Tissue Regeneration – From Basic Biology to Clinical Application Edited by Jamie Davies doc

TISSUE REGENERATION
–
FROM BASIC BIOLOGY
TO CLINICAL
APPLICATION


Edited by Jamie Davies











Tissue Regeneration – From Basic Biology to Clinical Application
Edited by Jamie Davies


Published by InTech
Janeza Trdine 9, 51000 Rijeka, Croatia

Copyright © 2012 InTech
All chapters are Open Access distributed under the Creative Commons Attribution 3.0
license, which allows users to download, copy and build upon published articles even for
commercial purposes, as long as the author and publisher are properly credited, which
ensures maximum dissemination and a wider impact of our publications. After this work

has been published by InTech, authors have the right to republish it, in whole or part, in
any publication of which they are the author, and to make other personal use of the
work. Any republication, referencing or personal use of the work must explicitly identify
the original source.

As for readers, this license allows users to download, copy and build upon published
chapters even for commercial purposes, as long as the author and publisher are properly
credited, which ensures maximum dissemination and a wider impact of our publications.

Notice
Statements and opinions expressed in the chapters are these of the individual contributors
and not necessarily those of the editors or publisher. No responsibility is accepted for the
accuracy of information contained in the published chapters. The publisher assumes no
responsibility for any damage or injury to persons or property arising out of the use of any
materials, instructions, methods or ideas contained in the book.

Publishing Process Manager Dejan Grgur
Technical Editor Teodora Smiljanic
Cover Designer InTech Design Team

First published March, 2012
Printed in Croatia

A free online edition of this book is available at www.intechopen.com
Additional hard copies can be obtained from


Tissue Regeneration – From Basic Biology to Clinical Application, Edited by Jamie Davies
p. cm.
ISBN 978-953-51-0387-5

Contents

Introductory Tissue Regeneration – A Clinical
Chapter Science Whose Time Has Come 1
Jamie Davies
Part 1 Understanding and Manipulating
Endogeneous Healing of Tissues 11
Chapter 1 The Role of Physical Factors
in Cell Differentiation,
Tissue Repair and Regeneration 13
Monica Monici and Francesca Cialdai
Chapter 2 Effect of Low-Intensity Pulsed
Ultrasound on Nerve Repair 35
Jiamou Li, Hua Zhang

and Cong Ren
Chapter 3 Disinfection of Human Tissues in Orthopedic
Surgical Oncology by High Hydrostatic Pressure 55
Peter Diehl, Johannes Schauwecker,
Hans Gollwitzer and Wolfram Mittelmeier
Chapter 4 Heparan Sulfate Proteoglycan Mimetics
Promote Tissue Regeneration: An Overview 69

Johan van Neck, Bastiaan Tuk, Denis Barritault and Miao Tong
Chapter 5 Angiogenesis in Wound Healing 93
Ricardo José de Mendonça
Chapter 6 Platelet and Liver Regeneration 109
Nobuhiro Ohkohchi, Soichiro Murata and Kazuhiro Takahashi
Chapter 7 Shared Triggering Mechanisms of Retinal
Regeneration in Lower Vertebrates
and Retinal Rescue in Higher Ones 145
Eleonora Grigoryan
VI Contents

Part 2 Application of Stem Cells 165
Chapter 8 The Therapeutic Potential of Stimulating
Endogenous Stem Cell Mobilization 167
Christian Drapeau, George Eufemio, Paola Mazzoni,
Gerhard D. Roth and Susan Strandberg
Chapter 9 Spermatogonial Stem Cells:
An Alternate Source of Pluripotent
Stem Cells for Regenerative Medicine 203
Liz Simon, Marie-Claude Hofmann and Paul S. Cooke
Chapter 10 Therapeutic Application of Allogeneic Fetal
Membrane-Derived Mesenchymal Stem Cell
Transplantation in Regenerative Medicine 221
Shin Ishikane, Hiroshi Hosoda and Tomoaki Ikeda
Chapter 11 Mesenchymal Stem Cells in CNS Regeneration 237
Arshak R. Alexanian
Chapter 12 Therapeutic Potential of MSCs
in Musculoskeletal Diseases (Osteoarthritis) 261
José Ramón Lamas, Pilar Tornero-Esteban
and Benjamín Fernández-Gutiérrez

