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Fundamental Biomedical Technologies
Alessandra Giuliani
Alessia Cedola Editors
Advanced
High-Resolution
Tomography in
Regenerative Medicine
Three-Dimensional Exploration into the
Interactions between Tissues, Cells, and
Biomaterials
Series Editor
Mauro Ferrari
The University of Texas
Houston, TX, USA
More information about this series at http://www.springer.com/series/7045

Alessandra Giuliani • Alessia Cedola
Editors
Advanced High-Resolution
Tomography in Regenerative
Medicine
Three-Dimensional Exploration
into the Interactions between Tissues,
Cells, and Biomaterials
Editors
Alessandra Giuliani Alessia Cedola
Department of Clinical Sciences Institute of Nanotechnology - CNR
Polytechnic University of Marche c/o Sapienza University
Ancona, Italy Rome, Italy
ISSN 1559-7083
ISBN 978-3-030-00367-8 ISBN 978-3-030-00368-5 (eBook)
https://doi.org/10.1007/978-3-030-00368-5
Library of Congress Control Number: 2018961740
© Springer Nature Switzerland AG 2018

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Alis volat propriis
It is used as the motto of the US state of
Oregon and its official English version is
“She flies with her wings,” in line with the
tradition of considering countries and
territories as women.
In fact, this book comes from an idea of the
two female co-editors and is the product of
high-level scientists, many of whom are
young women starting to fly with their wings
in the complex world of the scientific
research.
Preface
In the international scene over the last 20 years, the published papers related to the
many and varied studies of tissue engineering and regenerative medicine are
countless. Worthy of note are the two most authoritative journals in the sector, the
three sets of Tissue Engineering (Mary Ann Liebert Inc., USA) and the Journal of
Tissue Engineering and Regenerative Medicine (John Wiley & Sons Ltd., UK). Just
as wide and varied are the books produced. We should mention Fundamentals of
Tissue Engineering and Regenerative Medicine (Springer, 2009), Tissue Engineering
and Regenerative Medicine: A Nano Approach (CRC Press, 2012), and the open-
access productions of INTECH, but these are just a few non-exhaustive examples of
the international offer.
Among these productions, a certain number also contains imaging studies per-
formed using high-resolution tomography, but the information is usually extremely
synthesized into individual sections of volumes (as in Zhang Zhiyong, Jerry KY
Chan, and Teoh Swee Hin’s Computer Tomography and Micro-CT for Tissue
Engineering Application, on Imaging in Cellular and Tissue Engineering, edited by
Hanry Yu, Nur Aida Abdul Rahim, 2012) or often refers to high-resolution tomog-
raphy analysis of samples of interest for tissue engineering of specific anatomical
districts (like in Zehbe Rolf, Haibel Astrid, Franziska Schmidt, Riesemeier Heinrich,
James C. Kirkpatrick, Helmut Schubert, and Christoph Brochhausen’s High
Resolution X-Ray Tomography: 3D Imaging for Tissue Engineering Applications,
on Methods in Tissue Engineering, edited by Daniel Eberli, 2010).
There are also a fair number of books that focus on microtomography technique
and its applications; two notable examples are MicroComputed Tomography:
Methodology and Applications, edited by Stock, Stuart R., CRC Press 2009, and
Advanced Tomographic Methods in Materials Research and Engineering, edited by
John Banhart, Oxford University Press, 2008. Even in these cases, the information
related to regenerative medicine and tissue engineering applications is very limited,
often focused on the sole bone district.
An even more reduced number of scientific studies include results deriving
from synchrotron radiation experiments. Again, we are predominantly referring to
vii
viii Preface
journal papers or to individual chapters within broader treatises related to s pecific,

engineered sites that are focused on the techniques based on the use of synchrotron
radiation or, in numbers still lower, on the specific microtomography technique.
We have personally contributed, over the last 10 years, to some of these volumes
with chapters related to regenerative studies based on synchrotron radiation
microtomography. Among these works, we would like to mention Cedola A. et al.,
X-ray Micro and Nano-Probes: Fundamentals and Applications, Recent Res. Devel.
Applied Phys., 9(2006): 169–196; Fiori F. et al., Synchrotron Radiation and
Nanotechnology for Stem Cell Researchers, in Adult and Embryonic Stem Cells,
2012, New York, Dordrecht, Heidelberg, London, 81–102; Giuliani A. et al.,
Synchrotron Radiation and Nanotechnology for Stem Cell Research, in Stem Cells
in Clinic and Research, 2011, 683–708; and Giuliani A., Analysis of Bone Response
to Dental Bone Grafts by Advanced Physical Techniques, in Bone Response to
Dental Implant Materials, edited by A. Piattelli, 2016.
From this fragmentation of information forcefully emerges the need to produce a
book exclusively dealing with microtomography studies, mainly (but not exclusively)
based on synchrotron radiation, related to problems of regenerative medicine and
tissue engineering of several anatomical districts. De facto, there is the lack of a vol-
ume, similarly conceived, in the international scene, putting this book as a novelty in
the book market.
The goal of this book is even more ambitious: we would like to stimulate the
reader toward scientific and experimental multidisciplinary paths in regenerative
medicine. In fact, the project involves, as authors of the various chapters, highly
selected scientists with great experience at Synchrotron Facilities, some of them
working for years at the microtomography-dedicated beamlines. Furthermore, the
authors involved in this project have gained, over the years and within international
projects of scientific and popularizing character, great educational skills, mainly
directed at a heterogeneous audience. We believe that this is a solid premise to achieve,
in the multidisciplinary direction, an easy language that is also understandable for
readers with different educational backgrounds compared to physics, biology, and
clinical medicine. In fact, modern science teaches us that, without the creation of a
common and transversal language between the basic disciplines, further progress will
not be possible. This is convincingly demonstrated in Chap. 1, authored by Prof.
Franco Rustichelli, which we strongly wanted to start the full set of chapters, being
that it is a prerequisite for understanding the rationale behind this project. Indeed,
Prof. Rustichelli is an eminent scientist who, perhaps among the first in Europe,
believes in the importance of multidisciplinarity in science and in the role played by
the huge machines of physics in this context. It is no coincidence that, in the last
eight years, he scientifically coordinated two European COST Actions focused on
multidisciplinarity in regenerative medicine, i.e., the MP1005 – From nano to macro
biomaterials (design, processing, characterization, modelling) and applications to stem
cells regenerative orthopedics and dental medicine (NAMABIO), and the CA16122 –
Biomaterials and advanced physical techniques for regenerative cardiology and
neurology (BIONECA).
Preface ix
This book is a key manual for easy consultation that is intended for clinicians,
biologists, physicists, and biomedical engineers, but also for students who want to
continue their studies in the field of Regenerative Medicine and for ordinary people
who are intrigued by the fascinating world that revolves around synchrotron struc-
tures and their possible use in research, diagnostics, and medical therapy.
Ancona, Italy Alessandra Giuliani

Rome, Italy Alessia Cedola
Contents
1 The Huge Machines of Physics: The Bet

of the Multidisciplinary Research Teams
in Regenerative Medicine�� 1
Franco Rustichelli
2 Introduction to X-Ray Micro-tomography�� 19
Serena Pacilè and Giuliana Tromba
3 Role of Benchtop Microtomographic Systems
in Tissue Engineering�� 41
Rossella Bedini, Deborah Meleo, and Raffaella Pecci
4 Synchrotron Radiation X-Ray Phase-Contrast
Microtomography: What Opportunities More
for Regenerative Medicine?�� 51
Ginevra Begani Provinciali, Nicola Pieroni, and Inna Bukreeva
5 From Projections to the 3D Analysis of the Regenerated Tissue �� 69
Francesco Brun
6 In-Line X-Ray Phase Tomography of Bone
and Biomaterials for Regenerative Medicine�� 91
Max Langer
7 Inside the Bone: Tissue Engineering
and Regenerative Medicine Applications in Orthopedics�� 111
Marta Nardini, Maria Elisabetta Federica Palamà,
Alessio Romaldini, and Milena Mastrogiacomo
8 Bone Regeneration: Experiences in Dentistry�� 123
Serena Mazzoni
9 The Challenge of the Vascularization of Regenerated Tissues�� 139
Michela Fratini
xi
xii Contents
10 Synchrotron X-Ray-Based Functional and Anatomical

Lung Imaging Techniques �� 151
Sam Bayat, Christian Dullin, Marcus J. Kitchen, and Goran Lovric
11 Better Cartilage Imaging at Synchrotron Facilities�� 169
Annie Horng
12 Into the Heart: What Contributions to Cardiac Regeneration? �� 181
Alessandra Giuliani and Mara Mencarelli
13 Frontiers in Muscle Diseases: The X-ray
Microtomography Support to Latest Researches �� 195
Fabrizio Fiori
14 Nervous Tissue and Neuronal Cells: Patterning
by Electrophoresis for Highly Resolved 3D Images
in Tissue Engineering�� 205
Rolf Zehbe and Kerstin Zehbe
15 X-Ray Phase Contrast Tomography in Tissue
Engineering: Focus on Laboratory Implementations�� 217
Charlotte K. Hagen
Contributors
Sam Bayat Department of Clinical Physiology, Sleep and Exercise, Université de

