raman spectroscopy investigation of organic matrix orientation and
TRANSCRIPT
-
Raman spectroscopy investigation of organic matrix
orientation and chemistry changes during fracture healing
von Diplom-Ingenieur
Leonardo Galvis
aus Venezuela
von der Fakultt III Prozesswissenschaften
der Technischen Universitt Berlin
zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften
-Dr.-Ing-
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Roland Lauster
Gutachter: Prof. Dr.Ing. Claudia Fleck
Gutachter: Prof.Dr. Peter Fratzl
Gutachter: Prof. Dr.Georg Duda
Tag der wissenschaftlichen Aussprache: 16 Mai 2012
Berlin 2012
D83
-
Acknowledgements
I would like to express my gratitude to all the people who allowed the completion of this
work.
First of all, I would like to acknowledge Prof. Dr. Peter Fratzl and Prof. Dr. Georg Duda, not
only for giving me the incredible possibility of doing my PhD in the Biomaterials department
at the Max Planck Institute (MPI) as a graduate student of the Berlin-Brandenburg School of
Regenerative Therapies (BSRT), but also for their guidance. Many thanks for the all
discussions and support. Also, I would like to acknowledge my supervisor in the Technical
University Prof. Dr. Claudia Fleck for her guidance.
I am indebted as well to my supervisor, Dr. John Dunlop, for his guidance, patience and
support in every aspect during these years. I was his first PhD student and I truly think he did
a good job. Thanks a lot John.
Also, I would like to acknowledge the supervision of Dr. Admir Masic, who taught me all I
know about the Raman universe, the basis of my project, and for all the time dedicated to
the explanation of complex experimental details.
Many thanks to Dr. Philip Kollmannsberger, Dr. Daniel Kopetzki, Roman Schtz, Felix Repp
also to my colleagues from the Charit Carolin Schwarz, and Manav Mehta, for sharing their
knowledge on fracture healing.
To my group colleagues and friends working at the institute, thank you for the friendly
environment and the good moments. I will miss our mid-day lunches in the wonderful mensa
and our coffee breaks.
I would also like to thank my family: my mother Maria Yolanda, my grandmother Amelia and
my brother Giovannito. Many thanks for all your love and constant support throughout the
year. All what I have done is because of you. I would like to thank Inecita for all our
-
marvelous years together, the haircuts, the beautiful Benetton sweaters and text revisions.
Thanks Inecita!
Last but not least, I would like to dedicate this work to the memory of my father Teodoro,
who passed away before I started my European adventure.
-
Table of content
Raman spectroscopy investigation of organic matrix orientation and chemistry changes during
fracture healing. ............................................................................................................................
Abstract ........................................................................................................................................ Zusammenfassung ........................................................................................................................ 1. Introduction ........................................................................................................................ 1 2. Review of the literature ...................................................................................................... 5
2.1 Bone as a living material ............................................................................................. 5
2.1.1 Structural organization: Mineral and organic matrix ........................................... 6 2.1.2 Extracellular matrix .............................................................................................. 9 2.1.3 Collagen ............................................................................................................... 9
2.2 Fracture healing and callus formation ....................................................................... 12 2.2.1 Secondary bone healing ..................................................................................... 13 2.2.2 Overview of structural studies on fracture callus. .............................................. 16
2.3 Raman Scattering ....................................................................................................... 19
2.3.1 Polarized Raman spectroscopy (PRS) ................................................................ 21 2.3.2 Polarizability tensor and Raman tensor .............................................................. 22
2.3.3 Raman scattering intensity ................................................................................. 23 2.3.4 Orientation measurements and scattering geometry. ......................................... 25
2.4 Raman spectroscopy in bone ..................................................................................... 26
3. Objectives ......................................................................................................................... 31
4. Theoretical response of the amide I band according to the classical theory .................... 33
5. Methodology to obtain collagen orientation in collagen based materials and callus samples ..................................................................................................................................... 45
5.1 Raman spectra of bone, collagen type I, and embedded medium and the selection of the integrating range. ............................................................................................................ 45
6. Material and Methods ....................................................................................................... 51 7. Application of PRS in standard materials ........................................................................ 55
7.1 Gelatine ...................................................................................................................... 55 7.2 Rat tail tendon ............................................................................................................ 56 7.3 Secondary Osteon ...................................................................................................... 60
8. Application of PRS in fracture healing callus samples .................................................... 65 8.1 Cortex 2nd week (sample I) ........................................................................................ 68
8.2 Cortex 2nd week (sample II) ....................................................................................... 72
8.3 Cortex 6th week (sample I) ........................................................................................ 74
8.4 Cortex 6th week (sample II) ....................................................................................... 76 8.5 Raman ratios in the ROI located on the cortex .......................................................... 78 8.6 Mineralized cartilage and woven bone at early stage 2nd week (sample I) ............... 84
9. Discussion ........................................................................................................................ 89 10. Conclusions / Outlook ................................................................................................... 95
11. References ..................................................................................................................... 99 Appendix 1: 3
rd and 4
th week of fracture healing in callus cortex.......................................... 107
Cortex 3rd
week (sample I) ............................................................................................. 107 Cortex 3
rd week (sample II) ............................................................................................ 109
Cortex 4th
week (sample I) ............................................................................................. 111
-
Cortex 4th
week (sample II) ............................................................................................ 113
Appendix 2 : Mapped areas in the endosteal areas ................................................................ 115 2
nd week (sample I), general overview ........................................................................... 115
2nd
week (sample I) Area E. ........................................................................................... 118
2nd
week (sample II), general overview of the mapped areas ........................................ 121 2
nd week (sample II) Area B. .......................................................................................... 122
2nd
week (sample II) Area C ........................................................................................... 124 2
nd week (sample II) Area D .......................................................................................... 126
3rd
week (sample II), general overview of the mapped areas ......................................... 129
3rd
week (sample II) Area E ........................................................................................... 136 4
th week (sample I), general overview of the mapped areas .......................................... 137
4th
week (sample I) Area B ............................................................................................. 138 4
th week (sample I) Area C ............................................................................................. 140
4th
week (sample I) Area D ............................................................................................ 142
6th
week (sample I) Area B ............................................................................................. 147 6
th week (sample I) Area B ............................................................................................. 148
6th
week (sample I) Area C ............................................................................................. 150 6
th week (sample I) Area D ............................................................................................ 152
List of figures ......................................................................................................................... 156
-
List of Tables
Table 1 Statistics of the b values calculated on different collagen based material ................. 60
Table 2 Description of the rat callus samples used in the present work and the areas where
Raman imaging and collagen orientation determination was performed. ................................ 67
Table 3 Numerical integration of the bands related to mineral content and organic matrix in
the areas 1 and 3 in the sample II of the 6 weeks healing ........................................................ 82
-
List of abbreviations
PRS Polarized Raman Spectroscopy
PDB Protein data bank
ROI Region of interest
PMMA Poly (methyl methacrylate)
SAXS: small angle X-ray scattering
NI nanoindentation
qBEI quantitative scanning electron imaging
SAM scanning acoustic microscope
SHG second harmonic generation
TPF two-photon fluorescence
BMUs basic multicellular units
MMPs matrix metaloproteinases
FTIR Fourier transform Infrared spectroscopy
-
Raman spectroscopy investigation of organic matrix orientation and chemistry changes during fracture healing.
Abstract
Fracture healing is a complex process that involves cellular and biochemical response to
reestablish the biomechanical properties and original geometry of tissues in damaged bone.
In order to stabilize and eventually close the fracture gap, a heterogeneous material called
callus is generated during healing. Collagen as the primary building material of the organic
matrix in callus is highly organized in a variety of meaningful ways. Despite its importance,
research has been mainly focused on the mineral phase and there is only a limited
understanding of how collagen fibrils orient themselves during fracture healing. One tool that
is ideally suited to studying fracture healing in the early stages is Polarized Raman
Spectroscopy (PRS) as chemical and orientation of the materials can be obtained with high
resolution (0.6-1m) in a non-destructive manner.
In the present work, a methodology to obtain the collagen fibril orientation in fracture callus
was developed. As a first step, a theoretical analysis of the Raman Amide I band intensity
response for collagen structures was performed following the classical Raman theory.