Chapter 13 Stem Cell-Mediated
Intervertebral Disc Regeneration 283
Namath S. Hussain, Vickram Tejwani and Mick Perez-Cruet
Chapter 14 Towards Clinical Application of Mesenchymal Stromal Cells:
Perspectives and Requirements
for Orthopaedic Applications 305
Marianna Karagianni, Torsten J. Schulze and Karen Bieback
Chapter 15 Oral Tissues as Source for Bone
Regeneration in Dental Implantology 325
Dilaware Khan, Claudia Kleinfeld, Martin Winter and Edda Tobiasch
Chapter 16 Technologies Applied to Stimulate Bone Regeneration 339
Arnaldo Rodrigues Santos Jr., Christiane Bertachini Lombello
and Selma Candelária Genari
Part 3 Use of Scaffolds 367
Chapter 17 Preparation of Deproteinized Human Bone and Its Mixtures
with Bio-Glass and Tricalcium Phosphate – Innovative
Bioactive Materials for Skeletal Tissue Regeneration 369
Magdalena Cieslik, Jacek Nocoń, Jan Rauch, Tadeusz Cieslik,
Anna Ślósarczyk, Maria Borczuch-Łączka and Aleksander Owczarek
Contents VII

Chapter 18 Endochondral Bone Formation
as Blueprint for Regenerative Medicine 399
Peter J. Emans, Marjolein M.J. Caron,
Lodewijk W. van Rhijn and Tim J.M. Welting
Chapter 19 Tissue Engineering in Low
Urinary Tract Reconstruction 425
Chao Feng and Yue-min Xu
Chapter 20 Novel Promises of Nanotechnology
for Tissue Regeneration 453

Abir El-Sadik
Part 4 Modeling and Assessment of Regeneration 471
Chapter 21 Non-Invasive Evaluation Method for Cartilage
Tissue Regeneration Using Quantitative-MRI 473
Shogo Miyata
Chapter 22 A Mathematical Model for Wound
Contraction and Angiogenesis 489
Fred Vermolen and Olmer van Rijn


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 21
st
century discipline. The graph shows the
number of publications returned by a Pubmed search for ' “tissue engineering” <year>'.

Tissue Regeneration – From Basic Biology to Clinical Application

2
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.

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,
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). High-
frequency 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 – From Basic Biology to Clinical Application

4
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, regeneration-
promoting 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

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
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
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 cell-
mediated 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 – From Basic Biology to Clinical Application

6
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.

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
as those described in section 3 below, will actually prove more useful.
4. Construction of scaffolds
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 scaffold-
based 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 – From Basic Biology to Clinical Application

8
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 high-
resolution 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

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

Endourology / Endourological Society, 17(8), pp.659-666.
Hamid, S. & Hayek, R., 2008. Role of electrical stimulation for rehabilitation and
regeneration after spinal cord injury: an overview. European Spine Journal: Official
Publication of the European Spine Society, the European Spinal Deformity Society, and the
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
mediator for mechano-signal transduction in endothelial cells in situ. Vascular
Health and Risk Management, 4(6), pp.1273-1282.
Kenkre, J.E. et al., 1996. A randomized controlled trial of electromagnetic therapy in the
primary care management of venous leg ulceration. Family Practice, 13(3), pp.236-
241.
McCulley, J.P. & Petroll, W.M., 2008. Quantitative assessment of corneal wound healing
following IntraLASIK using in vivo confocal microscopy. Transactions of the
American Ophthalmological Society, 106, pp.84-90; discussion 90-92.
Owens, B.D., Stickles, B.J. & Busconi, B.D., 2003. Radiofrequency energy: applications and
basic science. American Journal of Orthopedics (Belle Mead, N.J.), 32(3), pp.117-120;
discussion 120-121.
Poelmann, R.E., Gittenberger-de Groot, A.C. & Hierck, B.P., 2008. The development of the
heart and microcirculation: role of shear stress. Medical & Biological Engineering &
Computing, 46(5), pp.479-484.
Simon, C.J., Dupuy, D.E. & Mayo-Smith, W.W., 2005. Microwave ablation: principles and
applications. Radiographics: A Review Publication of the Radiological Society of North
America, Inc, 25 Suppl 1, pp.S69-83.
Trounson, A. et al., 2011. Clinical trials for stem cell therapies. BMC Medicine, 9, p.52.