Grenoble Alpes & Grenoble University Hospital, Grenoble, France
Department of Anaesthesia, Pharmacology and Intensive Care, Geneva University
Hospital, Geneva, Switzerland
Rossella Bedini National Center of Innovative Technologies in Public Health,
Italian National Institute of Health, Rome, Italy
Francesco Brun Department of Engineering and Architecture, University of
Trieste, Trieste, Italy
National Institute for Nuclear Physics (INFN) – Trieste Division, Trieste, Italy
Inna Bukreeva Institute of Nanotechnology, CNR, Rome, Italy
Christian Dullin Institute for Diagnostic and Interventional Radiology, University
Hospital Goettingen, Goettingen, Germany
Italian Synchrotron Light Source “Elettra”, Trieste, Italy
Translational Molecular Imaging, Max-Plank-Institute for Experimental Medicine,
Goettingen, Germany
Fabrizio Fiori Department of Clinical Science, Polytechnic University of Marche,
Ancona, Italy
Michela Fratini Institute of Nanotechnology, CNR, Rome, Italy
Santa Lucia Foundation, Rome, Italy
Alessandra Giuliani Department of Clinical Sciences, Polytechnic University of
Marche, Ancona, Italy
Charlotte K. Hagen Department of Medical Physics and Biomedical Engineering,
University College London, London, UK
Annie Horng RZM – Radiologische Zentrum München, Pasing, Germany
xiii
xiv Contributors
Marcus J. Kitchen School of Physics and Astronomy, Monash University,

Clayton, VIC, Australia
Max Langer Creatis, CNRS UMR 5220, INSERM U1206, INSA-Lyon, Université
Claude Bernard Lyon 1, Université de Lyon, Villeurbanne, France
Goran Lovric Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland
Milena Mastrogiacomo Department of Experimental Medicine, University of
Genoa, Genoa, Italy
Serena Mazzoni Department of Clinical Sciences, Polytechnic University of
Deborah Meleo Rome, Italy
Mara Mencarelli Department of Clinical Sciences, Polytechnic University of
M. Nardini Department of Experimental Medicine, University of Genoa, Genoa, Italy
Serena Pacilè Department of Engineering and Architecture, University of Trieste,
Trieste, Italy
Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy
M. E. F. Palamà Department of Experimental Medicine, University of Genoa,
Genoa, Italy
Raffaella Pecci National Center of Innovative Technologies in Public Health,
Italian National Institute of Health, Rome, Italy
Nicola Pieroni Institute of Nanotechnology, CNR, Rome, Italy
Ginevra Begani Provinciali Institute of Nanotechnology, CNR, Rome, Italy
A. Romaldini Department of Experimental Medicine, University of Genoa,
Genoa, Italy
Franco Rustichelli National Institute of Biostructures and Biosystems, Rome, Italy
Giuliana Tromba Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy
Kerstin Zehbe University of Potsdam, Department of Chemistry, Potsdam,
Germany
Rolf Zehbe University of Potsdam, Institute of Physics and Astronomy, Potsdam,
Germany
Chapter 1
The Huge Machines of Physics: The Bet
of the Multidisciplinary Research Teams
in Regenerative Medicine
Franco Rustichelli
There are more things in heaven and earth, Horatio,

Than are dreamt in your philosophy.
– Hamlet Act 1, William Shakespeare
Abstract In the last decades the biology and medicine made great progresses
thanks, on one side to the expoitation of basic discoveries of physical phenomena,
and on the other side on the development of physical techniques applied to the
medical diagnostic area, like for instance the Nuclear Magnetic Resonance. This
chapter presents the Huge Machines of Physics, starting with large accelerators for
sub-nuclear physics, like the Large Hadron Collider, and continuing with synchro-
trons and free-electron lasers. It will be shown that the last two types of facilities can
provide very useful information both for biology and medicine. In particular it will
be emphasized the great help provided by the X-ray synchrotron radiation to the
regenerative medicine, with examples of tracking of stem cells in investigations of
pathologies of cardiological and neurological nature.
Many persons believe that, as the twentieth century was characterized by the splen-
dour of the discoveries in the field of Physics, with their numerous consequences in
our practical life, the twenty-first century will be characterized by the discoveries in
the field of biology, with a straightforward impact on Medicine and human health.
Actually already in the last decades, the biology (and as a consequence the medi-
cine) made surprising progresses, very often thanks to the basic knowledges pro-
duced by Physics and to the help provided by the innovative physical experimental
techniques.
F. Rustichelli (*)
National Institute of Biostructures and Biosystems, Rome, Italy
e-mail: [email protected]
© Springer Nature Switzerland AG 2018 1

A. Giuliani, A. Cedola (eds.), Advanced High-Resolution Tomography
in Regenerative Medicine, Fundamental Biomedical Technologies,
https://doi.org/10.1007/978-3-030-00368-5_1
2 F. Rustichelli
In this context, we will mention only two examples of milestone discoveries in

the field of biology, which were possible thanks to the use of a classical physical
technique, namely, the X-ray diffraction.
The first discovery was performed by Perutz and Kendrew, who were able to
elucidate for the first time the structure, at atomic level, of a protein, namely, the
haemoglobin, for which they received in 1962 the Nobel Prize in Chemistry. In the
same year, the Nobel Prize in Medicine was assigned to Watson, Wilkins and Crick
for having discovered the double helix structure of DNA, which was based on the
X-ray diffraction experiment performed by Franklin in the Physics laboratory of
Wilkins. Also if the paternity of this epoch-making discovery is often attributed
only to Watson and Crick, we must be aware that without the experimental contribu-
tion of the two physicists Franklin and Wilkins, and in particular without their
complex mathematical elaboration of the data, this leap forward in biological sci-
ence would have been impossible.
Since the time of Archimedes, humans, fascinated by the unknown, tried to enter
the mysteries of our real world.
Paradoxically, the scientific instrumentation has become bigger as the objects to
be studied have become smaller. Thus, the huge machines of Physics were gener-
ated, like tower of Babel or Pyramids in Egypt, which were erected to pursue the
eternal dream of immortality.
Many advances in the discovery of the physical properties of the world have been
made in the last few decades by building huge machines, some of which have
allowed the development of very useful characterization techniques in interdisci-
plinary research, including regenerative medicine, as will be discussed in this book.
Large accelerators built by physicists can be divided into two categories:
(a) Machines for producing high-energy collisions between subnuclear particles in
order to understand the microscopic world.
(b) Machines for the production of very intense X-rays to perform basic scientific
experiments and applied in various scientific disciplines, including biology and
medicine.
Although this book is focused on the second type of machines, we consider it
useful, for the sake of completeness and in some way for historical reasons, to spend
some words also on the first category of machines.
Let us consider the Large Hadron Collider (LHC) at the European Organization
for Nuclear Research Centre in Geneva, Switzerland (Figs. 1.1 and 1.2), which
has been realized to reach a new knowledge frontier on subnuclear elementary
particles [1].
It is a huge ring, 27 km long, where protons with incredibly high energy are
made to turn in opposite directions at nearly the speed of light and are then made to
collide. The existence of atoms, the first step to understanding the structure of mat-
ter, had been foreseen and advocated already at the time of ancient Greece (BC IV
sec), but only more than 2000 years later, atomic theory began to encounter scien-
tifically clear evidence.
1 The Huge Machines of Physics: The Bet of the Multidisciplinary Research Teams… 3
Fig. 1.1 The Large Hadron Collider (LHC) at the European Organization for Nuclear Research
Centre in Geneva. (Courtesy CERN)
Fig. 1.2 Internal view of the LHC in Geneva. (Courtesy CERN)

4 F. Rustichelli
Now, after a century of unwavering research in atomic and nuclear physics, we

hope to obtain fundamental information on the interaction forces of particles at
distances much smaller than the size of atomic nuclei, through these proton
collisions.
The study of the smallest interaction distance, in fact, can bring us information
about the most basic features of matter in our universe: the aim is to understand the
origin of the atomic nuclei components and then of all existing matter.
We still do not know the origin of the mysterious energy associated with mass, as
described by the hundred-year-old, well-known equation E = mc2. We also have no
clear idea as to the origin of energy and invisible matter, which contribute to 96% of
the total mass of the universe. Maybe even our numbers and the present mathemat-
ics are not adequate to describe the infinitesimal forms of our world. So something
new must happen when these proton collide in the gigantic accelerator of Geneva!
From such collisions a myriad of unstable elementary particles are obtained. A
very important experiment was already performed, namely, the discovery of the
Higgs boson [2], which was foreseen theoretically several decades ago by the physi-
cist, who gave to it his name and who was awarded recently by the Nobel Prize in
Physics (Fig. 1.3).
Qualitative leaps in physical knowledge have always been the origin of innova-
tive technological applications. Future experiments at the LHC could help us to
answer important questions and perhaps to understand the fundamental rules gov-
erning the evolution of the entire universe. Probably we will also learn more about
the nature of space itself, making a qualitative leap in our knowledge of nature.
Fig. 1.3 Image related to the discovery of the Higgs boson at the LHC. (Courtesy CERN)
Geneva’s machine is indeed an incredible effort to push ahead the frontiers of