Collagen-like peptide structures obtained in the protein data bank (PDB) were rotated in
plane and out of plane to evaluate the Amide I response at different polarization angles of
the incident light. Collagen oriented in plane exhibit sinusoidal anisotropic response of the
Amide I band where the maximal intensity is obtained when the polarization angle of the
incident light is perpendicular to the molecule and minimal when parallel. Once the molecule
is rotated out of plane the response becomes less anisotropic and when the molecule is
positioned perpendicular to the plane of observation the response is isotropic.
The methodology proposed to evaluate the collagen orientation using PRS is based on the
fitting the anisotropic response of the Amide I band at different polarization angles of the
laser. The fitting procedure allows us to obtain parameters related to the orientation of the
collagen fibres and the degree of alignment. This procedure was tested in collagen-based
materials that are completely isotropic (gelatine) and highly anisotropic (human osteon).
Finally, the PRS was employed in embedded rat callus samples to evaluate the collagen
orientation and chemical evolution in the fracture callus. The fracture callus samples were
harvested at the time points of 2, 3, 4 and 6 week of the healing (two samples per time point).
Selected regions of interest (ROI) were chosen on the cortex close to the fracture gap
-
throughout the healing evolution, as well as areas of cartilage and mineralized tissue in the
endosteal area were also investigated. It has been possible to obtain Raman images of the
most relevant molecular groups on a rat callus sample embedded in PMMA. This has revealed
interesting histological features like chondrocytes structures in the cartilage, as well as
hydroxyapatite and collagen distribution in soft and hard tissues within the callus. The
orientation mapping of collagen fibres on mineralized tissue and cartilage in the endosteal
region has revealed a high level of organization at the early stages of fracture healing. Highly
oriented collagen fibers found around chondrocytes suggest a templating process of the
extracellular matrix in soft tissue. The cortex region close to the fracture gap shows the first
signs of the remodeling on the 3rd
week of the healing. At the 6th
week collagen fibres are
orienting in different directions than longitudinal to the cortex exhibiting already an active
remodeling process.
-
Ramanspektroskopische Untersuchung der Orientierung organischer Matrices und chemischer Vernderungen whrend der Knochenheilung
Zusammenfassung
Knochenheilung ist ein komplexer Prozess, bei dem zunchst ein Kallus aufgebaut wird, der
die Bruchstelle provisorisch verbindet, bevor in einem Umbauprozess der ursprngliche
Aufbau des Knochengewebes wieder hergestellt wird. In der vorliegenden Arbeit wurde
polarisierte Ramanspektroskopie (PRS) benutzt, um die Orientierung der Kollagenfasern im
Kallus zu bestimmen. In einem ersten Schritt wurde eine theoretische Bewertung der
Intensitt der Amid I Ramanbande fr Kollagenstrukturen mit Hilfe klassischer Ramantheorie
vorgenommen. Kollagenhnliche Peptidstrukturen aus der Proteindatenbank (PDB) wurden
benutzt, um den Einfluss unterschiedlicher Polarisationswinkel des einfallenden Lichts auf die
Amid I Bande zu berechnen. In dieser Arbeit wird eine neue Methode vorgeschlagen, um die
Orientierung des Kollagens mittels PRS zu ermitteln. Diese basiert auf einer numerischen
Analyse des anisotropen Signals der Amid I Bande fr unterschiedliche Polarisationswinkel
des Lasers. Dieses Verfahren wurde fr kollagenreiche Materialien benutzt, die sowohl
vollstndig isotrop (Gelatine) als auch hochgradig anisotrop (Osteon im menschlichen
Knochen) waren. Schlielich wurde die PRS benutzt, um die Kollagenorientierung und
chemische Vernderungen whrend des Heilungsverlaufs zu bestimmen. Die untersuchten
Proben, welche von einem zuvor an der Charit durchgefhrten Versuch mit Ratten stammen,
wurden nach Intervallen von 2,3,4 und 6 Wochen Heilung entnommen (2 Proben pro
Zeitpunkt). Die Abbildung der Orientierung von Kollagenfasern im mineralisierten Gewebe
und im Knorpel im endostealen Bereich lie einen hohen Grad an Ausrichtung whrend des
frhen Heilungsprozesses erkennen. Die Anwesenheit von hochgeordneten Kollagenfasern
um die Chondrozyten im Knorpelgewebe deutet auf einen Templatierungsprozess der
extrazellulren Matrix des Weichgewebes hin. Der Kortex in der Nhe des Bruchs zeigt die
ersten Anzeichen einer Umgestaltung in der dritten Woche des Heilungsprozesses. Ab der
sechsten Woche orientieren sich die Kollagenfasern neu und der kortikale Knochen weist
bereits Zeichen eines aktiven Umbauprozesses auf. Die neu entwickelte Methode kann daher
benutzt werden, um die verschiedenen Stadien des Knochenheilungsprozesses bezglich der
Struktur und der Zusammensetzung des Gewebes zu bewerten.
-
1
1. Introduction
Every year, millions of people fracture bones. In the United States of America alone, over 6
million patients look for medical care due to bone injury. In most cases fractures heal without
complications, but 5 to 10% require subsequent surgical treatment [1, 2]. In impaired or
delayed bone healing, reparative tissues may not bind strongly enough to the existing bone or
the united ends of the bone may be misaligned. In extreme situations a fracture may fail to
heal, which leads to the so-called non-union [3]. For this reason, it is of great importance to
understand the genetic, cellular and biochemical mechanisms of normal and impaired healing
in order to develop effective treatments [4, 5]. During the fracture healing a complex cellular
and biochemical cascade is initiated to re-establish the original bone structure at all levels of
hierarchy. The implementation of scanning techniques that are sensitive to the nano and
micron scale are important, as they can provide tissue structural information during the
various stages of fracture healing. Such understanding of native material formation provides
the basis for stimulated healing in regeneration.
One interesting feature of bone is its ability to repair itself that allows full mechanical
competence after fracture [6, 7]. This process can occur in two ways: primary bone repair,
that takes place without callus formation, and endochondral ossification that is mediated by a
callus tissue formation. The distribution and type of osteogenesis during fracture healing
depends on mechanical stability as well as biochemical signaling and cellular composition on
the fracture gap [8]. Most clinically used fracture stabilization methods lead to moderate
interfragmentary movements of the fracture gap that initiate endochondral ossification. This
type of ossification is conventionally divided into four overlapping stages: inflammation, soft
callus formation, hard callus formation and bone remodeling. The inflammation stage is
characterized by hematoma formation due to the disruption of soft tissue and interruption of
vascular function. In response to the mechanical instability of the fracture gap, cartilaginous
tissue (soft callus) is formed by chondrocytes and fibroblasts generate fibrous tissue in
regions where the cartilage is deficient. Chondrocytes then become hypertrophic and
mineralize the extracellular matrix before undergoing apoptosis. Subsequently, the soft callus
is replaced by woven bone (hard callus) and at the final stage the callus is remodeled into
cortical bone, without leaving scarring tissue at the phase when trabecular bone is formed. A
more detailed overview of bone healing biology will be given in section 3.2.
-
2
Introduction
From the material science point of view, mineralized callus tissue, newly formed bone and
soft tissues experience continuous changes in their structural and mechanical properties.
Recently, the mechanical properties, chemistry and organization of tissues in fracture callus
have been studied with different techniques: small angle X-ray scattering (SAXS), which was
used to obtain maps of the orientation and morphology evolution of the mineral crystallites in
sheep callus; nanoindentation (NI) was used to measure indentation modulus of callus sheep
with less than one micrometer spatial resolution by monitoring the mechanical interaction
between an indenter and the surface layer of the material; quantitative scanning electron
imaging (qBEI) to measure the bone mineral density distribution (BMDD) that reveals
differences in the degree of mineralization and finally by scanning acoustic microscope
(SAM), that enabled the mapping of elastic stiffness coefficient of the mineralized tissue [7-
9].
Despite the importance of the organic extracellular matrix in bone mechanical competence,
these aforementioned studies on the fracture callus have focused mainly on the mineral phase.
There are several techniques available to perform studies in the organic matrix of the callus
such as histology /histomorphometric analysis. These are standard tools to evaluate
quantitatively the distribution of the different tissues during fracture healing [10-12].
Multiphoton techniques like second harmonic generation (SHG) and two-photon fluorescence
(TPF) have also been found to be suitable for detection of collagen orientation in diseased
bone and articular cartilage [13, 14]. On the other hand, spectroscopic techniques such as
infrared (IR) and Polarized Raman spectroscopy (PRS) can provide chemical information and
are sensitive to the orientation of the molecules in materials. PRS in particular has been
employed in the study of biological materials to determine cellulose fibrils orientation [15],
protein organization from spider silk [16-18], anisotropic response of Raman bands in
collagen bundles, hydroxyapatite crystallite orientation in human enamel [19] and spatial
changes in composition and orientation in cortical bone.
