivity with 8-nitroguanine conjugate did not disappear when the antibody was preincubated with 3-nitrotyrosine, guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), deoxyguanosine, 8-bromoguanosine, and xanthosine (Fig. 2B).

Fig. 2. Dot immunobinding assay and absorption test of anti-8-nitroguanine antibody.

8-Nitroguanine, a Potential Biomarker to

Paraformaldehyde (Nacalai, Kyoto, Japan)

Sliding microtome (RM2265, Leica, Germany)

**2.4.2 Preparation of animal tissues for IHC**

6. Make a small incision in the right atrium.

is no response, then proceed with the surgery.

side of the animal to allow proper drainage of perfusate.

Ethanol (Nacalai, Kyoto, Japan) Xylene (Nacalai, Kyoto, Japan)

**2.4 Detailed procedure for IHC**

pentobarbital.

forceps.

respectively.

end of this step.

give access to the heart.

Evaluate the Risk of Inflammation-Related Carcinogenesis 205

Monobasic and dibasic salts of sodium and potassium phosphate (Nacalai, Kyoto, Japan)

Average time for immunohistochemical (IHC) localization of 8-nitroguanine is 2-3 days, not including preparation and mounting of tissues. Albeit, time varies with duration of antibody incubation of second antibody in double fluorescent immunohistochemistry.

1. Animal is weighed and anesthetized by intraperitoneal injection of 50 mg/kg sodium

2. After the animal has fallen asleep, firmly pinch the foot with a pair of tweezers. If there

3. Position the animal on its back. Open the abdominal cavity with a midline incision to the sternum. Make a diagonal cut to each side of the sternum through the rib cage extending to either side of the neck. Care should be taken not to sever any vessels or puncture the heart. The sternum may now be clamped back with hemostatic forceps to

4. Cut the connective tissue surrounding the diaphragm and make a lateral cut on each

5. Lift the lungs to expose the descending aorta and occlude the vessel using hemostatic

7. Insert an i.v. catheter into the left ventricle. Remove the needle from the catheter. Position so that the tip of the catheter resides within the aortic arch. The optimal size of the catheter for transcardial perfusion is 18 and 22 gauge for rat and mouse,

8. Connect the catheter to the perfusion apparatus and begin the flow of 0.9% NaCl into the animal. For an adult rat 80 ml 0.9% saline should be infused over 3 min. For a mouse 20 ml saline should be infused over 2 min. Progress of the perfusion may be monitored by assessment of the eyes and gums which should be blanched toward the

9. Switch perfusion to 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde. It is common to see turgor and twitching of the upper extremities at the initial flow of fixative into the animal. Perfuse an adult rat, 300 ml of fixative over 10 min should be

Confocal laser scanning microscopy (FV-1000D, Olympus, Tokyo, Japan)

**2.4.1 Time required for immunohistochemistry of 8-nitroguanine**

Fluorescent light microscope (BX53, Olympus, Tokyo, Japan)

Antigen–antibody reactions were visualized by the peroxidase-anti-peroxidase method. Purified antibody gave a strong immunostaining only on the spot of 8-nitroguanine conjugate (A). 8-Nitroguanine, 3-nitrotyrosine, guanosine, 8-oxodG and deoxyguanosine were incubated with the antibody at the concentration of 0.7 g/ml, and were applied to 8 nitroguanine conjugate. Immunoreactivity disappeared only when the antibody was pretreated with 8-nitroguanine (B). 8-NG, 8-nitroguanine; 3-NT, 3-nitrotyrosine; G, guanosine; dG, deoxyguanosine and 8-BromoG, 8-bromoguanosine.


**2. Results**

#### *2.1. Yields and Chemical Composition of the Polysaccharide Extracts and Fractions*

Based on the compositional analysis (AOAC methods) *S. polycystum* indicated 2.75 ± 0.08% moisture, 17.62 ± 0.16% ash, 52.46 ± 0.52% carbohydrate, 17.36 ± 0.04% protein, and 1.85 ± 0.06% lipids on a dry basis. The yield of ethanol precipitate was 6.88 ± 0.52%. The ash content of the ethanol precipitate was 0.08 ± 0.01% (relative to the dry weight of raw material).