knowledge. Whether you consider it a proud Babel Tower or a cathedral to celebrate
the mysteries of nature, the researches carried out there will certainly have great
cultural implications, impossible to foresee four centuries ago when we were dis-
cussing whether the sun was turning around the earth or vice versa.
Now in Geneva, we can make discoveries that are fulfilling at large extent the
dream of Einstein: I want to know God’s thoughts. The rest are details, also if some-
body like Woody Allen seems to have said: Can we actually know the Universe? My
God, it’s hard enough finding your way around Chinatown.
However, very important scientific contributions are continuously produced also
by machines devoted to basic and applied science experiments, through the X-ray
radiation delivered by large accelerating machines called synchrotrons. A relevant
example is the European Synchrotron Radiation Facility (ESRF), built in Grenoble
(France) and shown in Fig. 1.4, constituting the most intense and brilliant X-ray
source existing today worldwide [3].
ESRF consists in a ring (called storage ring) which is a tube having a circumfer-
ence of 844 m, where the electrons travel for hours at a velocity close to that of light
(300 km/s). If they travelled around the earth, they would complete the circle more
than seven times per second. During their ultrafast travel, the electrons emit X-Ray
radiation of very large intensity, which is extracted through several tens of tangen-
tial beams, as reported in the scheme of Fig. 1.5. In any beam, several experimental
facilities are installed.
Fig. 1.4 The European Synchrotron Radiation Facility (ESRF) in Grenoble

6 F. Rustichelli
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Fig. 1.5 Scheme of the tangential X-ray beams at ESRF
Synchrotron light in addition to very high intensity and brilliance offer the
advantage of covering a wide range of frequencies through the electromagnetic
spectrum from the infrared, through the ultraviolet, to the X-ray region, allowing
also time-resolved experiments in the sub-nanosecond range.
The ESRF is used by several thousands of scientists every year, coming mainly,
but not only, from all Europe, operating in the fields of physics, chemistry, biology,
medicine (in particular in different areas of regenerative medicine as we will see
below), Earth sciences, material sciences and engineering, nanosciences and
nanotechnologies.
Another Synchrotron Facility, built very recently, is the Synchrotron-Light for
Experimental Science and Applications in the Middle East (SESAME). It is located
in Jordan and is the largest international scientific laboratory existing in this area. Its
members are Jordan, Iran, Pakistan, Israel, Turkey, Cyprus, Egypt, the Palestinian
Authority and Bahrain [4].
It is an installation of great social interest because its existence shows that it is
possible to build scientific and cultural bridges between different countries in a
region often tormented by violence, conflict and extremism, contributing to a cul-
ture of peace through international scientific cooperation.
Fig. 1.6 Inner view of the European X-ray free-electron laser (XFEL) in Hamburg
It is expected that, by fostering scientific and technical development in the