In the present work PRS was used to study callus fracture because it provides simultaneous
information on mineral and organic content and allows high resolution mapping (0.6-1m) in
a non-destructive manner [20, 21]. Moreover, the orientation of the molecules can be
determined by measuring the anisotropic response of the appropriate Raman bands at different
polarization angles of the light source [22, 23]. To do so, a methodology to obtain chemical
-
3
Introduction
information and orientation maps of the organic matrix of rat callus at the micron scale was
developed. This orientation Raman mapping is a non-standard procedure that required the
adaption of appropriate image and spectral analysis tools. Firstly, theoretical evaluation of the
anisotropic response of the Amide I Raman band of five collagen like-peptide was performed
by using the classical Raman theory. These results are shown in section 4. Secondly, the
methodology was tested by mapping the orientation of standard collagen-based materials.
This included gelatin (isotropic), stretched rat-tail tendon (anisotropic with highly oriented
collagen fibers) and a secondary human osteon. The resulting maps are shown in section 7.
Thirdly, PRS was used in the study of structural and chemical changes during fracture healing
in a rat callus. Raman images of organic/ mineralized phases, as well as the embedding media
of the regions of interest (ROI) and orientation maps of the organic matrix at different time
points (2, 3, 4 and 6 weeks) were obtained. These results are shows in the section 8 and 9.
Finally, this thesis intends to highlight the utility of PRS as a valid and useful method in the
study of orientation and chemical distributions in complex hierarchical tissues to understand
the relation structure/function in natural materials ranging from soft to hard native tissues.
-
5
2. Review of the literature
2.1 Bone as a living material
Bone is a dense and specialized form of vascularised connective tissue consisting of cells and
mineralized extracellular matrix, essential for body support, protection of vital organs such as
the brain and spinal cord and as site of attachment for ligaments and muscles [24, 25]. The
microstructure of cortical bone is represented in Figure 1
Figure 1 Microstructures present in cortical bone: Haversian canals that contain blood vessels, and
nerves, Volkmanns canals (transverse canals connecting Haversian canals) .Surrounding the Haversian
canals are the osteons that consist of concentric lamellae of bone tissue with a diameter from 100 to 300
m. Osteocytes reside in holes filled with extracellular fluid called lacunae within the bone matrix.
Lacunae are interconnected by channels running the through the calcified bone matrix called canaliculi.
(SEER training modules, structure of bone tissue. U.S. National Institute of Health, National Cancer
Institute .http://training.seer.cancer.gov/.)
Bones can grow, modify their shape through external remodelling or modelling and
continuously renew themselves [26]. Remodelling allows during the adulthood the repair of
damage (micro-cracks), the storage and release of calcium and phosphorus, growth factors,
cytokines and the maintenance of the acid-base balance [27]. The cells responsible for the
remodelling process of bone are: the osteocytes which are thought to act as mechanosensors
and initiate the remodelling, the osteoclasts, responsible for the disintegration of old bone
matrix and the osteoblasts, which produce new bone [26, 28-30]. This mechanism allows a
continuous turn over and the replacement of the matrix in bone [25].The remodelling process
-
6
Review of the literature
is not performed individually by each type of cells, but by a group of cells functioning like
organized unit named basic multicellular unit (BMUs) [31]. They operate on bone
periosteum, endosteum, trabecular surfaces and cortical bone, replacing the old bone by new
bone in discrete packets [26]. Some osteoblasts remain free at the surface while others
become embedded in the matrix they have secreted. The newly formed material, composed
mainly of collagen type I, is called osteoid. This structure is rapidly mineralized by the
deposition of hydroxyapatite crystals. Once the osteoblasts are imprisoned in the hard matrix,
the cell differentiates to become an osteocyte and is unable to divide itself further [25]. The
osteocytes occupies a small cavity or lacuna in the matrix and tiny channels or canaliculi
radiate from each lacuna, enabling the formation of gap junctions with the adjacent osteocytes
[25, 26] . Other types of cells present in bone are bone lining cells which are inactivated
osteoblasts (not buried in the new bone) located on the surface of newly formed tissue when
bone formation stops. They can be reactivated in response to chemical and/or mechanical
stimuli [26].
2.1.1 Structural organization: Mineral and organic matrix
Bone is a hierarchically structured material (see Figure 2) in which both high strength and
fracture toughness can be achieved by the unique architecture of its organic-inorganic phases
[26, 32-36]. Bone as a collagen-mineral composite contains nanosized mineral platelets
(essentially carbonated hydroxyapatite), protein (mainly collagen type I) and water [29, 32].
The organic matrix of bone consists of collagen and other non-collagenous proteins and
lipids. Around 85-90 % of the total protein content is fibrous collagen.
Figure 2 Hierarchical structure of a human femur showing the building blocks on the different size scale.
a) Human femur b) Osteon structure (contained in the red ellipse) c) Lamella d) Collagen fibre bundle e)
Mineralized collagen fibril f) Collagen molecules and mineral particles. Reprinted with permission of
ref.47, 2007 Elvesier.
At the organ scale, bone can adopt different shapes depending on function, long bones like the
femur or tibia provide stability against bending and local buckling by being usually thick-
-
7
Review of the literature
walled, hollowed tubes, and expanded at the end, whereas in short compact bones like
vertebrae the applied load is mainly compressive [37] [32].
Each bone is composed of a varying amount of trabecular bone (cancellous bone) and cortical
bone depending on its function [27]. The walls of tube-like long bones and the walls
surrounding the trabecular bone regions are made up of cortical bone and can be several
tenths of millimetres thick in vertebra or several millimetres or centimetres in long bones [32].
Cortical bone is covered with a fibrous envelope on its outer surface called the periosteum.
Cavities and canals within the cortical bone are covered with an intracortical envelope
(Haversian envelope). On the inner surface of the cortical bone the fibrous tissue facing the
bone marrow is called the endosteum [27]. Cortical bone is arranged in Haversian systems
(also called osteons): a simple osteons are 100 to 300 m in diameter and consist of
concentric layers or lamellae of bone tissue surrounding the central canal (Haversian canal)
[26, 27]. It has been proposed that lamellar bone corresponds to a rotated plywood structure,
where the fibres are found within a thin sub layer rotating around an axis perpendicular to the
layer [32]. Trabecular bone is found in the end of the long bones, in vertebrae and in flat
bones [26, 32]. It has an open sponge-like structure, made up of small structures called
trabeculae, which are generally classified as rods and plates. Trabeculae are usually less than
200 m thick and 1000 m and are themselves composed by BSUs (basic structural unit),
which are lamellar and about 50 m thick and 1000 m long [27].
Mineralized collagen fibrils of about 100 nm in diameter are the basic building blocks of the
bone material [29]. The fibrils are assemblies of collagen around 300nm long and 1.5 nm
thick, which are deposited by osteoblasts (see Figure 3) [32, 38]. Molecules adjacent in the
fibrils are staggered along the axial direction by D 67 nm, generating a characteristic pattern
of gap zones with 35nm length and 32nm long overlap zones [32, 33, 39]. The collagen fibrils
are filled and coated by tiny mineral crystals [32]. These crystals are mainly flat plates
arranged in a parallel fashion to each other and to the long axis of the collagen fibrils [40-42].
The crystals occur at regular intervals along the fibrils, with an approximate repeat distance of
67nm, which corresponds to the distance by which adjacent collagen molecules are staggered.
After nucleation, the crystals usually grow in plate-like manner [32]. In different mammalian
and non-mammalian species, bone mineral crystals have a thickness of 1.5-4.5 nm [43-45].
The simple hydroxyapatite Ca5 (PO4)3 OH often contains other chemical elements that can
replace calcium, phosphate or hydroxyl groups, the most common being the replacement of
-
8
Review of the literature
the phosphate group by a carbonate group [29, 32]. Subsequently, the mineralized collagen
fibrils then organize themselves into bundles that lead to the formation of fibres that are
further packed in regularly dense and ordered networks [29, 46].