Tissue Regeneration – From Basic Biology to Clinical Application

10

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
Understanding and Manipulating
Endogeneous Healing of Tissues

1
The Role of Physical Factors
in Cell Differentiation,
Tissue Repair and Regeneration
Monica Monici and Francesca Cialdai
ASAcampus Joint Laboratory, ASA Res. Div.
Dept. of Clinical Physiopathology, University of Florence
Italy
1. Introduction
Physical factors may induce significant biological effects, therefore they can be applied in
biomedical and biotechnological fields in order to drive and modulate biological processes.
It is well known that both humoral and physical factors (in particular, but not limited to the
mechanical ones) are necessary for maintaining tissue homeostasis. Both biochemical and
physical factors can induce the cells to reprogram their functions to adapt dynamically to
the environmental conditions.
It is evident, therefore, that the only way to approach functional tissue regeneration and
repair is to supply combined humoral and physical stimuli in a dose- and time-dependent
manner. For example, in vitro studies have shown that a biomimetic environment
simulating pulsatile flow is an indispensable condition for the tissue engineering of
functional trileaflet heart valves from human marrow stromal cells. Static controls show
morphological alterations and weaker mechanical properties (Hoerstrup et al., 2002).
Studies on the role of physical factors in tissue repair and regeneration cover a very broad
field that extends from investigations aimed at deepening our understanding of the
physiological mechanisms of tissue repair and regeneration to biotech advances in tissue

engineering, such as development of biocompatible scaffolds, 3D cell culture systems and
bioreactors, which in the future must integrate the delivery of biochemical factors with the
provision of physical stimuli that are equally necessary. In this chapter, far from
providing a comprehensive overview of this field of studies, we introduce some issues
concerning the application of physical factors in biomedicine and biotechnology and
report the results of our research on the application of various physical stimuli
(gravitational and mechanical stresses, laser radiation, electromagnetic fields (EMF)) for
modulating cell commitment and differentiation, cell adhesion/migration, production
and assembly of extracellular matrix (ECM) components, with the final aim of
understanding when and how physical stimuli can be useful for promoting tissue repair
and formation of functional tissue constructs. We also briefly mention how, in past
centuries, the role of physical factors in biological processes has been understood and
physical stimuli have been applied for therapeutic purposes.

Tissue Regeneration – From Basic Biology to Clinical Application

14
2. Mechanical stresses
The importance of gravitational and mechanical factors in modulating biological processes
has been known for a long time: from Galileo Galilei onwards, studies on functional
adaptation of the skeleton demonstrated that bone loss or gain is related to the magnitude,
direction and frequency of the stress acting upon the skeleton during application of loads
(Galilei, 1632; Wolff, 1985; Rubin, 1985; Frost, 1988; Rubin, 1984; Ingber, 1998).
Within the body, cells are subject to mechanical stimulation, caused by blood circulation,
ambulation, respiration, etc., which give rise to a variety of biochemical responses. It has
been demonstrated that changes in inertial conditions, shear stress, stretching, etc. can
strongly affect cell machinery. Cells may sense mechanical stresses through changes in the
balance of forces

that are transmitted across transmembrane adhesion receptors

that link the
cytoskeleton to the ECM and to

other cells. These changes, in turn, alter the ECM mechanics,
cell shape and cytoskeletal organization (Ingber, 1998, 1999). A great deal of information has
revealed that the ECM is a highly dynamic and elastic structure which undergoes
continuous remodelling, in particular during development, angiogenesis, wound healing
and other tissue repair processes. The ECM interacts with cells to provide relevant
microenvironmental information, biochemically through the release of stored soluble and
insoluble factors, and physically through imposition of structural and mechanical
constraints (Carson, 2004). On the other hand, mechanical stimuli modulate ECM
homeostasis: mechanical forces strictly regulate the production of ECM proteins indirectly,
by stimulating the release of paracrine growth factors, or directly, by triggering intracellular
signalling pathways leading to the activation of genes involved in ECM turnover (Chiquet,
2003).
Mechanical stimuli affect cells through poorly understood mechanotransductive pathways
that lead to changes in morphology and orientation, modulation of gene expression,
reorganization of cell structures and intercellular communication through both secretion of
soluble factors and direct intercellular contact (Maul et al., 2011; Kang et al., 2011; Park et al.,
2006; Papachroni et al., 2009; Wall & Banes, 2005; Bacabac et al., 2010; Hughes-Fulford &
Boonstra, 2010). Over the past decade, in vitro studies have indicated that the transduction
of physical stimuli involves the ECM-integrin-cytoskeleton network and also calcium
channels, guanosine triphosphatases (GTPases), adenylate cyclase, phospholipase C (PLC)
and mitogen-activated protein kinases (MAPKs), all of which play important roles in early
signaling (Rubin et al., 2006; Hoberg et al., 2005; Adachi et al., 2033; Mobasheri et al., 2005;
Chiquet et al., 2009; Bacabac et al., 2010; Hughes-Fulford & Boonstra, 2010). It has been
demonstrated that, in endothelial cells, different genetic programs leading to growth,
differentiation and apoptosis can be mechanically switched. Cells grow when they spread,
die when fully retracted, and differentiate into capillary tubes if maintained at a moderate