Middle East and neighbouring countries, the brain drain will reverse in the near
future. The SESAME creation was sponsored by UNESCO, the general director of
which was present together with the King of Jordan to its inauguration on May 16,
2017. On November 22, 2017, scientists saw the first monochromatic X-ray radia-
tion, signalling the start of the laboratory experimental program. In SESAME, like
in ESRF, electrons circulate around a circular tube (having a circumference of
130 m), guided by magnets and other equipment, close to the speed of light.
The electrons produce X-rays, having an energy of 2.5 GeV, which are extracted
through tangential beams, where several experimental facilities can be located allow-
ing researches in structure of matter, material science, cultural heritage, archaeom-
etry, biology and medical sciences. In the future 20 beamlines are foreseen but, at
present, only 3 of them are operative: they are mainly devoted to study living cells
and tissues, protein crystallography, structure of viruses and developing of drugs.
Another category of huge machines are X-ray free-electron lasers (Fig. 1.6),
which constitute extremely bright X-ray sources, having an enormous impact also
on biological research. They generate very short X-ray pulses, of a few hundred
femtoseconds, which are billions of times more intense than synchrotron radiation.
The succession of these very short pulses of radiation impinging very rapidly mov-
ing molecules allows to obtain, in spite of their high speed, a series of snapshots
of them, like they would be frozen in time, as it happens in discotheques when the
dancers of rock and roll are illuminated by stroboscopic light and appear frozen.
By using this technique, the Pearson American Dance Company obtained recently
very impressive effects by showing dancers appearing suddenly in the dark sus-
8 F. Rustichelli
pended and immobile at a considerable height above the floor, giving the illusion
of a levitation.
Moreover, the wavelength of the produced X-ray pulses is small enough allow-
ing to obtain pictures of the investigated samples at atomic resolution.
At the end of 2017, the 3.4-km-long European X-ray free-electron laser (XFEL)
started running near Hamburg (Germany), and it is, at present, the most performant
one of the world, producing 27,000 pulses per second [5]. The intensity of its pulses
is so high that they destroy the investigated sample; however, before sample destruc-
tion, enough photons are diffused in the detector, allowing to study the 3D atomic
structure of the sample.
In a second, it is possible to collect more than 3000 X-ray pictures compared to
about 100 of other facilities. In structure-determination experiments based on con-
ventional X-ray sources, one must obtain a single crystal of the investigated mole-
cule. Indeed, the pulses of XFEL are so bright that it sufficient to obtain single
crystals of very reduced sizes, up to the order of magnitude of few nanometres. In
addition, even non-crystalline clusters of molecules (for instance, in the case of
proteins difficult to crystallize, like some membrane proteins) are suitable. The
XFEL facilities allow investigations of the structure and dynamics of atomic and
molecular systems, previously not accessible. Indeed, by XFEL it is possible to
obtain movies showing the movements of enzymes and viruses by combining thou-
sands of snapshots and even investigating the dynamical aspects of catalysts.
X-rays are electromagnetic waves, like visible light, but characterized by a much
smaller wavelength, that is, of the order of tenths of nanometre, which corresponds
to the atomic diameter.
The nanometre is defined as 1 billionth of a metre. To remember this definition
and better understand the meaning of many frequently used words like Nanoscience,
Nanotechnology and Nanomedicine, let us consider the following practical example.
Suppose that shortly before the presentation ceremony of this book at the
University of Rome by editors Alessia Cedola and Alessandra Giuliani, they receive
a call from the Ambassador of China to Rome who says that one billion Chinese
would like to participate in this very interesting event. It is possible to satisfy the
request by hosting the Chinese in the hills around Rome and using large screens, but
the problem arises of how to feed them at lunchtime. It is very easy to buy an Italian
salami “mortadella”, 1 m long, and cut a billion slices, producing an equal number
of sandwiches. The thickness of each slice is 1 nm!
Using X-ray diffraction it was possible to determine the atomic-level structure of
the different chemical compounds and therefore also of biological molecules. To
understand why X-rays can “see” the position of atoms, while radiation with a lon-
ger wavelength, such as visible light, cannot, let us imagine having a row of water-
melons on a table.
If we enter the room with an eye patch so that we cannot see anything and try to
guess the structure of the line of objects on the table by touching the line with a
1-m-long bar, we will discover a flat structure. On the other hand, if we use a pencil
(roughly with the same length as the diameter of the watermelons) as a probe, we
Fig. 1.7 Nobel Prizes obtained by experiments based on X-rays
will obtain a correct structural information on the curvature and on the position of
the watermelons.
In practice, the position in space of the different atoms of a given compound or
molecule is obtained by first recording the X-ray intensities diffused in the different
directions of space by the electrons belonging to all the atoms contained in the
sample studied, then performing very complex mathematical treatments.
Figure 1.7 reports the list of Nobel Prizes assigned before 2009 for researches
using X-rays. The physicist W.C. Roentgen discovered such type of radiation in
1895 and performed the first radiography on the hand of his wife.
Von Laue and Knipping obtained the first X-ray image diffracted by a crystal in
1912. Fifty years later, the Nobel Prize in Chemistry was assigned to Perutz and
Kendrew, as previously mentioned, for having clarified for the first time the struc-
ture of a protein, i.e. the haemoglobin, after a very intense work due to the high
number of atoms contained in it. Since then, the structure of a large number of
increasingly complex proteins has been determined.
In the same year, the Nobel Prize in Medicine was awarded to Watson, Wilkins
and Crick for discovering the double helix structure of DNA.
Several structural biology experiments have been performed at the European
Synchrotron Radiation Facility (ESRF) over the past 15 years. We will mention
some examples, in detail what we consider the most brilliant, namely, the determi-
nation of the structure, at the atomic level, of the ribosome, which was awarded
10 F. Rustichelli
with the Nobel Prize in Chemistry awarded in 2009 to Yonath, Steitz and
Ramakrishnan [6].
The ribosome has the fundamental role of reading the information contained in
the RNA messenger and using it to produce proteins.
Ribosomes exist in all living systems, from bacteria to humans, and are ideal
targets in bacteria for antibiotic drugs. The adventure of this extraordinary discov-
ery began at the end of the seventh decade of the twentieth century, when an Israeli
researcher, Ada Yonath, decided to bravely face this challenge, overcoming a series
of problems that were considered impossible to solve by the great majority of the
scientific community. In fact, the ribosome is one of the most complex proteins/
RNA, consisting of a “small subunit” and a “large subunit”. In the human ribosome,
the “small subunit” consists of a DNA molecule surrounded by 32 proteins, while
the “large unit” is made up of 3 DNA molecules surrounded by 46 proteins.
Therefore, each subunit is made up of thousands of nucleotides and thousands of
amino acids, for a total of hundreds of thousands of atoms: the challenge for Ada
was to find for each of these atoms the exact values of its Cartesian coordinates.
The first critical problem which Ada was obliged to solve was the production of
a single crystal, of good quality, in which the ribosomes must be located in space in
an extremely ordered way, namely, according to a triply periodic structure along the
three Cartesian axes. After 10 years of intense research, she succeeded to obtain a
perfect crystal from the thermophilic bacteria of the Dead Sea.
However, 10 years more were necessary to overcome other important difficul-
ties, also of mathematical–physical nature, for which she took advantage of the
cooperation with other outstanding scientists. Finally, in 2000, they were able to
publish the structures at atomic level, obtained at the ESRF, of the “large subunit”
of ribosome of the Haloarcula marismortui and of the “small subunit” from the
Thermus thermophilus [6].
It is known that to combat bacteria, which are becoming progressively more
resistant, it is necessary to produce new antibiotics. Researchers from the various
pharmaceutical companies take advantage of the knowledge of the ribosome struc-
ture to understand the mechanisms of action of antibiotics and to develop new
drugs [7].
In addition to the studies mentioned above, related to the structure of the ribo-
some, other researches carried out at the ESRF, many essentially related to the area
of biology, have allowed the involved scientists to be awarded, some of them with
Nobel Prizes, as a result of the exceptional experimental opportunities offered by
this European instrument. For example, the Nobel Prize awarded to Kobilka and
Lefkowitz for having elucidated the structure of “G-protein-coupled receptors” [8]
or the Nobel Prize in Chemistry assigned in 2003 to Roderick Mackinnon for struc-
tural and mechanistic studies of ion channels in cell membranes [9].
In the last section of this chapter, I would like to present the potential offered by
X-ray synchrotron radiation for investigations related to regenerative medicine.
Since the numerous chapters of this book deal with, also from a theoretical point
of view, the different tomographic opportunities available for the different areas of
the medical sciences, here only a general sample of these potentials is presented,
supported by some examples, with the objective to convince the reader to continue
the immersion in reading the topics of specific interest.
Most of the examples presented in this book are based on the use of X-ray
microtomography, in particular in phase-contrast microtomography, for applica-
tions in regenerative orthopaedics, regenerative dentistry, degenerative neurology
and regenerative cardiology.
A revolution occurred in the area of imaging for medical diagnostic purposes
when, at the beginning of the 1970s, the first equipment for X-ray computed tomog-
raphy became available in hospitals [10]. The new technique presented enormous
advantages as compared to the conventional X-ray radiography technique, by deliv-
ering 3D structural information on the investigated organ, as compared to the pro-
jection on the 2D photographic plate of the inner structures of the organ.
The principle of this imaging technique is very simple: hundreds of conventional
2D projection radiographs are recorded, in correspondence to an equal number of
rotation angles of the sample with respect to the direction of the impinging X-ray
beam. The obtained images are based on the absorption contrast. Then, by using
proper mathematical algorithms, the so-called tomographic image reconstruction is
performed, delivering volumetric structural information on the investigated sample.
A crucial problem in regenerative orthopaedics is the repair and the regeneration
of damaged tissues produced by different causes, including diseases like cancer.
The classical approach for repairing the bone lesions makes use of ceramic scaf-
folds loaded with bone marrow stromal cells (BMSCs). Several studies [11–14]
have shown that this procedure generates new-engineered bone in a shorter time
than with cells or scaffold alone.
These experiments had the objective to find scaffolds with ideal characteristics,
namely, enough large surface area and pores for bone growth and high interconnec-
tivity in order to achieve the blood vessels penetration into the pores [15–25].
Several scaffolds materials were considered: metals, ceramics, glasses, natural
and artificial polymers, combinations to form composites and recently nanostruc-
tured materials.
X-ray microCT was shown to provide not only interesting images of the internal
microstructure but also quantitative structural data, applying different parametriza-
tion methods [23–25]. In particular, scaffold surface, volume, pore wall thickness
and diameter were evaluated.
The microCT technique was also used to investigated in non-destructive way
tissue-engineered constructs obtaining quantitative information on the newly
formed bone. Pioneer experiments were performed in small animal models [15, 26].
For instance, at the microCT beamlines of ESRF, the kinetics of bone growth in an
immunodeficient murine model was determined by analysing the structural param-
eters at different time points after implantation.
Moreover, more recently, microCT was also used to quantify the resorption rate
of the scaffolds by engineered bone [27].
The same team of scientists was able to visualize, without the need for any con-
trast agent, the microvascular network of an engineered bone derived from 24 weeks
of implantation in a mouse [28]. The used technique, based on phase contrast, is
12 F. Rustichelli
Fig. 1.8 (a, b) MicroCT imaging of the 3D vessel network and (c) 2D microCT sampling slice.
(d) Histogram of the vessel diameter distribution measured by phase-contrast-based microCT
(open circle) and histology (solid circle). (From Ref. [28])
able to image thin low absorbing structures, like blood vessels, which are transpar-
ent when using conventional microCT based on X-ray absorption contrast.
Figure 1.8 shows the 3D blood vessel network and the histogram of the vessel diam-
eter distribution in comparison with the data obtained by histology.
An application of the phase-sensitive microCT for a clinical investiga-
tion related to regenerative dentistry was recently performed at ESRF [29].
Mesenchymal stem cells derived from human dental pulp were seeded on colla-
gen I scaffolds and grafted in human mandible defects. Three years after grafting,
some biopsies were extracted from these sites and investigated by conventional
methods and by propagation-based phase-contrast microCT. The regenerated tis-
sue appeared more compact than the control alveolar bone. Figure 1.9 shows a
microCT 3D reconstruction where the regenerated bone (white) and the blood
vessels (red) are clearly visible.
It should be noted that both of the dental research mentioned above were con-
ducted in the context of the European Project “MPNS Action COST MP1005 –
From nano to macro biomaterials (design, processing, characterization, modelling)
and applications to stem cells regenerative orthopaedic and dental medicine
(NAMABIO)”.
Fig. 1.9 Three-dimensional image of a portion of human regenerated mandible observed by syn-
chrotron radiation-based phase-contrast microCT. The newly formed bone is virtually rendered in
white and the newly formed blood vessels in red
Another European Project recently funded and deserving to be mentioned in this