Figure 3 Diagram of a mineralized collagen fibril showing the arrangement of the mineral crystals parallel
to each other and parallel to the collagen fibrils in a staggered arrangement. Adapted from [41]
Bones as a natural composite combine the strength and stiffness of the mineral particles with
toughness and viscoelastic properties of the organic components [39]. The organization of the
mineral platelets and extracellular matrix are responsible for mechanical competence of the
bone tissue [35]. The inorganic components are responsible mainly for the compressive
strength and stiffness, while organic components provide toughness [26, 35]. The toughness
of a material is typically determine by the ability of it microstructure to dissipate deformation
energy without propagation of a crack [47]. Theoretical analysis of the mineralized collagen
fibrils have also shown that arrangement/dimensions of the mineral particles and the thickness
of the organic layers play a crucial role in the bone mechanical properties, in this regard the
organic layer must be thin to ensure the stiffness of the tissue and the mineral particles must
be thin to reduce the brittleness of the tissue [36, 48].
-
9
Review of the literature
2.1.2 Extracellular matrix
A substantial part of organ volume is the extracellular space that is composed of an intricate
network of macromolecules (collagens, fibronectin, laminins and tenascins), soluble proteins,
and polysaccharides (glycosaminoglycans and proteoglycan) that are secreted locally and
assembled into an organized network in close association with the surface of the cell that
produce them [49-51]. Variations in the relative amount of the different type of matrix
macromolecules as well as their organisation leads to a great diversity of forms, each adapted
to the functional requirements of a particular tissue, for example the matrix can become
mineralized to form hard structures in bone or teeth, or form a transparent matrix in the cornea
[25]. The extracellular matrix is not only important to stabilize the physical structure of
tissues; it also plays a complex role in regulating cellular behaviour by influencing their
survival, development, proliferation, shape and function [52]. The extracellular matrix is
mainly produced locally and oriented by the cells in the matrix [25].
2.1.3 Collagen
The family of collagens comprises several fibrous proteins that are found in all multicellular
animals [25]. They are secreted by connective tissue cells, as well as by a variety of other cell
types. Collagens are the major structural element of all connective tissues, contributing to the
stability of tissues and organs and maintain their structural integrity [25, 53-55]. Additionally
to their structural aspects, collagens are involved in different functions such as local storage
and delivery of growth factors and cytokines, being therefore fundamental for organ
development, wound healing and tissue repair [53]. Initially collagens were defined as
proteins of the extracellular matrix that contain large domains of repeating Gly-X-Y
sequences (X corresponds frequently to Proline (Pro) and Y to 4-Hydroxyproline (Hyp)),
which have a triple helical structure but genomic DNA libraries and screening of cDNA
techniques have revealed a large number of related proteins with a varying length of Gly-X-Y
sequences, even in some cases with a globular structure [59-61].
Based on the number/position of the triple helical regions and on its supramolecular
organization, collagens can be classified into classes of fibril forming collagens (FACIT),
fibril associated collagens, network forming collagens, anchoring fibrils, transmembrane
collagens and others with unique functions [53, 56]. Members of this family of molecules
-
10
Review of the literature
contain at least one triple-helical domain, are located in the extracellular matrix and are able
to produce supramolecular matrix assemblies [57]. The triple helical domain is composed of
three polypeptide chains (-chains) that are each coiled into a left-handed helix. Each
polypeptide chain of collagen consist approximately of 300 units of Gly-X-Y [58]. The three
chains are then wrapped around each other into a right handed super-helix so that the final
structure is a rope -like rod [59]. So far, 25 distinct types of chains have been identified,
each encoded by different genes [25]. In theory more than 10000 types of triple stranded
collagen molecules could be assembled with the combination of several of these chains but
only 26 types of collagen molecules have been found until today [25].
The rod-like triple helical structure is important for fibril formation and structural integrity
[60]. Collagen molecules require the presence of Glycine (Gly) every third residue because a
larger amino acid will not fit in the centre of the triple helix where the three chains come
together. The Proline (Pro) and particularly the 4-Hydroxyproline (Hyp) imino acids limit the
rotation of the polypeptide chains, and 4-hydroxyproline plays an essential role in the
formation of intramolecular hydrogen bonds that contribute to the stability of the triple helical
conformation [53, 59]. Melting studies of two triple helical synthetic peptides (Pro-Pro-Gly)
and (Pro-Hyp-Gly) demonstrated a distinct disparity in the melting temperature [61]. All the
residues in the X and Y positions are highly exposed to solvent, making the triple helical
structure suitable to interact with other molecules, as well as to self-association [59, 60]. The
geometrical features of a triple helix do not allow intrachain hydrogen bonds. A direct
N-H (Gly)O=C (X position) interchain hydrogen bond can exist for all the sequences and
has been confirmed by X-ray crystal structure determination of model peptides [59, 60].
-
11
Review of the literature
Figure 4 A) Image of a collagen fragment with some of the iminoacid positions (Gly),(Pro),(Hyp) and (Ala)
on one of the three molecule chains (adapted from the protein data base PDB) B) Sketch of the protein
backbone and the peptide unit where C correspond to carbon atoms, N nitrogen , C carbons , O oxygen
and R the iminoacids attached.
2.1.3.1 Fibril-forming collagens
Collagens type I, II, III, V and XI are called fibril-forming collagens and they can assemble
into highly oriented supramolecular aggregates, the typical quarter staggered fibril array with
diameters between 25 and 400 nm [55, 57, 59]. These fibril-forming collagens are the main
component of skin and bone and are by far the most common type [25]. The abundant fibril
forming collagens have the strictest requirement for an interrupted pattern: each polypeptide
chain contains a contiguous stretch of 1000 residues with a Gly-X-Y repeating sequence [56].
Such regularity may be critical for the formation of quarter staggered fibrils with a 67 nm
axial repeating period (D- period) that is characteristic for the assemblies of these collagens
[56]. In contrast, non-fibrillar collagens typically contain one or more breaks in the Gly-X-Y
repeating pattern under normal and non-pathological conditions [56].
The fibrillar collagens comprise the following members: Collagen type I is the most
significant and abundant fibrillar collagen. Monomeric type I collagen consist of two 1
chains and one 2 chain [62]. In most organs, tendons and facia, type I collagen provides
tensile stiffness and defines considerable biomechanical properties in bone [53].
Collagen type II is predominant in hyaline cartilage but is not restricted only to cartilage, the
triple helix of the collagen type II is composed of three 1(II) chains forming homotrimeric
molecule similar in size and biomechanical properties to that of collagen type I [53].
A B
-
12
Review of the literature
Compared to collagen type I, collagen type II contains more hydroxylysine as well as glucosyl
and galactosyl residues that mediate the interaction with proteoglycans [53].
Collagen type III is a homotrimer of three 1 (III) chains and is widely distributed in collagen
I containing tissues with the exception of bone. This type of collagen is similar to collagen
type I in its ability to form 67 nm axially periodic fibrils in blood vessels, skin and other
tissues that require extensibility [53].
Collagen type V and XI are formed as heterotrimers of the three different chains (1, 2,
3). The 3-chain of the type XI collagen is encoded by the same gene as the 1 chain of type
II collagen and only the extent of glycosylation differs from 1 (II) [53].
On the other hand, collagens types IX and XII are called collagen fibril associated because
they are found on the surface of the collagens fibrils and are thought to link these fibrils to
one another and to other components of the extracellular matrix. The structures of these
collagens are characterized by collagenous domains interrupted by short non-helical
domains [53].
2.1.3.2 Collagen assemblies
The fibril forming collagens I, II, III, V, and XI spontaneously aggregate after processing
procollagens into ordered fibrillar structures in vitro, comparable to crystallization processes
with initial nucleation and subsequently organized aggregation [53]. Hydrophobic and
electrostatic interactions of the collagen monomer are involved in the quarter-staggered
arrangement of collagen, which in turn can aggregate into five stranded fibrils and
subsequently in larger fibrils [53]. These fibrils can be more or less oriented depending on the
type of tissue. Collagen type I fibrils present in tendons, for example, are parallel to each
other and form bundles or fibres, whereas in skin they are randomly oriented in a complex
network of interlace fibrils [53].
2.2 Fracture healing and callus formation
Bone regeneration is an important function of living organisms as it provides reparative power
to the vertebrate organisms, including the ability of unite fractured bones and refill defects
[63]. The fracture healing process recapitulates certain aspects of skeletal development and is
governed by a variety of cellular elements and signalling molecules [6, 64]. The regeneration
process of bone healing is initiated in response to injury and must re-establish the original
-
13
Review of the literature
geometry, the biomechanical competence, and function of the damaged structure [65]. The
repair of the bone tissue involves the same set of structural proteins found in normal bone
development: collagen type I, collagen type II and X and also many other regulators of
chemotaxis, mitosis and differentiation [65-67].