degree of extension (Chen, 1997).
The in vitro application of mechanical stretch, simulating the mechanical load to whom
heart cells are exposed in vivo, initiated in adherent cultures of neonatal cardiomyocytes
morphological alterations similar to those occurring during in vivo heart growth
(Vandenburg, 1996). Stem cell commitment, the process by which a cell chooses its fate, and
differentiation, the resulting development of lineage-specific characteristics, have been

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration

15
shown to be affected by cell shape (Roskelley, 1994; McBeath, 2044; Watt 1988; Spiegelman,
1983). Internal and external forces regulate cell shape and studies have shown that cell shape
can control apoptosis, gene expression, and protein synthesis, in addition to stem cell fate
(Chen, 1997; Thomas, 2002).
The cells belonging to tissues that resist the effects of gravity are particularly sensitive to
mechanical and gravitational stimuli, which play a key role in the development and
homeostasis of these tissues. Lack of gravitational and mechanical stresses leads to the
formation of impaired tissues with lower mechanical properties and reduced function.
It is well established that bone adapts its mass and architecture in accordance with the
external mechanical loads applied and osteocytes, terminally differentiated cells of the
osteoblastic lineage, may be considered “mechanosensory cells” (Vatsa et al., 2010). They are
sensitive to both stretching and fluid flow. Mechanical stimulation of osteocytes induces
intercellular signaling which results in the modulation of osteoblast and osteoclast activity
(Chow et al., 1998; Turner et al., 1997). Interestingly, it has been shown that the stimulation
of a single osteocyte activates many surrounding cells (Vatsa et al., 2007).
Osteoblastic differentiation can be induced by applying mechanical stress, for example by
stretching the surface on which the cells are attached (Cavalcanti-Adam et al., 2002). Many
studies revealed that the micromotions at the interface between bone and artificial scaffolds
play a key role in scaffold integration: they can promote tissue differentiation or induce
bone resorption (Prendergast et al., 1997; Carter et al., 1998; Buchler et al., 2003; Stadelmann

et al., 2008; Jasty et al.,1997). In a recent paper on biomechanics of scaffolds for bone tissue
engineering applications, it has been stated that in the development of a scaffold it is
important to take in account not only the structural integrity but also the load transmitted to
the cells via the scaffold deformation (Pioletti, 2011).
A recent review of studies which investigated the importance of loading in maintaining the
balance of matrix turnover in the intervertebral disk, reported about the possible role of
overloading in the initiation and progression of disc degeneration and proposed a
physiological/beneficial loading range as a basis on which to design loading regimes for
testing tissue constructs or favouring differentiation of stem cells towards “discogenic” cells
for tissue engineering (Chan et al., 2011)
An overview of studies on the role of mechanical stimuli in chondrogenesis showed that
uniaxial loading induces the upregulation of genes associated with a chondrogenic
phenotype while multiaxial loading results in a broader pattern of chondrogenic gene
upregulation, revealing that not only intensity, but also direction and other parameters
which characterize the stimulation are relevant for the achievement of the final effect. The
physiological multiaxial pattern of loading within articulating joints is so complex that
currently, even with the most sophisticated bioreactors, it would be impossible to simulate
the in vivo situation. Therefore, it has been suggested to use the body as an “in vivo
bioreactor”(Grad et al., 2011).
Conditions of gravitational unloading, both real and modeled by a Random Positioning
Machine (RPM), negatively affect cellular organization and ECM production in cartilage
constructs, even if at different extent. (Stamenkovic et al., 2010).