context is the COST Action CA16122 “BIONECA – Biomaterials and advanced
physical techniques for regenerative cardiology and neurology”.
The principal BIONECA challenge is to establish an intensive interaction among
the top-level European institutions of different scientific background, with the main
goal to reduce the number of deaths and costs associated with neurological and
cardiological diseases.
Indeed, in the field of neurological disorders, cell-based treatment appeared as a
very promising approach to ameliorate (or at least to slow) the course of different
diseases. Positive results were obtained by using the primary foetal tissue for clini-
cal trials related to the Parkinson’s disease. Moreover, recent in vivo studies have
shown that neural stem/precursors cells show a satisfactory survival, counteracting
different neurological pathologies.
Another type of adult stem cells, the mesenchymal stem cells (MSC), show great
promise in controlling the inflammatory activity and repairing of damaged tissues in
the presence of different pathologies, including ALS, Huntington’s, Alzheimer’s
and Parkinson’s diseases, as well as spinal cord injuries, stroke and multiple
sclerosis.
In this context, one of the major challenges is the treatment of Duchenne muscu-
lar dystrophy, a pathology inducing severe muscular damage in the affected patients.
14 F. Rustichelli
Recent pioneering investigations [30, 31] demonstrated that cellular therapy is able
to produce an improvement of the pathology by repairing the muscle damage. In
particular, it was observed in muscular dystrophy mice models that a subpopulation
of human stem cells, through an arterial route delivering, can reach the sites of
muscle regeneration more efficiently than using myogenic progenitors.
After a first ex vivo experiment [32], another in vivo investigation [33] was per-
formed by using microCT at ESRF, having labelled the injected stem cells with
highly radiopaque iron oxide nanoparticles. This study allowed to elucidate the
kinetics of the diffusion of the stem cells from the blood stream to the different
skeletal muscle sites, obtaining results undoubtedly of great interest to attempt cel-
lular treatment for muscular tissue repair.
Recently, the X-ray high-resolution phase-contrast tomography was also used
to investigate, in ex vivo conditions, the microstructure of spinal cord in mice [34].
In particular, the 3D vascular and neuronal systems were imaged at scales from mil-
limetres to hundreds of nanometres. Similar investigations were also performed by
comparing the healthy mouse neuronal architecture with one of the mouse models
of multiple sclerosis [35].
Finally, the same team was able to investigate, by phase-contrast nanotomog-
raphy, the alterations induced by autoimmune encephalomyelitis in the vascular
and neuronal networks of mice and possible treatments with mesenchymal stem
cells [36].
Cardiovascular diseases (CDVs), in particular congestive heart failure (CHF),
are other leading causes of death worldwide, in a population progressively aged.
Indeed, in spite of important advances in the treatment of ischemic and non-
ischemic cardiovascular diseases [37], a poor understanding exists of the basic
mechanism of CHF, justifying the search for new therapeutic options.
In this context, the regenerative cardiology constitutes one of the most promising
strategies, where a key role is played by stem cells, with their unique ability of self-
renewal and multipotency. Stem cell-based treatments to regenerate the myocar-
dium can be accomplished by local injection of cells in the myocardium and
systemic mobilization by cytokines and by tissue engineering strategies [38–42].
Primitive cells, with stem/progenitor characteristics, were isolated and expanded.
Several clinical trials have shown that they represent a suitable autologous source to
repair damaged myocardium by cellular therapy [43, 44].
In preclinical research, high-resolution tomographic imaging based on the syn-
chrotron radiation was shown to offer a valid support in cell tracking, as demonstrated
by a pioneering experiment performed at the ESRF [45]. This study, based on cellular
therapy [46], showed that X-ray microCT is able to visualize, with high resolution
and definition, the 3D spatial distribution of rat cardiac progenitor cells inside the
infarcted heart, in ex vivo conditions and after short time from their injection. Thus,
microCT imaging represents a considerable stride forward as compared to 2D histo-
logical analysis and constitutes a promising tool in view of future clinical experiments.
All of these studies, most of which will be reported in more detail in the next
chapters of this book, demonstrate the prospective usefulness of the considered
tomographic techniques in the study of the damage induced by various diseases and
traumas and their repair with regenerative medicine approaches.
In conclusion, a first paradoxical consideration that springs from this first chapter
is the need to build ever larger machines, the modern cathedrals of these last two
centuries, to explore the secrets of nature and life on an ever smaller scale.
A second consideration is linked to the importance of X-rays and in recent times
of synchrotron radiation, to provide experimental instruments more and more suit-
able to produce knowledge not only in the fields of structure and dynamics of matter
but also in more complex areas such as biology and medicine that even led to the
awarding of the Nobel Prize in several brilliant discoveries!
The last consideration is for the Physics’ discipline that, beyond the importance
that it has in the interdisciplinary research, is at the base of all this magical world of
enormous machines and scientific applications. If we just restrict our considerations
to the brilliant results already obtained in biology and to the enormous prospects of
the contributions of synchrotron radiation for clinical progress in the innovative area
of regenerative medicine, we can conclude that we are operating in accordance with
the philosophy of one of the first great physicists of modern time, Galileo Galilei,
who said:
I am looking for the light of science and its benefits.
La Luce della Scienza cerco e ‘l Beneficio
Galileo Galilei
Acknowledgements It is a pleasure to thank Prof. Federico Quaini and Dr. Mario Stefanon for
their precious scientific contributions and to Mr. Mario Pergolini for his outstanding technical
support.
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The girl had risen to her feet. Quite cowed as she was for the
moment, a joy was in her heart to hear herself so repudiated in that
company. Her worst fears were laid: her venom was turned to honey.
She whimpered a little, in a panic half feigned, half felt,—
“There, I don’t want to. I’m going, for sure.” Then a spit of courage
came to her—“and I’ll tell the other he may just die for all you care”—
and she turned.
But, before she could reach the door, a swift step followed, and a
soft white hand, ringed and scented, was placed upon her shoulder.
She hesitated an instant, faced round, and the next moment the two,
high saint and lowly sinner, were clasped together weeping.
Poor Molly knew her place. She sunk at the other’s feet again, till
Yolande knelt beside her, and put her arms about the shameful
head.
“Poor child! poor sinful woman,” she said, to a flurry of sighs and
sobs. “O, what was I to hold you so apart! But you don’t understand
—you can’t, God pity you. The worse for him that killed your
innocence.”
“He—”
“I’ll not hear his name.”
“He was my only one; and—and, for your sake, he’s been wanting
to make me good.”
“Has he? There’s a way.”
“Maybe. But not the way you mean. That’s closed to such as us.”
“Alas! What way, then?”
“Make yourself impossible to him.”
“I? Sweet saints, give me patience with this poor ignorance! How
can I make her comprehend that I could never be more impossible to
him than I am.”
“O, yes! you could.”
“There, there, child! How?”
“O, mistress! don’t you know?”
“Know what? Why am I letting you talk to me like this? I’m all
groping in a maze. O! haven’t you a father?”
“Yes, for sure.”
“Give up your sin. Go back to him and ask his pardon.”
“You don’t know him. His pride’s above his station. He’d ne’er
suffer me again to come anigh him.”
“Wouldn’t he? What a thing’s this pride in men!—a vengeance, not
a judge! Fatherless, then! O, O! that’s to be lost and helpless—crying
to a void—sinking, sinking; and not a straw to hold by!”
“Ah, hush ye, pretty one—hush ye!”
The Magdalen, with winking wondering eyes, was become the
comforter. She clasped the cold hands within her own warm palms,
and mumbled them, and loved their softness. Yolande, her head
bowed, sat grieving still a little.
“To look all round, and not to know where to turn—no guide, no
help out of this maze!”
She snuffled, and mopped her eyes; then struggled to regain her
estate. “There, child! my heart bleeds for you! What is your name?
O! I forgot; you haven’t one”—for, indeed, to this sweet orthodoxy, an
unchurched passion was a nameless thing—a maiden title forfeited
to anonymity.
“I’m Molly Bramble, please my lady.”
She hung her head. The other pursed her lips a moment.
“Well, well, child—we’ll call you as we call our dog or parrot—
terms for distinguishment.”
Then the moth plunged for the light, about which she had been
desperately fluttering this nervous while.
“You mentioned of your nursing someone? or perhaps I confused
your meaning?”
“Ay, did I. You know him. Saint-Péray.”
The other put her away and got hurriedly to her feet.
“You’re nursing him! You?”
“He brought him to me—told me to; told me to help him back to be
a man, and win you yet.”
“Who brought him? Who told you?”
“There: I wasn’t to speak his name.”
“Nursing him? Where?”
“Why, in the little villa that he keeps for me.”
“That he keeps? O, my love, my Louis!”
“Ah, ah! you love him still. You make my heart sing, you do!”
“O, Louis! O, mon bien aimé! que les artifices des méchants t’ont
environné! You must not be left: you must not stay there: you do not
know. The villain! the false friend!”
“O, O, my lady!”
“Is he not? He dared to ask my hand.”
“O! it’s true then!”
“Two nights ago.”
“Ah, me! that explains it.”
“What?”
“Why, what he told me before he left next morning. ‘I’ve changed
my mind,’ says he. ‘She’s not for him no more. What you’ve said
you’ve to unsay. They must be kept apart at any price.’ They were
his last words before he went.”
“Were they?—those?”
“His very words.”
“Yesterday?”
“Yesterday morning.”
“O, my child! give up this wicked man, to save your soul!”
“No, I’ll stick to him.”
“Poor prodigal, enamoured of the husks.”
“He said he would be good for your sake. You owe him that.”
“For my sake?”
“Ay, even for true love’s sake, maybe—though it wounds my heart
to speak it. There’s a way you could show him.”
“A way? I? to what?”
“To mend a wrong. O, dear good lady, I’ve seen your eyes
confess!—never deny it. One marriage brings another—it might, it
might even lead to that—O, mistress, mistress!”
“You are mad. You don’t know what you say—you know nothing.”