Bone repair can occur by different specific mechanisms that depend on the biophysical and
mechanical environment. Bone synthesis is always mediated by mesenchymal stem cells and/
or surface osteoblasts to produce bone in woven and lamellar matrix conformation [65]. These
mesenchymal stem cells are thought to have different origins: in the inner osteogenic layer of
periosteum, [68], the perivascular mesenchymal [69], the endosteal cells (endosteum), the
undifferentiated mesenchymal cells of the bone marrow and the soft tissue. Studies have
shown that a number of different pre-myogenic cell lines can be induced to differentiate into
chondrogenic or osteogenic cells when treated with bone morphogenetics proteins (BMPs)
[70].
During fracture repair the following bone formation mechanisms can be observed [65]:
Primary bone repair (direct bone repair): mediated exclusively by intraosseous
Harvesian system osteoblasts and osteoclasts without undergoing the cartilage phase.
In primary bone repair osteoclasts first absorb necrotic bone on either sides of the
fracture and are followed by osteoblast intervention .The mechanical stability of the
fracture that leads to a primary bone repair are extremely rigid fixation and a gap size
< 500 m [71].
Secondary bone healing (Endochondral repair): mediated by inner periosteal layer
and marrow tissues, synthesizing cartilage and the woven bone in the environment of
relative micro-motion between the fragments. Most of the stabilization methods used
in the clinical treatment allow the inter-fragmentary movement that leads to secondary
bone healing. This repair process will now be described in detail due to its importance
to the understanding of the present thesis.
2.2.1 Secondary bone healing
As previously mentioned, secondary bone healing is conventionally divided into four
somewhat overlapping stages namely inflammation, soft callus formation, hard callus
formation and remodelling [2, 6]. The location of important areas on callus and that will be
-
14
Review of the literature
referred during this section are shown in Figure 5 and the illustration of the complete healing
process is shown in Figure 6.
Figure 5 Histological section of rat callus stained with methyl green-Van Gieson where the blue areas
correspond to cartilage and deep red to mineralized tissues. The area A correspond to cortical bone close
to fracture gap, area B is mineralized callus in the endosteal region and area C is mineralized callus on the
periosteal area
Stage 1- Inflammation: This stage is associated with the disruption of local soft tissue and
interruption of normal vascular function, as well as distortion of marrow architecture. The
bleeding into the fracture site is contained by the surrounding tissue and develops into a
hematoma, which presents an essential role in regeneration due to its angiogenic and
osteogenic potential [64]. From an immunological point of view the fracture hematoma
represent the starting point of the inflammatory process, thus initiating the healing cascade
[72]. Hematoma formation signals the body to send macrophages to the wounded area to
breakdown and absorb necrotic tissues [63]. Degranulating platelets, macrophages and other
inflammatory cells infiltrate the hematoma between the fracture fragments, which leads to the
formation of a fibrous thrombus. Over time capillaries grow into the clot that is then
converted into granulation tissue. Also, secretion of cytokines and growth factors by the
inflammatory cells induce migration of multipotent mesenchymal stem cells in the
granulation tissue. Some studies indicate that these undifferentiated cells may have originated
from injured connective tissue or adjacent muscle, tendon and fascia [6, 65].
-
15
Review of the literature
Figure 6 Endochondral osssification phases in the fracture healing a) Inflammation: the disruption of soft
tissues and bleeding generates an hematoma.This phase is characterized by an acute inflamatory response
b) Soft callusformation:Cartilagenous tissue is generated to stabilized the fracture fragments. c) Hard
callus formation: In this phase the chondrocytes mineralize the cartilagenous matrix characterized by the
formation of temporaty woven bone d) Bone remodelling:the irregular woven bone is replaced by lamellar
bone and the standart cortical structure restored. The boxes in the figure correspond to areas of
investigation in the literature and should be ignored. Adapted with permission of ref [73], 2001 Wiley.
Stage 2- Soft callus (fibrocartilage) formation: The relative movement of the environment
closer to the fracture gap and the relative hypoxia lead to the formation of a large
cartilaginous mass not only outside the cortices but also within the cortices [65]. During this
stage chondrocytes and fibroblasts are predominant. The cartilage is mainly avascular,
although the subsequent replacement with woven bone involves vascular invasion [6].
Chondrocytes derived from mesenchymal stem cell progenitors proliferate and synthesize
cartilaginous matrix until all fibrous tissue is replaced by cartilage [6]. Chondrocytes secrete
mainly collagen type II, unlike hypertrophic chondrocytes which are collagen type X
producers [74]. On the areas where cartilage production is deficient fibroblasts synthesize
fibrous tissue. In the final stages of soft callus production all chondrocytes become
hypertrophic and mineralize the cartilage before undergoing apoptosis [75].
Stage 3- Hard callus formation: In this phase the unorganized bone is replaced by a more
organized structure that signals the complete restoration of damaged bone [68]. It is
characterized by a high level of osteoblast activity and the formation of mineralized bone
matrix, which arises directly from the peripheral callus [6]. This woven bone matrix contains
-
16
Review of the literature
a combination of proteinaceous and mineralized extracellular matrix synthesized by mature
osteoblasts, which differentiate from osteoprogenitors in the presence of osteogenic factors
[6].
Stage 4- Bone remodelling: Bone remodelling is the final stage of fracture healing, when the
hard callus is remodelled into mature cortical and/or trabecular bone. The remodelling process
is driven by orderly bone resorption, which is followed by formation of lamellar bone [6]. The
cells involved in the resorption of mineralized tissue are osteoclasts, multinucleated cells
formed by the differentiation and fusion of haematopoietic precursors. To remodel, the
osteoclasts polarize and adhere to the mineralized surface and form a tight seal with the bone
matrix and create an acid microenvironment containing proteolytic enzymes. The acidic
environment demineralizes the matrix, while proteinases degrade organic components such as
collagen. Once this process is completed, the osteoblasts are able to lay down new bone on
the eroded surface [42]. A group of proteases known as matrix metaloproteinases (MMPs)
like collagenases, gelatinases and stromelysins that are responsible for extracellular matrix
degradation. The MPPs have been also implicated in driving angiogenesis during tumor
invasion and bone development [6, 68, 76]. Resorption of woven bone releases non
collagenous proteins such as BMP and transforming growth factor (TGF-) which are thought
to stimulate osteoblastic activity [66, 68]. The previously mentioned BMPs are signalling
molecules that regulate a myriad of cellular process including differentiation, proliferation
and growth. The TGF- proteins appear to control growth and differentiation during
development and injury repair in humans and other species. In addition to their importance in
recruiting cell function, TGF- proteins are also potent promoters of collagen production,
without which hydroxapatite production and therefore ossification cannot occur [66].
2.2.2 Overview of structural studies on fracture callus.
In this section a brief overview of studies performed on fracture callus is given, starting from
a macroscopic scale and going down to more microscopic followed by computer simulation.
This is intended to reveal the current status of the structural and chemical callus research and
to help place the results of the present work into the context of the mechanical properties and
structural information provided by previous studies.
One fundamental point in the experimental research of the fracture healing is the selection of
the appropriate animal model. In the study of healing under different mechanical conditions
-
17
Review of the literature
small animal (mice and rats) and large animal models (sheep) are commonly used [77, 78],
although distinct dynamics of the healing are observed between the models [79]. In the field
of tissue engineering large animal models are preferred because the bone biology, mineral
composition, turnover and the mechanical loading environment are similar to human
conditions [78]. One way to experimentally generate a fracture healing response is a surgical
cut through the bone called osteotomy after which the bone fragments are stabilized by
different type of fixation devices that allow to control the stability, interfragmentary strain and
size of the bone gap to be [71].
Excellent reviews are available in the study of non-pathological conditions of fracture healing
that comprises the role of extracellular matrix [76], cellular and molecular mechanisms [6, 65,
80-82], biochemical signalling [66] and biomechanical forces [83]. At the macroscopic scale,
the most common approach to study fracture healing is the combination of histology,
radiology and mechanical testing (torsional strength and stiffness) to evaluate the course of
the healing influenced by the mechanical stimulation [11, 84, 85]. In the last decades,
different studies at the micron and nano scale have been performed: Morgan et al. [86]
studied mice calluss by micro-computed tomography (CT) at different time points and
different conditions that alter the normal fracture healing. The outcome values from (CT)
like total callus volume (TV), mineralized callus volume (BV), callus mineralized volume
fraction (BV/TV), bone mineral content (BMC), tissue mineral density (TMD), standard
deviation of the mineral density (TMD) were evaluated statistically with data obtained from
torsional experiments to elucidate which are strongest predictors of the mechanical properties
in callus. TMD, BMC, BV/TV and TMD explained 70% of the variation in torsional strength.