#### *2.2. Purification of Polysaccharides Molecular Weight Distribution and Monosaccharide Composition*

As indicated in Figure 1a, anion exchange purification resolved the polysaccharide precipitate into five fractions (F1–F5). The recovery yield of F1 and F5 was higher than other fractions. The molecular weight distribution of fractions (F1–F5) indicated a decreasing trend and was estimated to be centered on 77.0, 65.5, 59.5, 60.0, and 39.5 kDa, respectively (Figure 1b). The monosaccharide composition of each fraction is given in Table 1. F1 had a higher galactose and mannose content, whereas the other successive fractions indicated increasing amounts of fucose. Fucose content was highest in F5 (Figure 1c) with galactose and a minor amount of glucose.

**Figure 1.** Purification of precipitated polysaccharides by DEAE-cellulose anion exchange chromatography. (**a**) Separating the collected column eluates into five fractions based on their polysaccharide content, (**b**) molecular weight distribution of the eluted column fractions (F1–F5), and (**c**) the monosaccharide composition analysis of the fraction F5.

**Table 1.** The composition of column fractions.

Chemical composition was calculated based on triplicate determinations. Results are given as the means ± SD.

#### *2.3. Characterization of Polysaccharide Structure (FTIR and NMR Analysis)*

Wavenumber range 400–2000 cm−1 of FTIR spectra covering the fingerprint region of polysaccharides were used to characterize the structural properties of polysaccharides. All FTIR spectra, including column fractions and the commercial fucoidan standard, indicated prominent peaks at 845, 1035, 1616 cm−1, and a broad peak between 1220–1270 cm−1. A clear di fference was seen for the intensity of 1220–1270 cm−1 peak between each spectrum, although the intensity of the peak at 1035 cm−1 was similar between all. The peak at 1733 cm−1 was absent in the commercial fucoidan, whereas a minor peak was recorded for F5. However, 1733 cm−1 peak was prominent in all other fractions (F1–F4). 1H and 13C NMR spectra of deuterium exchanged F5 fraction are presented in Figure 2b,C. In the 1H spectrum, solvent peaks were observed at 2.50 ppm for dimethyl sulfoxide and 4.80 ppm for deuterium oxide. Unresolved peaks between 5.1–5.4 ppm could be assigned to protons of α-l-fucopyranosyl units. Prominent peaks observed at 4.46, 3.90, and 3.71 ppm were respectively assigned to H-1, H-5, and H-3 of D-galactons. The two overlapping peaks at 1.15, and 1.4 ppm were assigned to methyl groups in fucose [1,2]. In the 13C spectrum, prominent peak 101.6 and peaks between 65–80 are arising from the (1–6)-β-d-linked galactons [2].

**Figure 2.** Structural characterization of polysaccharide fractions. (**a**) FTIR spectra of the fractions (F1–F5) provided in comparison to the commercial fucoidan, (**b**) 1H NMR spectrum of F5, and (**c**) 13 C NMR spectrum of F5. The NMR spectra were obtained for the deuterium exchanged polysaccharides.

#### *2.4. Variation of Antiproliferative Activity of the Column Fractions*

As shown in Figure 3, F4 and F5 promptly reduced the viability of both HL-60 and MCF-7 cells compared to other fractions. The antiproliferative e ffects of F5 were stronger than F4. The IC50 values related to F5 treatment was 84.63 ± 0.08 μg mL−1 and 93.62 ± 3.53 μg mL−1 respectively on HL-60 and MCF-7 cells. The viability of Vero cells (Figure 3c) was simultaneously evaluated to compare the cytotoxic e ffects of these polysaccharides on non-cancerous cells. The viability of Vero cells was 89.83 ± 1.76% at 100 μg mL−1 concentration.

**Figure 3.** Antiproliferative activity of polysaccharide fractions as a measure of cell viability. (**a**) HL-60, (**b**) MCF-7, and (**c**) Vero cells. Cells were pre-seeded in 96 well plates for 24 h and incubated with samples for another 24 h. Cell viability was measured by MTT assay. Results are given as the means ± SD (n = 3). Significant differences from the control were identified at \* *p* < 0.05 and \*\* *p* < 0.001.