“I know your love is dying there for love of you.”
“Dying? No, no!”
“Come to him, and see.”
“I cannot.”
“He must die then. He’ll not last till morning else. ’Twas for that I
dared this all.”
“O, what am I to do?”
“No one need know: a great lady like you.”
“You say he’d marry you?”
“I say one marriage brings another.”
“O! Sweet saints, direct me! Lead my distracted mind! I cannot
come with you, I say!—Wait while I fetch my cloak!”
********
Fiorentina, bidden to hold her tongue to Louis-Marie, told him
everything—under promise of secrecy: how that one was coming in
a little to break his brain’s web and kill the wicked spider—a
physician, maybe: maybe a wise woman; for indeed physicians were
not “her,” and the signorina had stated distinctly, in answer to his
cries, that she was going that moment to fetch her to cure him.
Fortunately or not, he heard her without comprehending. He was
lying apathetic by then, quit of the “fellow in the cellarage.” That
thundering whisper silenced, all commoner voices served him but as
opiates. By-and-by he fell into a doze; and the little camerista drew
his curtains, and lit his candles, and went below to gossip with her
house-mate.
The storm laboured up and over, mingling with the sick man’s
dreams. The rush of tempest smote on ice. He was alone in a
surging darkness. It cracked, with a roar of thunder, and spilled a
dead body at his feet. Madly he strove to spurn the thing—into
monstrous-seeming abysses—for all their blackness they were
shallow troughs. Or else the glacier rolled like water, and threw it up.
He trampled it in fury—it writhed away, reshaping. Then it took to
laughing; and the laugh was echoed from hard by—and there was
Bonito hiding in a drift. He woke with a scream.
But he was sleeping again, when the little camerista hurried up,
and looked into his pale exhausted face, and touched some pillows
into comfort before leaving him.
Sweet dreams this time, but still of weeping rains. Only they fell
softly on a Chapel roof. She was not there beside him, and he
wondered why she lingered. Till, glancing at the coloured statue of
the virgin, he saw it stir and smile, and stretch out wistful arms to
him, and heard it breathe his name—“Louis, Louis!” And it was she
herself, descending and coming to him; but, before they could reach
and touch, she had vanished.
“Louis, Louis!” Her voice wept far remote, an infinite yearning, faint
and always fainter; till suddenly, with a crash, the roof was rent, and
a flood of fire rushed in, revealing her—quite close to him—a
breathing apparition—all love and sorrow paining her sweet eyes.
He lay and did not stir. “Yolande!” he whispered.
She sighed, and clasped her hands; she answered with the plaint,
if not in the words, of love-lorn Madeline:—
“O, leave me not in this eternal woe,
For if thou diest, my Love, I know not where to go.”
She moved, and was kneeling by him, pleading with hurrying
sighs,—
“The sin was mine—the sin was mine! And, O! a fruitless sacrifice!
So pale, so worn—O, thing without a heart, to have caused this cruel
sickness in my love!”
“Yolande!” A wilder thrill gave out the word.
“Louis; if thou couldst still find that in me worth living for! Ah, do
not die! I would be so loving and so penitent. Not forward—no. The
shame in me’s an ecstasy. I cry to have you humble me.”
“Lily of Savoy—the white lily—and mine!”
A gloating transport whispered in his voice.
“Thine still, dear love; and, for all her shame—inviolate.”
She hid her face to speak it. This was no swooning vision, but
reality. No matter whence she had come, or at what instigation—the
death-warrant was cancelled. Life at her words flowed back to him,
lapped in a sensuous dream. Doubts, fears, proscriptions were all
forgotten. His pulses beat to madness: a delirious hunger of her
swelled his veins. This sweet fruit of his desire! It were as if the
heavy-bosomed grapes, made animate by Love, had drooped of
their own pity to the lips of Tantalus. Should he not crush them in his
mouth? unquestioning, praising the heavenly mercy, not abusing it
with one self-scruple as to his deserts? It was characteristic of him,
at least, so to surrender his will to circumstance. He flushed as if
intoxicated. He leaned impassioned towards her: “My wife!” he
whispered, and drew her to his heart.
She raised her streaming eyes,—
“What you have suffered for my sake—and not the least to find
you here.”
“Here, Yolande? the best that could have happened to me.”
“O, my love! you must not say it. It is a wicked house.”
“Yolande!”
“O, God! my saint is innocent! Louis! this man, your friend, and the
poor girl—!”
“What of them?”
“They live in sin together—O, my lamb among the wolves!”
Old tremors, old lost scruples seized him at the words. He clung to
her.
“Take me away, Yolande. I am so sick and helpless.”
“Yes, yes, my love, my husband! Come with me.”
“No, I am too ill. To-morrow. Don’t leave me, now you’ve come.”
“O, I must! Louis!”
“Then I shall die. ’Tis only you can save me—make me a man
again.”
“O, love! you kill my heart!”
“To save me, Yolande! To save yourself that new self-reproach if I
died without.”
“And if you were to die in spite?”
“O, love! that cried to me to humble it! We will be man and wife to-
morrow. I shall live for that—I must. The thought will lay the spectres
that would kill me else. Yolande! you will not let me die?”
“O, Louis! let me rather.”
“Come to me, my dear, my love, my wife—there, sweet, my wife,
this seal upon your lips!”
********
In the grey of the dawn, cold and austere after tempest, the
signorina Brambello hurried forth to procure an accommodating
priest. He was easily found, easily bribed, easily persuaded into
quick conclusions. The two were joined before the altar of San
Maddalena, a dingy chapel in an obscure neighbourhood, and Molly
and Fiorentina were the witnesses.
At the end, in the sombre porch, the pale bride turned upon the
English girl.
“God, in His mercy, so give thy sin to mend itself—my sister!”
She hesitated an instant, then threw her arms about the other’s
neck, kissed her on the mouth, and hanging her sweet head, went
with her husband down the steps into the silent street. And his face
also was bowed, as he walked feebly beside her.
CHAPTER XIII
Cartouche, released, at the end of a week, from his inaugural
business in the Le Prieuré Prefecture, returned forthwith to Turin—
and to the re-encountering a problem, whose difficulties, one had
thought, he might have studied more profitably at a distance. But a
characteristic precipitancy, in deed and word—as much acquired as
born of self-reliance in him—compelled him from hesitating on the
brink of things. When angels and devils were at contest in his
interests, he was not going to miss the excitement, nor the chance of
applauding, or perhaps damning, the victors.
But he had had a more wearing time of it than he would have
cared to admit, even to himself. He was not apt at moral
conundrums; and one had come to consume his peace
confoundedly. He felt it always smouldering in his breast, ready to
break out into flame at any moment.
And he had really laid out its premises very impartially for his own
consideration. He was an eclectic by nature; as, alas! is the case
with a number of naughty people. It is unfortunate, indeed, that
righteousness so often lacks the sense of humour, which is the
faculty for seeing both sides of a question. The want seems to give
obliquity such a superiority—though it is a specious one, of course.
He could admit, then, the inevitableness of a deed, which had
preserved an honour most dear and sacred to himself. He could not
admit a claim to that honour personified, as the price of blood. Louis,
the slayer of a woman’s husband, could not take that husband’s
place. Were she, knowingly, to let him, her honour would be forfeit:
were he to take advantage of her ignorance, he would be doing a
vile thing. She was not for Louis: could never be, in any scheme of
moral purifications.
For whom, then? Why, scarcely less vile were he, Cartouche, to
seek to take advantage of his friend’s hard fortune (It will be
observed that he somehow inferred for that problematic vileness its
problematic opportunity—the ineradicable instinct, perhaps, of an
amoroso, experienced in the ways of audacity, to whom a rebuff had
always stood, and likely been always justified in standing, for an
incitement to fresh aggression).
As to another question, that of his own relationship to the dead
man, he utterly declined to recognise it as one involving his personal
interdiction. The marriage had been a mere conditional contract, of
the essence of a betrothal, and the conditions had not been
observed. No moral prohibition, such as touched upon the forbidden
degrees, was implied by it, he told himself: and told himself so, he
insisted, merely to emphasise the singleness of his renunciation. He
would have the full credit for his self-sacrifice. His responsibility was
not to a sentimental scruple, but to his ideal of an immaculate honour
in the woman he worshipped.
Remained the question of his attitude towards the murderer of his
father, and of his royal commission to hunt down that unknown
assassin. Well, he had both discovered and exonerated him; but the
offence was still officially un crime qualifié. To condone it were to
make himself an accessory.
He would condone it, however, since by so doing he testified to his
loyalty to his ideal. Yolande’s eternal fame should owe him that
sacrifice of his duty to his nobler conscience. By so little, at least, he
would justify himself in the thankless wardenship of her honour; by
so little he would make himself the right to claim her into an
association with himself.
So far and so good for his solution of the problem. This dear prize
was not for Louis; it was not for him. What, then, was to be its
destiny?
There was his ideal. Eternal maid, by virtue of her deathless
bondage to the past, she was to exist the unattainable goddess of all
desire. He might not reach to her; but he might enforce his own
precedence in her worship. He would be the high-priest of that altar,
winning to his place by heart’s-devotion. He pictured her, a virgin for
ever unfulfilled, the flying figure on the vase, and himself, the
passionate shepherd, stricken to an endless rapture of pursuit. What
sweeter, more idealistic heaven?
“She cannot fade, though thou hast not thy bliss;
For ever wilt thou love, and she be fair.”
A pretty, pretty romance! But was it practical?
His soul, at least, flamed out to it. It gave him a mad wild joy to
think that circumstance, and by no contrivance of his own, had
removed the one mortal bar to its attainment.
Whence, now, and wherefore, his return to Turin—to make himself
secure of his transfigured idol—to confirm Louis-Marie, if necessary,
in his renunciation of an untenable claim. For knowing the man, he
could not but have his doubts of his resolution. So much of him was
based on emotion—a treacherous foundation.
And, for the rest—his own title, by way of redemption, to that
priesthood? Why, Molly, of course, was to be included in the