Leong et al.[87] measured the indentation modulus of different tissue types using one rat
callus fracture. The indentation modulus was found to be an indication of the mineral content
and increased along the periosteal aspect with distance from the fracture gap. Manjubala et al.
[8] used a sheep osteotomy model in combination with nanoindentation (NI) and quantitative
backscattered electron imaging (qBEI) to measure indentation modulus and mineral content
respectively. It was found that the indentation modulus in the mineralized tissue in the
periosteal region is far lower that of the cortical bone, even at the final stage of the healing,
although the mineral content in both tissues is similar. As a general trend on the periosteal
region, the initially quite heterogeneous primary woven bone becomes more homogeneous,
stiff and mineralized with the fracture healing evolution. Liu et al.[88] used small angle X ray
scattering (SAXS) and environmental scanning electron microscopy to map the distribution of
-
18
Review of the literature
mineral particle size and orientation in different areas in sheep callus. It was shown that the
mineral particles present in the periosteal callus increased in their mean thickness with the
healing progression but did not change their degree of alignment. Moreover, the mean
thickness of the mineral particles in the cortex close to the osteotomy gap was smaller and
less aligned than the deep cortex at the late stage of the healing. Based on the distribution on
the mineral thickness and the degree of alignment at the different time points, a two wave
bone formation theory was proposed where a woven deposition is followed by a lamellar bone
apposition. In the first wave, unorganized mineralized tissue is first formed to stabilize the
fracture followed by a second wave of bony tissue with lamellar-like structure, having more
aligned mineral particles.
Preiniger et al. [89] have studied sheep callus by SAM obtaining maps of the elastic stiffness
coefficient and porosity in the cortical bone close to the osteotomy gap and in the mineralized
callus. This study shows a decrease in the cortical tissue stiffness and an increase in the
porosity in regions adjacent to the osteotomy gap. In contrast there is an increase in the
periosteal mineralized callus stiffness. The remodelling process indicates that the change in
material properties of the tissues in the healing area lead to a homogenization to minimize
strains in the boundaries between the tissues.
Finally, a finite element calculation of the fracture healing was performed by Vetter et al. [90,
91]. In this study the influence of the mineral heterogeneity on the strains within fracture
callus was evaluated (using the experimental values of the elastic modulus of newly formed
bone during the healing) and quantification of spatial tissue pattern performed. The result of
this simulation showed an overall increase in the elastic modulus of the mineralized tissue in
callus over time (mean value), simultaneously with a reduction of the stiffness of the cortical
bone close to the cortex due to resorption process that takes place in this area. Additionally,
was proposed by the same author a six stages fracture healing model, by generating average
images of each stage. This was intended to classify the fracture stages by the topology
observed in the tissue distribution and not for time point of the healing where the single
sample only reflects the healing situation of a particular animal.
This literature revision shows that the mechanical properties of the fracture callus have been
studied at the micro scale by different techniques. The structural and chemical information
that explain the evolution of the mechanical properties during healing in fracture callus has
been obtained mainly from the mineral phase leaving aside valuable information of the
-
19
Review of the literature
organic component. Therefore, one of the aims of this work is to obtain chemical and
structural information mainly from the organic phase by using PRS.
2.3 Raman Scattering
In the present subsection, an overview on the physical principles of Raman scattering,
considerations about the Raman tensor and the classical Raman theory will be provided.
These concepts are used to evaluate the anisotropic response a collagen-like peptide and the
organic matrix orientation in collagenous materials.
Raman Scattering is the inelastic energy scattering of light interacting with molecules [21]. In
this process, the electric field of the light induces a dipole moment in the molecule due to its
polarizability [21, 22]. The oscillation of the electric field of the incident light induces
oscillation of the dipole moment that can absorb or emit energy by transitions between
different oscillation energy levels (the energy absorption process is used in Infrared (IR)
spectroscopy). The molecules that are excited to a higher energy level return to the ground
state by the emission of scattered light in elastic process called Rayleigh scattering. However,
the scattering process may involve the transition of the excited molecules to different energy
levels from their ground state. The scattering associated with this inelastic energy transitions
is called Raman scattering and can be divided in Stokes Raman scattering that involves
vibrational energy transitions of the molecule to a higher energy level and anti-Stokes Raman
scattering that involves energy transitions to lower energy levels [21]. These different
transition processes can be observed in Figure 7.
-
20
Review of the literature
Figure 7 Diagram of the energy transitions in Raman scattering. In the Rayleigh transition, a photon
interacts with the molecule polarizing its electron cloud, raising the molecule from ground state to a
virtual level and return releasing a photon with the same energy as the initial one. The Stokes Raman
scattering is a transition of the molecule from the ground state to a virtual level and return releasing a
photon with less energy than then initial exciting photon .In the Anti-Stokes transition the molecule is
promoted from an excited vibrational state to the virtual level and return to the ground state, emitting a
photon with more energy than the initial one.
In normal Raman scattering by molecules in isotropic media, the dipoles are simply those that
result from the action of the electric field component E of the incident light on the molecules
expressed in the following equation.
E.
Equation 1
Where is the molecular polarizability, and is the induced dipole moment.
The electric field is an oscillating function that depends on the frequency of the light o
according to
)2cos( tEE oo
Equation 2
Where Eo is the impinging electric field, and t is the time.
The substitution of Equation 2 on Equation 1 gives:
)2cos(. tE oo
Equation 3
The polarizability depends on the motion of the nuclei in the molecule. The motion of
nuclei can be expressed in terms of the normal coordinate of the vibration q. The dependence
of on q (the change in intermolecular separation with the vibration) can be approximated by
a series expansion to second-order [92].
-
21
Review of the literature
ji
jiqjqiji
k
k qk
qqqq
qq
k
,0
2
0
02
1
Equation 4
The displacement kq concerning the molecular vibration is considered to be very small
meaning higher orders in the expansion can be neglected (from the third term).
The normal mode is a time dependant vibration with a frequency vm. This can be expressed for
the k-th atom as
)2(0 tCosqq mkk
Equation 5
where qk0
is the equilibrium position
Combining equations 3, 4 and 5 gives
)2cos()2cos()2(
0
ttEqq
tCosE mookqk k
ooo
k
ttEqq
tCosE momookqk k
ooo
k
)(2cos)(2cos2
1)2(
0
Equation 6
The 0 term represent the light that has not been shifted in frequency (Rayleigh scattering). In
the second part the term (v0-vm) is related to the Stokes Raman scattering and the term
(v0+vm) to the anti-Stokes Raman scattering.
From Equation 6 the condition to produce Raman lines is shown by
0
0
kqkq
Equation 7
This equation states that the molecular polarizability of the vibrational mode should be neither
maximum nor minimum when the atoms are in the equilibrium position [21, 92].
2.3.1 Polarized Raman spectroscopy (PRS)
The vibrational spectroscopies (IR and PRS) are some of the few molecular level probes that
offer chemical information of the materials and are inherently sensitive to the orientation of
the molecules. PRS in particular can be used to determine the molecular orientation within the
sample by measuring the anisotropic Raman response of certain chemical bonds at different
the polarization of the incident radiation [93]. Moreover, when PRS is coupled with confocal
microscopy, spectroscopic maps of the samples at micron level can be obtained [21, 94, 95].
PRS has been extensively used in molecular orientation studies of polymers and organic films
-
22
Review of the literature
where the molecular orientation plays a critical role in the material properties [96-99]. The
orientation of the molecules is frequently described in terms of a statistical distribution of
specific sub-elements with respect to a macroscopic frame of reference. This orientation
distribution function (ODF) is written as series of expansion of mathematical functions, of
which pre-factors are the parameters to be experimentally determined [100]. For example,
PRS have been used in biological materials like silkworm and spider silk fibres to determine
the order parameters and the associated ODF of the amide I band mainly associated with the
C=O stretching vibration of the peptide group and clarify the organization of the -sheet and
crystallites that give rise to silks extraordinary mechanical properties [16-18].