#### *2.5. E*ff*ect of F5 upon Apoptotic Body Formation in HL-60 and MCF-7 Cells and Pathway Studies by Western Blot Analysis*

As shown in Figure 4a, under the Hoechst 33342 staining, HL-60 and MCF-7 cells indicated increased nuclear fragmentation and condensation (intensified spots) for increasing F5 concentrations. Nuclear double staining method (Figure 4c) assists in distinguishing early and late apoptosis or necrosis. Green fragmented nuclei (early apoptosis) were seen under 25 μg mL−1 of F5 treatment after a 24 h incubation period, which increased with increasing F5 concentrations. The presence of orange color spots together with green color fragmented nuclei (late apoptosis) were detected under 50 μg mL−1 concentration. Increased late apoptosis events were observed with 100 μg mL−1 of F5 concentration, whereas a few necrotic HL-60 cells were also detected. Western blot results indicated increased production of Bax, caspases, and p53 (only MCF-7) levels with increasing F5 concentrations together with increased PARP cleavage. Alternatively, reducing levels of Bcl-xL were also detected with increasing F5 concentrations.

#### *2.6. F5 Increased the DNA Damage in HL-60 and MCF-7 Cells and the Population of Sub-G1 Hyperploid Cells*

Comet assay serves as a tool in identifying cellular DNA damage. As depicted in Figure 5a,b, the length of the comet tail increased in both HL-60 and MCF-7 cells with increasing F5 concentrations. Flow cytometric analysis with propidium iodide (PI) is widely employed in identifying the accumulation of cells in different phases of cell cycle based on their DNA content [14]. According to Figure 5c,d, the proportion of Sub-G1 apoptotic cells increased with increasing F5 concentrations.

**Figure 4.** Effects of F5 in inducing apoptotic body formation in HL-60 and MCF-7 cells and analysis of the levels of molecular mediators. (**a**) Cells under Hoechst 33342 staining, (**b**) Western blot analysis of the levels of apoptosis-related molecular mediators in HL-60 cells, (**c**) cells under nuclear double staining, and (**d**) Western blot analysis of the levels of apoptosis-related molecular mediators in MCF-7 cells. Experiments were repeated three times to confirm the reproducibility.

**Figure 5.** Effects of F5 in inducing single-cell DNA damage in HL-60 and MCF-7 cells and cell cycle analysis. Comet assay (**a**) HL-60 cells, (**b**) MCF-7 cells. Cell cycle analysis of (**c**) HL-60 cells, and (**d**) MCF-7 cells. Pre-seeded cells were exposed to different concentrations of F5 for 24 h. Experiments were repeated three times to confirm the reproducibility.


**3. Fuel cell thermodynamic efficiency**

In the process of energy conversion in a fuel cell, the initial chemical energy between the enthalpy of the products and reactants is converted into electrical energy and thermal energy, as stated in the first law of thermodynamics. The efficiency of any energy conversion device is defined as the ratio between useful energy output and energy input [19–22]:

$$
\eta = \frac{\text{actual electrical work}}{\text{maximum available work}}
$$

$$
\eta = \frac{\Delta \mathbf{G}}{\Delta \mathbf{H}} = \frac{\Delta \mathbf{H} - \mathbf{T} \Delta \mathbf{S}}{\Delta \mathbf{H}} \tag{43}
$$

Themaximum possible thermodynamic efficiency of a fuel cell can be written as [9]:

$$\eta == 1 - \frac{\text{TAS}}{\Delta \text{H}} \tag{44}$$

In the case of a fuel cell, the useful energy output is the electrical energy produced, and the energy input is the enthalpy of hydrogen, that is, hydrogen's HHV. Assuming that all of the Gibbs free energy can be converted into electrical energy (the reaction is reversible), the maximum theoretical efficiency of a fuel cell is [23, 24] (**Figure 3**):

$$\eta = \frac{\Delta \mathbf{G}}{\Delta \mathbf{H}} = \frac{237.34}{286.02} \ge 100\% = \\$3\%$$

For hydrogen's LHV, the fuel cell efficiency would be [26]:

$$\eta = \frac{\Delta \mathcal{G}}{\Delta \mathcal{H}} = \frac{228.74}{241.98} \times 100\% = 94.5\%$$

The LHV has higher efficiency compared to HHV, because the reversible efficiency of the fuel cell decreases as the operating temperature increases [27].

The expected fuel cell efficiency is not always achieved due to thermodynamic and electrochemical irreversible losses [28].