transcendent scheme. She was to share his atonement, and be
appointed a vestal to the altar of his love. He would pension her off
for that purpose; he would—
O, “a mad world, my masters,” where love could not legalise itself
without making a scapegoat of somebody!
And there was even another flaw—his promise to Yolande. But he
had been obliged to forget all about that.
As he walked, in a sort of sombre self-complacency (as of a martyr
about to testify) through the streets, his mind was busy over those
first practical solutions of his problem which he was about to face. It
would be necessary, he had decided, to inform his friend—restored,
he hoped, by now to reason—of the impossible situation which his
appointment had brought about, and to urge him to resolve its
insuperable difficulties by instant flight. That must be the first step.
And, afterwards—?
Alert, perspicacious by instinct, his eyes had become aware, as he
moved on, of something oddly inquisitorial, something droll and
furtive, in the glances of friends and acquaintances whom he met,
whether directed at himself, or slyly interchanged. He affected to
pass all by unconcerned, nodding brightly here and there without
stop or comment; but he made mental notes, abstractedly stroking
his sword-hilt, as if it were a pet terrier’s head. He felt, quietly, a little
wicked. His theory of self-reforms, it would appear, halted yet
something short of meekness and the second cheek to the smiter. At
the corner of a street he ran plump upon Dr Bonito.
The adverb is figurative. The Doctor was always as shrewd an
encounter as an edge of north wind. He cut into one’s meditations
like a draught. On the present occasion, it seemed, he cut to get
home into an adversary unprepared. His lean face kindled to the
unexpectedness and opportuneness of the meeting.
“Hail, hail, M. le Préfet!” he croaked, in hoarse glee. “Here’s a
magnetic conjunction! What man so much in my mind!—and, lo! I
look up; and the man himself! Have you despatched both rogues and
measures in your new Province? But doubtless you are returned
betimes to assay the truthfulness of the great report. Well, be
satisfied; it is true.”
Cartouche balanced on his heel, imperturbably conning the face of
his old familiar. He saw enough there to detain him a reflective
moment. The two had not met since their parting “Under the
Porticoes.”
“Father Bonito,” said he; “I do not want to possess your mind. You
can stick up a bill for a new tenant. I have grown a little particular in
my tastes. In the meanwhile, I am only this hour returned to Turin,
and greatly pressed for time. What, in a word, is this report, of which
you speak and I know nothing?”
The doctor sprawled up his hands in feigned astonishment.
“Gods! I believe he really hasn’t heard it! and the very stones of
the town babbling with it these days past. Not to have heard it—the
one most interested, with myself—he hasn’t! I’m my own first suitor
to his gratitude for this.”
“Well; the devil give you brevity!”
“No, no—one moment—stop! The Marchioness di Rocco, Mr Trix
—ah!”
He withdrew a detaining hand, grinned, took off his hat, and
mopped his forehead with a ropey clout, eying his halted prey the
while.
“A long throw that, Monsieur,” he said; “yet it hooked you. But, to
be sure, she’s a killing bait.”
Cartouche, just lifting his eyebrows, vouchsafed him no other
answer. He knew his man—was steeling himself quietly against
some blow which he felt was preparing, and which he saw would be
designed to take him off his guard. Let Bonito, in that case, extract
what satisfaction he could out of his manner.
In fact, when the stroke actually fell, his reception of it was so
apparently unconcerned as even to deceive the doctor into a doubt
of the effectiveness of his own home-thrust, and to aggravate his
malice proportionately.
“Yes, a killing bait—a—killing—bait,” he said; and threw his
handkerchief into his hat, and covered himself—all deliberately.
“Well,” he said, “congratulate me, Mr Trix. He was shy; but—he’s
taken her at last.”
Cartouche yawned.
“In the name of patience—who’s taken whom?” said he.
“Who? Why M. Saint-Péray has taken his Marchesa, that’s all.”
“Well, those are news, to be sure.”
“Are they not—eh? He-he! You are looking worn, Mr Trix. I’m
afraid you take your new duties too seriously. You shouldn’t forget
that all social office is a compromise—a figure representing the
balance between good and evil, to lower one of which unduly is to
exalt the other unduly. Yes, we’ve married our couple.”
“Have we, indeed? And who are ‘we,’ my Bonito?”
“There! these low levels tell on one coming from the heights. You
must be careful of your throat. I notice a huskiness in it already. Why,
indeed, save for a natural diffidence, I might say, Monsieur, that ‘we’
stands for ‘I’; seeing that, as a fact, the initiative was mine. In any
case, what we were one in desiring is, at this moment, an
accomplished thing. The two are married—not, as you may suppose,
a union regarded with favour in certain quarters.”
“No; I suppose not. And how did you bring it about?”
“Ah—ha! there’s the marrow! Why, how you flush and pale! I doubt
the prudence of exciting you, Mr Trix, in this present turbulent state
of your blood.”
“Exciting me? What do you mean? Why should I be excited? Have
I been hanging rogues so few as to start at the mention of a noose?
Tell me how you managed it, my dear excellent old devil.”
“Well, I will. There are points you mayn’t approve; but the end
must justify the means. Listen, then. I could not make our friend
eligible in the way I proposed. But still I was his matrimonial agent—
you remember the term, it was your own? As such my duty to him,
my duty to myself, demanded renewed enterprise on my part. You,
who have expressed an eagerness no less than mine to secure this
match, will, I hope, condone, even approve, the advantage I took of
a report concerning yourself to realise our common wish.”
“A report? What was that?”
“Why, that you yourself was a suitor for the hand of the lady.”
“Yes? and the advantage you took of that same veracious
legend?”
“It may have been a legend: it was certainly an opportunity. What
did I do? Why—forgive me, sir—I simply went and repeated it, for
what it was worth, to the Signorina Brambello, and left the leaven to
ferment. The result was quite astonishing. She ran straight off, it
appears, in a pet of jealousy to the lady; induced her to return with
her to the bedside of her stricken gallant (by which, or thereabouts, it
seems our Madam spent the night), and married the two incontinent
the next morning at a neighbouring Chapel (called, somewhat
appropriately, la Maddalena), giving herself and another for witness.
Now, am I to be congratulated or not? A word in season hath
accomplished what all your theories of pretty heartenings and
reassurances had failed to. You appealed to the signorina’s
sympathies; I to a baser but more practical sentiment. Acknowledge
who was the better sophist.”
Cartouche clapped him on the shoulder.
“You, you, my Bonito. The credit is all yours, and the triumph. I will
not forget it. I will not overlook your part in this happy
consummation.”
Bonito grinned.
“Nor your innamorata’s, eh, Mr Trix? Egad! she’s a name in Turin
to-day. She might command—but, there! these reports are not for my
lips.”
“Her price, you mean? Well, she shall have it. Now I must go. I
have business which can wait no longer.”
He went off, humming a little song. As once before, the doctor
stood conning his receding figure, until it had vanished round a
corner. Then he gave a short sudden laugh, and turned to his own
way.
“Well acted,” he thought; “and well out of the reckoning, he; and
well saved, my own skin—for the present—I’m a little afraid at the
expense of the dear signorina’s. But, bah! if the wind were to hold its
breath for fear a leaf or two might fall, there’d be no clearing the air
in this world of scruples.”
********
Cartouche walked straight to the little villa in the Lane of
Chestnuts. It was a glowing, lustful day. The white curtains in the
windows bosomed out to him like love’s own welcome; lizards
basked on the walls; the flowers in the garden hung sweet drowsy
heads. He was singing still when he reached the door: singing when
he greeted Fiorentina with a chin-chuck: he walked, with a song on
his lips, into the parlour. She was there, sure enough—a flushed
palpitating beauty, with a brave front of greeting, and a quaking heart
behind it. He had no idea of making many words about the thing. He
stopped in the middle of the room, smiling at her.
“What!” said he: “no kiss for me?”
She had never realised until this moment the fulness of her daring,
nor its madness. She gulped sickly, as she crept up to him without a
word, and put her lips to his cheek.
He had a purse of gold ready, and held it out to her.
“There are your wages, Judas.”
As if her legs had been knocked from under her, she went down at
his feet.
“No, no! He was dying, Cherry!”
“Better he had died.”
“O, don’t condemn me unheard!”
“Did you disobey me?”
“Yes; but—”
“That is enough.”
“O, my God! Am I to go?”
“Yes.”
“Think what it means to me?”
“I am thinking.”
“And you can do it?”
“And I can do it—a hundred times. And worse than that, if you
tempt me. Take your price, and go—back to England, if you are wise.
Do you see this in my hand? It’s my last mercy.”
He drew away from her, where she lay, cast upon her face and
moaning.
“I am going,” he said. “But I shall return in the afternoon at three
o’clock. If then I find you still here—understand what I say—your
chance to save yourself is past. I’ll kill you on our bed—I mean it.”
A wild desolate scream broke from her throat. He threw the purse
down beside her on the floor, and left the house without another
word.
At three o’clock to the minute he returned. Not till he had searched
into every corner of the villa, would he question the red-eyed
cameristas, huddled awaiting him in their kitchen. Then he learned
that she had gone indeed. They would have besieged his heart with
tearful clamour, telling of the scene—its rending piteousness; but he
stopped them peremptorily, paid them their wages, double and
treble, and dismissed them.
He had already seen that the purse of gold lay untouched where
he had thrown it down upon the floor. For all his gripping will, that
gave his heart a wrench. He stooped and took it in his hand—
hesitated—then, with a curse at his own weakness, thrust it into his
breast. He went from room to room, bolting the windows. In one
upstairs he paused—so long that ghosts began to stir and whisper in
the empty house. Something, he thought, was moving the curtains of
the bed to which his back was turned. Little slippers stole from
underneath a chair and walked without sound upon the floor. He
heard a sigh—it was himself sighing. With a mad oath, he turned and
tramped downstairs, resolutely, making all the noise he could. The
next moment he had clapped to the door behind him, and was in the
open air.
That night, pacing the streets, he passed a hospital for Magdalens.
A box, beseeching charity, was in the wall. He stopped, and taking