2.3.2 Polarizability tensor and Raman tensor
The polarizability is the measure of the change in the electron cloud distribution of the
molecules in response to an applied electric field and can be described by a symmetric second
rank tensor. In general the induced dipole moment of a molecule can be expressed as:
E
].[
Equation 8
Where [] is the polarizability tensor and E
is the electric field of the incident beam. The
Raman tensor is considered as well as a second rank tensor because it is the differential of the
polarizability tensor. The Raman tensor is described as:
'''
'''
'''
'
zzzyzx
yzyyyx
xzxyxx
Equation 9
The form of the Raman tensor is unique to the symmetry of the molecular vibration [21]. A
Raman tensor with spherical symmetry has only two parameters, whereas for no specific
symmetry six parameters are required. Since the principal axes of the Raman tensor do not
coincide necessarily with those of the molecular chain of the specimen, other coordinate
system x-y-z and X-Y-Z need to be introduced for the molecular chain coordinate system
and specimen coordinate respectively [21]. These coordinate systems are related to each other
by rotation through the Euler angles defined in Figure 8B.
-
23
Review of the literature
Figure 8 A) Ellipsoid representing the Raman tensor and B) Definition of the Euler angles(,,)
correlating Raman tensor axis (x,y,z) with the specimen axis (X,Y,Z)
The actual measurements to obtain molecular orientation distribution function are done based
on the specimen coordinate system and do not need absolute solutions of the tensor
components but only ratios of the diagonal components [21]. When the component 'ZZ is
much larger than the other components 'XX and '
YY the Raman tensor can be approximated
to have cylindrical symmetry [21].
2.3.3 Raman scattering intensity
The interaction between the incident radiation and the molecules depends on the angle
between the electric vector of the radiation and the shape of the polarizability tensor [21]. The
derivation of the equation correlating these parameters with the intensity of the Raman
scattering will be provided in this subsection. The oscillating electric field of the incident light
is able to induce a dipole moment in the molecules. These dipole moments in turn, induce and
scatter an electric field from the molecules. The induced electric field
scE by molecules at a
distance r from the molecule can be calculated by the Maxwell equation:
Sin
r
tvtE
sc
sc
)(4)(
22
Equation 10
where sc is the frequency of the scattering radiation, is the angle between the dipole axis
and the scattering radiation. The term )(t
is the induced dipole moment and is time
-
24
Review of the literature
dependant since the electric field vector, )(int tE
of the incident light changes with time [21]
according to:
)(.)( tEt in
Equation 11
The scattering intensity from the induce dipole moment, Isc is given by:
22
424422
2 SinErEI inscscsc
Equation 12
The Rayleigh scattering component of scI should be proportional to the value of
2 . On the
other hand the Stokes and anti-Stokes of the Raman scattering should be proportional to2
' .
Using the Raman tensor ' the scI for Stoke Raman scattering ( StokesscI ) over the solid angle
4 of can be written by [21]:
ij
ijminStockessc Ic
I2
4
042
57
)(3
2
Equation 13
where c is the speed of the light inI is the intensity of the incident light,'
ij is (i,j)-th
component of the Raman tensor, and the i,j are the directions of the scattered and incident
respectively. Based on the theory of electromagnetic waves [21].
2
8inin E
cI
Equation 14
This equation can be further simplified for a Raman scattering per solid angle sI and taking in
consideration the polarization direction of the scattered incident and scattered light,
2'
0 ' ggsllII
Equation 15
where 0I
summarises the constant terms in equation 6. The terms g and g are the polarised
direction of the incident beam and the analyser respectively, gl
and 'g
l
are the direction cosines
of the incident and the scattered beam along the g and g directions respectively. For the
molecules of all radiation volume in the sample this equation becomes [21, 96, 101]
2'
0 ' ggsllII
Equation 16
The last equation will be used in this work to evaluate the anisotropic response of the amide I
Raman band that related to proteins and peptides in biological materials.
-
25
Review of the literature
2.3.4 Orientation measurements and scattering geometry.
The most common optical geometries used in Raman scattering experiments are the right
angle scattering (RAS) and the back scattering geometry (BS) in consideration of the
geometrical angular dependence of the scattering intensity [21]. In Raman experiments the
description of the geometry is done by the so-called Portos notation A(BC)D where A gives
the direction of the incident beam, D the direction of propagation toward the detector, B the
polarization direction of the incident beam and C the polarization direction of the analyser. In
the BS geometry the same A and D axis should be chosen while in RAS A and D directions
are different. Defining the laboratory coordinate system as (XL, YL, ZL) as shown in Figure
9A, the Portos notation correspond to ZL (XL YL) ZL.
In the present work the direction of the incident beam and the direction of the scattered light
toward the detector are the same A=D, the polarization direction of the incident beam (laser)
B is changed during the orientation experiments but the scattered radiation is collected in all
the polarization directions.
Figure 9 A) Back-scattering geometry ZL (XL YL) ZL according to the Portos notation. B). Raman setup
to the estimation of the collagen fibre orientation in the present work. The laser excites the sample in the
direction Z at a particular polarization angle on the plane XY. The resulting Raman radiation is collected
again in the Z direction for all possible polarization directions of the Raman scattering light (no analyzer
is used in the optical pathway).
-
26
Review of the literature
2.4 Raman spectroscopy in bone
Vibrational spectroscopy has been used for several decades to characterize bony tissues.
Several reviews on the topic are available in the literature showing remarkable potential of
vibrational spectroscopy in both material and medical fields [102-104]. The earliest study
producing Raman spectra from intact mineralized tissues goes back to 1970 with the work
published by Walton et al [105]. The initial task was the assignment of the Raman bands in
bone to a particular chemical vibration of both the organic and inorganic components. This
was done primarily using already determined vibrations from infrared spectroscopy [103]. As
an example a Fourier transform Infrared spectroscopy (FTIR) spectrum of bone is shown in
Figure 10 A. The spectral assignment of bands the Amide I (1620-1680 cm -1
) and Amide II
(1520-1570 cm-1
) are vibrations from the carbonyl group and NH respectively in proteins
(mainly collagen). The mineral phase is mainly represented by the 1,3 phosphate group
contour (950-1200 cm -1
) and the 2 carbonate substitution in the mineral crystal (band centred
at~870 cm-1
)[106].
Characteristic Raman spectrum of bone (Figure 10B) shows the mineral associated bands
corresponding to 1 symmetric stretching vibration at 961cm-1
, and 2 and 4 phosphate
bending vibrations located at ~438 cm-1
and ~589 cm-1
respectively. The carbonate
substitution in bone shows a band at ~1075 cm-1
[107, 108]. The amide I band at~1666 cm-1
and Amide III~1243 and 1271 cm-1
in bone correspond mainly to the presence of collagen.
The Amide I region (~1620-1700 cm-1
) is mainly due to the C=O stretching while the Amide
III band (~1240-1320 cm-1
) arise from the combination of NH bending and CN stretching
of the peptide group [23].
Figure 10 A) Typical FTIR spectra of bone powder with the peaks related to the organic and inorganic
components of bone. The spectrum was taken from ref.[106] B) Raman spectra of cortical bone showing
the representative bands of the organic and mineral phases, the spectrum was taken in our laboratory.
Wavenumbers cm-1
-
27
Review of the literature
In 1995 Ohsaki et al. [109] used Raman spectroscopy to study the mineralization and
demineralization of a synthetic bone material that has been transplanted to the auditory ossicle
in rats. These process were followed by the changes in the mineral band 1 PO4-3
and showed
that bone destruction associated with cholesteatoma in the auditory ossicle is a form of de-
and remineralization. Chen et al. [110] have used Raman spectroscopy in combination with
other techniques to study the mineralization of collagen in a demineralized fish bone and
Wopenka et al. [111] have studied the gradual mineralization in the tendon to bone
connection of the rotator cuff. Apeldoorn et al. [112] have used confocal Raman spectroscopy
along with scanning electron microscopy revealing that the early in-vitro formed extracellular
matrix produced by rat osteoprogenitor cells resembles mature bone chemically. Tarnowsky
et al. [113] used Raman spectroscopy to characterize bone mineral development in mice
calvaria (from embryonic stages to six month age). This study suggested a different phosphate
environment throughout the tissue development, looking at the changes in the phosphate band
(1 PO4-3
.) The same study shows the presence of heterogeneous mineralized tissues in the
postnatal specimen suggesting the ionic incorporation and crystal perfection as the mouse
develops.