### **4. Irreversible losses**

Other than calculating energy quantities during the conversion of chemical energy to electrical energy, there is also the matter of electron flow through

materials in the fuel cell process. The single fuel cell provides a voltage dependent on operating conditions such as temperature, applied load and fuel/oxidant flow rates [29, 30]. If a fuel cell is supplied with reactant gases, but the electric current is not closed, it will not generate any current, and one would expect the cell potential to be at the theoretical cell potential for the given conditions (temperature, pressure and concentration of reactants). In reversible conditions, the energy loss is the heat lost towards the environment, TΔs, due to negative entropy [12].

E ¼ Etherm � ηact � ηohm � ηconc (45)

q ¼ TΔs � nFE ¼ ΔH � ΔG � nFE (47)

nFT (46)

where E is the cell potential, Etherm is the thermodynamic potential, ηact is the voltage loss due to activation polarisation, ηohm is the voltage loss due to ohmic polarisation and ηconc is the voltage loss due to mass transport polarisation and the

S ¼ E

In addition, the related heat lost for irreversibility can be calculated as (**Figure 4**):

Thermodynamics is used to understand the process of energy conversion in fuel cells. The determination of a fuel cell's performance depends on thermodynamic evaluation. The heat potential of a fuel is given by the enthalpy of the reaction. Not all heat potential of a fuel can be used to perform useful work; the reversible work of a fuel is defined by Gibbs free energy, which is the electrical work. The study of the electrical effects shows that the molar flow of the fuel used is proportional to the electric current and the reversible work is proportional to the reversible voltage. The cell voltage varies with temperature, pressure and reactant/product activities. Irreversible losses cause a difference in the efficiency of reversible and real processes, with efficiency of real processes always less than reversible processes. The losses are due to two major reasons, namely, irreversible kinetic losses and fuel utilisation losses.

We greatly appreciate the National Research Foundation and the Tertiary Education Support Programme for financial support as well as the Chemistry

Department at the University of the Western Cape.

*H2/O2 polarisation curve at equilibrium and voltage losses in fuel cell [30].*

entropy generation results [38–41]:

*DOI: http://dx.doi.org/10.5772/intechopen.90141*

*Fuel Cell Thermodynamics*

**5. Conclusion**

**Figure 4.**

**Acknowledgements**

**15**

However, in practice, the thermodynamic cell potential is decreased from its ideal potential, usually less than 1 V, due to irreversible losses known as overpotential or polarisation [17]. The fuel cell performance overpotential is due to [14, 31]:

The standard measure of performance is the polarisation curve, which represents the cell voltage behaviour against operating current density (**Figure 4**). From the figure, the voltage loss caused by mixed potential and crossover, activation polarisation, ohmic polarisation and mass transport losses is the most significant in the tail of the I-V curve. The maximum fuel cell is then examined through the reversible voltage of the system, which is calculated using thermodynamics and the actual voltage of the system [36]. The final voltage is lower than the thermodynamic voltage and is usually between 0.5 and 1.0 V. Although polarisations cannot be eliminated, material choice and electrode designs can contribute to their minimisation [37]:

*Fuel Cell Thermodynamics DOI: http://dx.doi.org/10.5772/intechopen.90141*

materials in the fuel cell process. The single fuel cell provides a voltage dependent on operating conditions such as temperature, applied load and fuel/oxidant flow rates [29, 30]. If a fuel cell is supplied with reactant gases, but the electric current is not closed, it will not generate any current, and one would expect the cell potential to be at the theoretical cell potential for the given conditions (temperature, pressure and concentration of reactants). In reversible conditions, the energy loss is the heat

However, in practice, the thermodynamic cell potential is decreased from its

overpotential or polarisation [17]. The fuel cell performance overpotential is due to

i. Activation overpotential: The activation polarisation is related to the charge transfer processes occurring during the electrochemical reactions on electrode surfaces. The losses are caused by the slowness of the reactions taking place on the surface of the electrodes [32]. Activation polarisation depends on the nature of type of electrode, ionic interactions, ion-solvent interactions and the

ii. Ohmic overpotential: In most fuel cells, the most important contribution to this resistance is the electrolyte, due to the ionic nature of its conductivity, resistance to the flow of electrons through the electrodes and the contact

iii. Mass transport (concentration) overpotential: Concentration polarisation occurs due to a decrease in the concentration of the reactants at the electrode-

electrolyte interface. Due to diffusion or convection problems in the

iv. Fuel crossover overpotential: 'Crossover' is one of the common effects occurring in alcohol fuel cells [22]. Although the electrolyte, a polymer membrane, is not electrically conductive and practically impermeable to reactant gases, some amount of fuel will diffuse from anode to cathode to react with oxygen, resulting in fewer electrons in the generated current of electrons that travel through an external circuit [34]. With this transit the cathode potential decreases, thus reducing the overall efficiency of a fuel cell. It occurs when the intermediates generated by fuel oxidation have higher concentration than oxygen at the cathode. The increase of temperature