the purse from his breast, dropped the coins from it, one by one, into
the slit.
Then he turned and disappeared into the darkness.
PART III
CHAPTER I
We mortals discuss the world as a subject of our common
understanding, and no two of us see it with the same eyes. To Victor-
Amadeus the third’s, for example, it was a stage for fêtes galantes;
to the Chevalier de France’s a ball fettered to the ankle of an heir-at-
law, infamously kept from his inheritance; to those of a certain “little
corporal,” as yet unaccredited, it was a potential family estate; to
Yolande’s and Louis-Marie’s a reformatory for original sin; to Bonito’s
it was a footstool to the stars, to Cartouche’s an absurd necessity, to
Jacques Balmat’s a glorious field for adventure.
In 1786 Jacques was the most famous man in Le Prieuré, and for
long distances beyond it. In notability he had outstripped all these
other claimants to our attention. For he had won his mountain and
his wife, and basked in the lustre and the reward of a great
enterprise greatly accomplished. Yet he took his reputation modestly,
as became one who had looked on Death too often and too close to
boast himself superior to that God. He’d propitiated, not defied him.
There was something very solemn, very sobering in having gained
that awful shadow for one’s friend. So he accepted his part without
arrogance, but without hypocrisy.
“Ah! monsieur,” he said to Saint-Péray, lord-consort to his lady of
the Manor: “you should have held on; you should not have lost heart;
you should have been with me. There are no heights so inaccessible
but that the good God will surrender them to our trust in Him as the
first guide of us all. There is no corner of His world of which He hath
said, ‘Faith shall not enter here.’”
Madame Saint-Péray (she had dropped—flung away, rather—her
title) looked up from her needle-work, with a little frown, like an acute
accent, nicked between her eyes. She was conscious, on this
occasion as she had been on others, of that half protective half
accusatory note in the young mountaineer’s respectful addressings
of her husband, which somehow touched a corresponding chord in
herself. It vibrated on a thought of weakness; it was the tremor in the
heart of dying dreams; its first movement in her had been co-
instantaneous with the fall of her saint from transcendent to merely
human heights. Something of discharm spoke in it; a sense as of an
idol convicted of petitioning his worshipper; a sense as of an
unwilling accessory to another’s secret sin; a sense as of a
responsibility incurred where help had been expected. These several
emotions she found suggested somehow in young Balmat’s tone.
Were they common to all sympathetic spirits brought into whatsoever
relations with her husband? She feared so. She feared, more, that
Louis-Marie liked it to be so. His caressing confidence in all others
than himself constituted at once his strength and his weakness. He
ruled by sweet dependence, and was satisfied to rule.
There were hints of a certain change in her in these days—signs
of an enforced self-emancipation, which, in its process, had a little
chilled the texture of her faith. It was, in its moral, like that hardening
of the grain which only a close observer can detect in the “fixing” of a
pastel. The bloom was a thought less virgin; the eyes less liquid-
clear; the lips had tightened to a scarce perceptible primness. Her
love was as single, as great, as self-sacrificing as ever. Only it had
altered its habit to a sterner garb. It ruled where it had served; it had
made a subject of him who had been its lord; it justified itself by
every concession to the loved one but that of self-abandonment. And
in such implied reproaches as those of honest Balmat’s it felt its
attitude vindicated. “You should have been with me,” he had said. He
should. If he had, if it had been in his nature to be, this twin history of
theirs, she believed, had never come to find its tragedy and
redemption. Louis at this moment had been her king—her tyrant,
even; their parts had never of necessity been reversed.
Of course, in all this, she only skimmed the truth. There was more
to be inferred, even than she supposed, from the young
mountaineer’s tone. It implied, in fact, a troubled conscience,
seeking to allay its own suspicions on the strength of a serenity in
their object which must surely, it told itself, be incompatible with guilt.
For, indeed, a certain serenity had come to succeed in Louis-Marie
the storms and anguish of a former state. His wife’s tender
ministrations; a year of utter peace, of utter immunity from
disturbance in their retreat, had restored him to a measure of self-
confidence—even to a point of view something broader than that in
which Cartouche had confirmed him. Now he was inclined to think
that his deed had been not only righteous, but heroic; that his
bearing of its burden in silence was a saintly discipline; that, in any
case, his confiding of his awful secret, like King Midas’s barber, to
the reeds, had acquitted him of the first responsibility to it. And the
last was, after all, his most characteristic comfort. He grew well on it,
as a worried schoolboy, quit of his imposition to a merciful parent,
forgets his troubles in a moment.
There remained only, to disturb his conscience, the question of his
conditional absolution, as decreed by Cartouche. Well, as to that, he
had assured and reassured himself, his friend was scarcely
matriculated in moral philosophy. But, even were he called upon by
him to answer for his act, he had still this to plead—that he had not
married Yolande, but Yolande him.
For the rest, slow growing sense of security had come to mend his
sickness of another shadow. A year had passed, and it had not yet
pursued him to his fastness in the Château di Rocco. He hoped now
it never would. He hoped he read, in the social exile which their own
mutinous act had decreed upon himself and Yolande, an
abandonment of any interest in their further fortunes. God grant they
might be permitted to make out their days in peace, justifying—as
they for ever strove, and intended for ever to strive to do—in their
devotion to their church, in a wide and noble beneficence, their
inheritance of a wicked man’s possessions. For to this end only had
they decided to take up the burden of an estate otherwise hateful to
them.
It was a mellow September noon. The three sat under the front of
the grim old Château in the quiet sunlight. Far off across the valley,
on a level with their eyes, great flakes of silver-white, spangling a
golden haze, were the huddled masses of the Alps, no less. Soft and
unsubstantial in appearance as the floating iridescences one sees in
water, they were still the native home and most austere dominion of
primordial rock and ice. It seemed impossible to realise it. The very
shadows on their slopes were traced so soft, they were no more
shadows than the blue veins in marble, than the blue inter-webbings
of running surf. Surely that mist of peaks must be descended cloud,
and the changing colours of it the bloom of angelic wings beating
within!
Below the sitters’ feet terrace declined upon terrace, until, halted
against a buttress wall, the cultivated land gave place beyond to
stony pastures, which descended to the lower verge of the estate
and the great wrought-iron gates of the entrance.
And between, poised high in the mid-ether of the valley, a
watching kestrel floated like a leaf.
Madame Saint-Péray, looking up, answered for her husband. Her
recognition that neither high achievement nor great failure was ever
for this dear weanling of her passion was not to find her loyalty to
him at fault—rather to confirm her jealousy for his reputation.
“That is a very right sentiment for a guide, M. Jacques,” she said;
“but there may be nobler conquests for duty even than those of
mountains. Monsieur owed his life to me; and he sacrificed his
ambitions to that debt.”
That was the thorn. Then she offered the rose.
“For you, you owed that conquest to your love; and bravely you
strove and gained. I hope the dear father recovers himself of your
naughtiness?”
Jacques laughed; then essayed his little gallantry. No Frenchman,
however primitive, lacks that essential grace,—
“I said, Monsieur should not have lost his heart for the enterprise. I
was a dog, an imbecile. What summit could equal that to which his
heart attained! I thought myself near heaven as I stood up there
alone—the first to get so near. Alas, Madame! Monsieur staying on
the ground had already gained it.”
Monsieur, lying comfortably back in his chair, smiled kindly.
“That is very true, Jacques; and I wish I could take credit for the
best deserts. But you have not answered Madama’s question.”
“Of Dr Paccard, Monsieur? The old man is almost himself again.
He can see his son-in-law at last.”
“It was cruel of you to force him to the summit,” said Madame.
“Why, what would you?” answered the mountaineer. “He would
never have believed else; and upon his belief depended my reward.”
“But, by all accounts, he could not see, even then.”
“That is true; but others could. My faith, he was bad! But it was his
bargain, not mine, that he should accompany me to witness. He
would have given up before we slept the first night on la Côte. There
had been enough and to spare already to terrify him. With dusk had
come an oppression of the air. Our axes sang like flutes. Suddenly,
as I climbed, holding my staff by the middle, it had a knob of light for
head—a thing like a luminous bladder, that palpitated, and swelled,
and shrunk and swelled again; till, in a moment, it detached itself and
floated away, far, far into the shadows, where it burst with a clap like
thunder. Then came the lightning, above, everywhere. One blaze
struck the ground, right in front of us. It was as if a bucket of fire had
been emptied from some window of the rocks. It splashed up and
was gone, leaving a stench—Mon Dieu! the fish they had been
gutting up there were not very fresh.”
“O, horrible, horrible!”
“Better than that our heads had received it. But I am fatiguing
Madame?”
“No, no. Go on. I have wanted so much to hear it from your lips.”
“He slept exhausted, for all his fright, wrapped in my blanket, and
moaning for the good roast chicken, which he had ordered at home
against his soon return. When he awoke, it was bright calm sunlight,
and he had gathered new heart of rest. We went on and up; but his
courage soon ebbed, running out at his heels, until, Mon Dieu! he
was crawling on his belly like a mole. That was laughable enough;
but even so, my merriment could urge him no further than the Dôme
du Goûter, where he sat down and refused to move a step further. I
gave him my glass, and told him to look how the villagers watched
us from below, and at Martha herself, the brave child, waving to us
with her handkerchief. It was all of no use. I had to leave him and go
on alone. The thin air suffocated me. The wind shaved my cheeks,
drawing blood from them like a clumsy barber. Every sweep of its

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