Tissue Regeneration – From Basic Biology to Clinical Application

16
Our group is conducting for several years research on the role of gravitational and
mechanical stimuli in cell differentiation, tissue repair and regeneration, with particular
attention to the remodelling phase.
Our studies demonstrated that gravitational unloading favours the differentiation of

osteoclastic precursors (FLG 29.1 cells). After 72 hours exposure to conditions of
microgravity, modelled by a RPM (angular velocity of rotation 60°/s), the cells showed a
dramatic increase in apoptosis, but the viable ones showed osteoclastic-like morphology,
cytoskeletal reorganization, significant changes in gene expression profile. The expression of
the major osteoclastic markers Receptor Activator of Nuclear Factor Kappa-B (RANK) and
Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) strongly increased and cells
showed the ability to resorb bone (Fig. 1) (Monici et al., 2006).

Fig. 1. Scanning electron microscopy of a bone slice exposed to FLG 29.1 cells cultured in
modelled microgravity. Adherent cells on the bone surface can be observed. Arrows
indicate the sealing zone.
Analysing the gene expression profile of human mesenchymal stem cells (HMSC) in loading
conditions (3 hours exposure to 10xg in hyperfuge), we found overexpression of genes
involved in osteoblastogenesis (GLI1, NF1, MEN1) and downregulation of genes involved in
adipogenesis (PPAR

, FABP4) (Tab. 1) (Monici et al., 2008a).

Gene
Control RPM 10 x g Nd:YAG
FABP4 27 304 3 9
PPARG 12 587 11 14
GLI1 47 45 789 547
NF1 25 9 241 258
MEN1 48 25 158 258
Table 1. Gene expression profile in HMSCs.

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration

17

These results, in agreement with those of other authors (Kaneuji et al., 2011; Wang et al.,
2010; Searby et al., 2005) reveal that mechanical/gravitational stresses induce osteoblastic
differentiation while gravitational unloading and loss of mechanical stress favour
adipogenesis, osteoclastogenesis and bone resorption.
In cultures of fibroblasts exposed for 3 hours to hypergravity (10xg), we observed enhanced
expression of collagen I and fibronectin (20% and 30% more than control, respectively),
while chondrocytes exposed to the same treatment showed a marked increase in collagen II,
aggrecan and Sox 9, a transcription factor which plays a key role in chondrogenesis.
Therefore, after definition of optimal range of intensity and force direction, loading can be
used to stimulate ECM production by cells of the connective tissues and to favour
chondrogenesis.
A series of experiments we carried out with the aim of studying the effect of gravitational
unloading on processes involved in tissue remodelling demonstrated that the loss of
mechanical stress causes a disregulation in laminin and fibronectin (FN) production by
fibroblasts and endothelial cells (Fig. 2B) (Monici et al., 2011). In particular, FN forms a
disordered and intricate network, reproducing the typical condition of fibrous scars. We
hypothesized that the altered FN fibrillogenesis could be a cause of impaired ECM
rebuilding and altered cell adhesion/migration and could contribute to the impairment of
wound healing observed in microgravity (Midura & Androjna, 2006; Delp, 2008).

Fig. 2. FN expression in CVECs (analysed by immunofluorescence microscopy): A) control,
B) exposed for 72 h to modelled microgravity and C) treated with pulsed Nd:YAG laser
(1064 nm). In figure B a tight network of FN fibrils appears while in figure C the FN fibrils
are parallel and ordered (see arrows).
Studying the behaviour of aortic endothelial cells cultured in micro- and hypergravity we
found that the exposure to simulated microgravity conditions for 72 hours (angular velocity
of rotation 60°/s) caused a reduction in coronary venular endothelial cell (CVEC) number.
Genomic analysis revealed that proapoptotic signals increased, while antiapoptotic and
proliferation/survival genes were downregulated by the absence of gravity. Activation of
apoptosis was accompanied by morphological changes, with mitochondrial disassembly

and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence
analysis. Moreover, cells were not able to respond to angiogenic stimuli in terms of
migration and proliferation (Morbidelli et al., 2005)