The standard measure of performance is the polarisation curve, which represents the cell voltage behaviour against operating current density (**Figure 4**). From the figure, the voltage loss caused by mixed potential and crossover, activation polarisation, ohmic polarisation and mass transport losses is the most significant in the tail of the I-V curve. The maximum fuel cell is then examined through the reversible voltage of the system, which is calculated using thermodynamics and the actual voltage of the system [36]. The final voltage is lower than the thermodynamic voltage and is usually between 0.5 and 1.0 V. Although polarisations cannot be eliminated, material choice and electrode designs can contribute to their

electrolyte, the concentration of the reactants is not maintained at the initial level. Reaction product accumulation can also cause a dilution of reactants. The concentration gradient thereby formed causes a drop in electrode

lost towards the environment, TΔs, due to negative entropy [12].

electrode-electrolyte interface [33].

*Thermodynamics and Energy Engineering*

resistance at the cell terminals.

activity, and the terminal voltage is reduced.

escalates the crossover effect [35].

minimisation [37]:

**14**

[14, 31]:

ideal potential, usually less than 1 V, due to irreversible losses known as

$$\mathbf{E} = \mathbf{E}\_{\text{therm}} - \eta\_{\text{act}} - \eta\_{\text{ohm}} - \eta\_{\text{conc}} \tag{45}$$

where E is the cell potential, Etherm is the thermodynamic potential, ηact is the voltage loss due to activation polarisation, ηohm is the voltage loss due to ohmic polarisation and ηconc is the voltage loss due to mass transport polarisation and the entropy generation results [38–41]:

$$\mathbf{S} = \frac{\mathbf{E}}{\mathbf{n} \mathbf{F} \mathbf{T}} \tag{46}$$

In addition, the related heat lost for irreversibility can be calculated as (**Figure 4**):

$$\mathbf{q} = \mathbf{T}\Delta\mathbf{s} - \mathbf{n}\mathbf{F}\mathbf{E} = \Delta\mathbf{H} - \Delta\mathbf{G} - \mathbf{n}\mathbf{F}\mathbf{E} \tag{47}$$

**Figure 4.** *H2/O2 polarisation curve at equilibrium and voltage losses in fuel cell [30].*

## **5. Conclusion**

Thermodynamics is used to understand the process of energy conversion in fuel cells. The determination of a fuel cell's performance depends on thermodynamic evaluation. The heat potential of a fuel is given by the enthalpy of the reaction. Not all heat potential of a fuel can be used to perform useful work; the reversible work of a fuel is defined by Gibbs free energy, which is the electrical work. The study of the electrical effects shows that the molar flow of the fuel used is proportional to the electric current and the reversible work is proportional to the reversible voltage. The cell voltage varies with temperature, pressure and reactant/product activities. Irreversible losses cause a difference in the efficiency of reversible and real processes, with efficiency of real processes always less than reversible processes. The losses are due to two major reasons, namely, irreversible kinetic losses and fuel utilisation losses.

### **Acknowledgements**

We greatly appreciate the National Research Foundation and the Tertiary Education Support Programme for financial support as well as the Chemistry Department at the University of the Western Cape.

*Thermodynamics and Energy Engineering*

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**1. Introduction**

Material irradiation, material bombardment with energetic particles in the electro-volt to mega-electro-volt range, generally promotes changes in the material microstructure as a result of the energy transference between the solid atoms and the particle used to bombing the solid. The induced microstructural changes by ion beam irradiation that have been investigated and reported during the past five decades are: surface erosion, cratering, atoms implantation at controlled depth, crystal lattice disorder, phase transformations, second phases precipitation, etcetera [1, 2].

Because ion irradiation can deposit high energy densities locally in relatively short time periods, it could be used to evade thermodynamic constraints and create microstructures containing non-thermodynamic equilibrium phases [3, 4].

• When a surface is bombarded by an energetic ion beam a rich variety of surface structures are developed at atomic, microscopic, and macroscopic scales. These structures include elevations, with respect to the surrounding surface, e.g., plateaus, cones, and pyramids among others, depressions, e.g., pores and cavities. These features may occur singly or in large numbers and often form repetitive patterns.

The knowledge acquired about the basic mechanisms of the phenomena that occur during the interaction between the ion beam with the material used as a target, the knowledge acquired as a result of the damage induced by the radiation has permitted the development of new technology to manufacture materials with specific properties due at the high control that could have over the material structure [8].

The most spectacular example of the use of materials irradiation is the analysis technique known as FIB (Focused Ion Beam). The ability of this technique to grind, image, and deposit material is critically dependent on the nature of the particle-solid beam interaction.


**3. Systematic literature review**

#### **3.1. Objectives**

The purpose of this study is to systematically review the existing literature that addresses the conservative treatment of muscle injuries in adult (skeletally mature) athletes.

#### **3.2. Methods**

Electronic databases searched for the purpose of this systematic review included: Medline and PubMed.

The research question (PICO) was defined with the following criteria:

P: Adult (skeletally mature) athletes; I: conservative treatment; C: other treatment or no treatment; O: recovery of pre-injury muscle parameters and functional activities.

The following search method was used:

("Athletic injuries"(Mesh) OR "Muscle, Skeletal/injuries"(Mesh) OR "Sprains and strains"(Mesh) OR Muscle injur\*(Tw)) AND ("Rehabilitation"(Mesh) OR "Rehabilitation programs"(TW) OR Treatment(Tw) OR "Physical therapy modalities"(Mesh) OR "Therapeu‐ tics"(Mesh)) AND ("Treatment outcome"(Mesh) OR "Muscle strength"(Mesh) OR "Recovery of function"(Mesh) OR "Evidence based medicine"(Mesh))

Filters: Randomized Controlled Trial; Systematic Reviews; Guideline; published in the last 10 years

For more details on the search strategy see Appendix 1.

### **3.3. Results**

**2. Context**

172 Muscle Injuries in Sport Medicine

injuries.

athlete.

**3.1. Objectives**

**3.2. Methods**

**P I C O**

The following search method was used:

PubMed.

**3. Systematic literature review**

Preparing this work we wanted to identify a context characterized by muscle injury in an adult person, skeletally mature, involved in sport activities. The target population was identified in high level professional athletes, but it is obvious that the fallout application in clinical practice involves above all amateur athletes, including different sports from soccer. It seems that the sports involving sprinting, acceleration, deceleration, rapid change of direction and jumping (e.g. soccer, American football, rugby, etc.) may exposed athletes to an increased risk of muscle

The choice of a conservative treatment program for muscle injuries should be based on its effectiveness, its cost-effectiveness analysis, as well as on the expectations and aims of the

The purpose of this study is to systematically review the existing literature that addresses the

Electronic databases searched for the purpose of this systematic review included: Medline and

P: Adult (skeletally mature) athletes; I: conservative treatment; C: other treatment or no

**I**ntervention Conservative treatment

("Athletic injuries"(Mesh) OR "Muscle, Skeletal/injuries"(Mesh) OR "Sprains and strains"(Mesh) OR Muscle injur\*(Tw)) AND ("Rehabilitation"(Mesh) OR "Rehabilitation

**P**opulations/**P**atient Adult (skeletally mature) athletes

**C**omparison intervention Other treatment or no treatment

**O**utcome Recovery of pre-injury muscle

parameters and functional activities

treatment; O: recovery of pre-injury muscle parameters and functional activities.

conservative treatment of muscle injuries in adult (skeletally mature) athletes.

The research question (PICO) was defined with the following criteria:

Initially, 488 papers were identified for potential inclusion. After an analysis about the internal requirements of the studies, only five reports met the inclusion searching criteria. "The Physiotherapy Evidence Database (PEDro) Scale was used by two independent reviewers to assess the methodological quality of each included full text article" (Centre for Evidence-Based Physiotherapy. Physiotherapy Evidence Database (PEDro) Scale. Available at: http:// www.pedro.fhs.usyd.edu.au/scale\_item.html.)

To view the studies included and excluded from this review of the literature see appendix 2.

From the analysis of the few items found arises that there is no research on therapeutic interventions of muscle injuries. There are only a few studies on hamstring muscle injuries, which may not provide strong scientific evidence.

A recent Cochrane review (Mason et al., 2012) highlights the lack of studies about the thera‐ peutic interventions of muscle injuries, especially that there are not right drawings studies, precisely Randomized Controlled Trials. The authors contend that at the moment there is limited evidence to suggest an increased frequency of daily stretching to reduce recovery time and the percentage of recurrence.

Some preliminary scientific evidence available, suggests exercises to improve movement dysfunction. Another recent systematic review (Reurink et al., 2012) demonstrates that the therapeutic interventions for acute hamstring injuries such as stretching, agility and trunk stabilization exercises have limited evidence.

Sherry and Best (2004) performed a prospective randomized study to compare two rehabili‐ tation programs for the treatment of acute hamstring muscle injury. The study concludes that a rehabilitation program which consisted of exercises of agility and trunk stabilization was more effective than a program emphasizing isolated stretching and selective strengthening exercises of the hamstring muscles.

#### **3.4. Discussion**

Following a comprehensive appraisal of the available literature five studies were included in this systematic review. Our findings suggest a lack of research studies that addresses the conservative management of muscle injuries. All five studies included in this review concern the hamstring muscle injuries. As a consequence this limits the possibility to effectively generalize these findings to the clinical settings. The limited number of relevant articles as well as their focus only on the hamstring muscle injuries, highlights the need for future welldesigned randomized controlled trials to conclusively evaluate the effectiveness of conserva‐ tive treatment in the management of muscle injuries, particularly for muscles other than the hamstrings.

Despite the inconclusive results of our review, we will try to provide the reader with the tools to build a reasoned approach to manage the muscle injuries, having as a benchmark the basic

Conservative Treatment of Muscle Injuries: From Scientific Evidence to Clinical Practice

http://dx.doi.org/10.5772/56550

175

**4. Conservative treatment: from scientific evidence to clinical practice**

�Describe the stages of tissue healing and the importance of application of this knowledge in

�Identify characteristics of the different grades of strains and application of this to rehabilitation.

�Design appropriate interventions for describe the conservative treatment from scientific evidence to

Although a variety of conservative treatment strategies exist for the management of muscle injuries, and the "RICE (Rest, Ice, Compression, Elevation) approach" is widely accepted, there is still no consensus regarding the best conservative treatment to offer to patients with muscle

Knowledge of basic principles of skeletal muscles regeneration and repair mechanisms may help to define a rational rehabilitation program. Many authors highlighted that the healing process of an injured skeletal muscle is characterized by three phases. The first phase or *destruction phase*, is characterized by disruption and subsequent necrosis of muscle myofibrils, by the formation of a hematoma between the stumps muscle and by the inflammatory reaction. The second phase or *repair phase* consists in the phagocytosis of necrotized tissue with the regeneration of myofibers and the concomitant production of connective tissue scar and revascularization into the injured area.The third phase or *remodeling phase*, characterized by the maturation of the regenerated myofibers, contraction and reorganization of the scar tissue and recovery of the functional capacity of the muscle. Relying on the basic knowledge on connective tissue healing, an ideal treatment and rehabilitation program of an acute soft-tissue

Firstly, immediately after the injury, the ideal treatment should follow RICE (rest, ice, com‐ pression, elevation) principles; allowing to minimized pain, swelling, inflammation, and

hemorrhage, to offering the best possible condition for the healing process.

sciences and the few available evidences from clinical studies.

**Keys points**

rehabilitation.

clinical practice.

injuries.

**4.1. End points of therapy**

�State aspects of the clinical evaluation.

injury has been formulated to fulfill some requirements.

### **IMPLICATION FOR RESEARCH**

In order to standardize the therapeutic approach and intervention strategies for muscular injuries should produce scientific literature characterized by an appropriate design of studies (RCT) as well as a high methodological quality.

In addition, researchers should also produce studies on alternatives muscle groups and not only hamstring muscles.

#### **3.5. Author's conclusions**

There is no consensus regarding treatment for muscle injuries. Most proposed conservative rehabilitation treatment of muscle injuries have not been assessed using randomized control‐ led trials. Even when randomized controlled trials have been conducted, most have low total numbers of injured athletes, which potentially explains the variability among study results. Most other studies do not provide a level of evidence greater than expert opinion. Although the initial treatment of rest, ice, compression, and elevation is accepted for muscle injuries, no consensus exists for their rehabilitation. Until further evidence is available, current practice and widely published rehabilitation protocols cannot either be supported or refuted. As medical science does not help us we have to turn our attention to the basic sciences to have an approach based on biomedical fundamentals. Thus, factors that impact skeletal muscle structure, function and regeneration are of great importance and interest not only scientifically but also clinically.

#### **TAKE HOME MESSAGE**

There is no consensus regarding the rehabilitation treatment for muscle injuries. There is a very low production of research studies about therapeutic interventions. The basic sciences are a benchmark for developing a rehabilitation program to manage the muscle injuries.

Despite the inconclusive results of our review, we will try to provide the reader with the tools to build a reasoned approach to manage the muscle injuries, having as a benchmark the basic sciences and the few available evidences from clinical studies.


*4.5. lpa1 Locus Molecular Genotyping*

A molecular analysis was performed using *ZmMRP4* sequence-specific amplification polymorphism (S-SAP) markers that allowed the identification of the *lpa1-1* versus *Lpa1* allele. The allele-specific forward primers were designed on the single nucleotide substitution polymorphism in the *ZmMRP4* 10th exon [29,36]. The *Lpa1* wild type–specific forward primer was ZmMRP430L (5'-GTACTCGATGAGGCGACAGC-3'), whereas *lpa1-1* mutation-specific forward primer was ZmMRP432L (5'-GTACTCGATGAGGCGACAGTG-3'). The reverse primer ZmMRP410R (5'-CCTCTCTATATACAGCTCGAC-3') was used to amplify both wild type and *lpa1-1* alleles.

The reaction mixture of the *Lpa1*/*lpa1-1* allele-specific amplifications contained an aliquot of genomic DNA, 1 × Green Go Taq buffer (Promega, Madison, WI), 2.5 μM MgCl2, 0.2 μM each of dATP, dCTP, dGTP, and dTTP, 0.3 μM of forward ZmMRP430L/ZmMRP432L-specific primer, 0.3 μM of reverse ZmMRP410R primer and 1.25 unit of Go Taq Flexy DNA polymerase (Promega), in a final volume of 25 μL.

The reaction mix underwent an initial denaturation step at 94 ◦C for 2 min, 37 cycles of denaturation at 94 ◦C for 45 s, annealing at 62 ◦C for 1 min, extension at 72 ◦C for 1.5 min. Extension at 72 ◦C for 5 min was performed to complete the reaction. The *Lpa1* and *lpa1-1* amplicons were 468 bp long. The amplicons were loaded on 1% (*w*/*v*) agarose gels and visualized by ethidium bromide staining under ultraviolet light.

### *4.6. Structural Analysis of ZmMRP4 Locus in Putative New lpa 1 Mutant*

*ZmMRP4* locus was amplified in wild type and in putative new lpa1 mutant using an high fidelity long range DNA polymerase (Platinum Super Fi DNA Polymerase, Invitrogen) with the primers 5L (5'-TGGTGAGGGGATCAGAGACG-3') (forward primer, position -313) and ZM 2R (5'-GAACTTCCAAAGGCAAGGGACA-3') (reverse primer, position +3413), ZM2F (5'-GGAAAAGTGAGCTCCAAAGTTTA-3') (forward primer, position +3218) and 51R (5'-AAGCATCAGCTTCGGGTAATGT-3') (reverse primer, position +6460). The primers positions are referred to the ATG on the genomic sequence.

The amplicons obtained were run on 1% agarose gel and visualized by ethidium bromide staining under UV light.

**Author Contributions:** Conceptualization, R.P.; methodology, R.P. and M.L.; data curation, G.B., C.R. and E.C.; writing—original draft preparation, G.B. and C.R.; writing—review and editing, R.P. and M.L.; funding acquisition, R.P.

**Funding:** This research was funded by Regione Lombardia, BIOGESTECA project, grant number 15083/RCC.

**Acknowledgments:** We thank V. Raboy (USDA ARS, Aberdeen, ID, USA) and T.P. Brutnell (Shandong Agricultural University, China) for the generous gifts of lpa1-1 and *Ac* line seeds, and Davide Reginelli for his hard work in the field.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.