Raman spectroscopy has also been used to study bone quality that refers to the ensemble of
composition and architectural properties of bone that together determine its material
properties and its ability to perform its mechanical function [114]. Most of the experimental
Raman determinations of mineral crystallinity are based on the measure of the width of the
primary phosphate band near 959 cm-1
. This is because for example in synthetic carbonated
apatites, increased mineral crystallinity yields narrower phosphate bandwidths, which is
mirrored by reduced carbonate substitution [114]. In this regard Freeman et al. [115] showed
that the crystallinity of the hydroxyapatite, the type B carbonate substitution (substitution of
carbonate ions in phosphate positions) and the brittleness of the bone material increase with
age in mouse femora. This paper also showed spectral and mechanical properties changes
associated with the incorporation of fluoride into the mineral crystal (the 1 PO4-3
band is
shifted from 961 to 964 cm-1
, possibly due to the exchange of F- and OH
-1 in the lattice).
Akkus et al. [116] studied the age-related changes in the crystallinity (1 PO4-3
bandwith),
mineralization (relative amounts of mineral and organic matrix), and the substitution of
carbonate ions in phosphate positions (type-B carbonate substitution) by Raman spectroscopy
in rat bones. Here it was shown that crystallinity, carbonate type B substitution and the
mineral to collagen ratio all increased due to rat aging. Crystallinity of bone mineral has been
-
28
Review of the literature
extensively explored by studying the 1-3 contour in IR spectrum. It was found clear
correlation between intensity ratio of bands centered at 1030 and 1020 cm-1 with maturation
and dimensions of apatite crystallites [117, 118].
The effect of mechanical stress in bones has been studied also by Raman spectroscopy. In the
study of Decarmejane et al. [119], murine cortical bone was subjected to hydrostatic pressure
and the rate of vibrational change in mineral and organic bands as function of the applied
pressure was evaluated. It was observed that hydrostatic pressures induce a large shift in the
collagen bands,(the CH2 stretched band was shifted from ~2935 cm-1
to ~2970 cm-1
, CH2 wag
shifted from 1460 cm-1
to ~1490 cm-1
, the Amide I band from~1655 cm-1 to 1645 cm-1
). The
CH2 shifts were reversible contrary to the Amide I band. It was suggested that the irreversible
shift in the Amide I band is due to changes in the secondary protein structure. The phosphate
and carbonate bands were affected as well by the pressure showing shifting and broadening of
the bands (phosphate from ~960 cm-1
to ~977 cm-1
and carbonate from ~1070 cm-1
to ~1082
cm-1
) being both reversible. Carden et al. [120] used a cylindrical indenter to deform
permanently bovine cortical bone and studied if this induced an alteration of the Raman
spectra of the organic and inorganic components of bone. It was found that at the edge of the
indents there is an increasing in the low-frequency component of the Amide III band and high
frequency component of the amide I band, that might indicate the rupture of the collagen
crosslinking.
Raman spectroscopy has proven to be not only useful in the characterization of bone from the
chemical composition but also orientation information of the mineral and organic phases in
bone can be obtained. Lasers used to excite the sample are inherently polarized meaning that
orientation effects in cannot be ignored when compositional analysis is done. Microstructural
and spatial differences in local mineral and protein composition across individual osteons
have been also well studied. Carden et al. [121] have shown amorphous type of calcium
phosphate that is thought to be precursor of the hydroxyapatite in the older interstitial bone
material rather than in the recently remodelled bone. Timlin et al. [122] have observed
phosphate PO4-3
and mono-hydrogen phosphate (HPO42-
) species in newly from trabecular
bone. Polarized Raman spectroscopy has been used in conjunction with SAM and NI to
evaluate the composition and mechanical properties of individual osteonal lamella. Hofmann
et al. [123] used the 1 PO4-3
band and the Amide I band to evaluate the spatial distribution of
organic and mineral phase in combination with acoustic SAM images. Kazanci . et al. [20]
-
29
Review of the literature
[124] have performed confocal Raman mapping of osteonal tissues, where spatial changes in
the amount of mineral and organic matrix, and the variation of the mineral content was
imaged as function of the polarization angle of the laser. As result it was determined that the
1 PO3-4
band and the Amide I band are quite sensitive to the orientation and polarization
angle of the incident laser, while Amide II and 2 PO4-3
and 4 PO4-3
are less sensitive thus
making them suitable for compositional analysis of bone structures. Additionally, the Raman
images of the ratios 2 PO4-3
to Amide III band has revealed higher values in the interstitial
bone regions. Gamsjaeger et al. [125] have performed Raman polarization experiments in
normal cortical bone in the transversal and longitudinal plane of the mouse femur allowing
the determination of the orientation and composition changes as function of the animal age.
More recently Falgayrac G.[126] et al studied the fibril alignment of the lamellar bone in
cortex by using PRS, taking in consideration specific Raman ratios (3 PO4-3
/ 1 PO4-3
),
Amide III (1271cm-1
) / Amide III (1243cm-1
) and Amide I / Amide III (1243cm-1
). This study
shows simultaneous tilting in intra-lamellar collagen fibril and mineral crystal organization
that is consistent with a twisted plywood organization in the Haversian bone structure.
In conclusion, vibrational spectroscopy has been used to study chemical changes in bone in
normal and pathological conditions using different models, mainly related to the study of the
organic phase. More recently, the use of polarized Raman spectroscopy in confocal
microscopy have offered the possibility to study not only the chemistry, but also structural
organization of bone at micron scale. The present work used the advantages offered by PRS to
study chemical and orientation evolution of rat callus samples, particularly on the organic
phase following the recently work made in the microstructural organization of bone.
-
31
3. Objectives
The main goal of the present work was to study the organic matrix orientation and chemical
evolution of the fracture healing in a rat model. In order to accomplish this general objective
several partial objectives were defined:
To evaluate the theoretical response of amide I in different collagen-like peptide
molecules according to the classical theory of the Raman scattering.
To develop the PRS imaging method to extract chemical and structural information
from collagenous tissue.
To test the technique on well-characterized materials like gelatine, rat tail tendon and
secondary human osteon.
To use the PRS to extract chemical and orientation information during the fracture
healing in a rat model.
-
33
4. Theoretical response of the amide I band according to the classical theory
One important aspect determining the orientation of the organic matrix in different materials
is to understand the Raman anisotropic response of collagen which is one of the major
components of the extracellular matrix in bone (almost 90%) [76]. In doing so, we have used
the classical Raman scattering theory to evaluate the anisotropic response of the Amide I
band, in helical protein structures: a single collagen-like peptide and -helix peptide molecule
as shown in Figure 11.
Figure 11 A) Structure of the collagen like-peptide (ID:1cag) showing the characteristic triple helical
structure. B) Alpha helix(ID:1djf) generated by the twisting of a single polypeptide around itself forming a
cylinder. The crystal structures are taken from the RCSB PDB (www.pdb.org) and the images generated
by Pymol.
The Amide I band is assigned to the vibration of the trans peptide group (CONH) present in
proteins and peptides. The position of this band is between 1640-1680 cm-1
[23, 127]. Tsuboi
et al. derived a relative Raman tensor of Amide I vibration making a Raman study of a single
crystal of the artificial dipeptide sweetener aspartame (-Laspartyl-Lphenylalanine methyl
ester) [23, 128-131]. This tensor was described to have its largest polarizability along the line
that is oriented from C=O bond in the sample plane that the peptide group as shown in Figure
12A.
A B
http://www.pdb.org/
-
34
Theoretical response of the Amide I band
Figure 12 a) Principal Raman tensors axis and relative values of the tensor components r1=(xx/zz)
r2=(yy/zz) of the aspartame trans peptide group [23]. The amide marker occurs at 1667 cm-1 b)
Principal axis (XYZ) and relative values of the amide I Raman tensor components R1=(XX/ZZ) and R2
=(YY/ZZ) of a idealized - helix. This amide I marker of - helix is expected near 1651 cm-1
.
The x-axis of the tensor is taken to be the line of the largest polarizability; the y-axis is in the
peptide plane perpendicular to the x-axis and the principal z-axis perpendicular to the peptide
plane. The local Raman tensor has the following ratios of its diagonalised components:
(xx/zz=20) and (yy/zz=4).
Tsuboi et al [23] proposed that Amide I Raman tensor from a single peptide molecule
(aspartame) might be transferable to more complex multi-peptide structures. Following this
approach, a relative Raman tensor for a continuous-idealized -helix was described to have
the Z axis of the tensor parallel to the axis of the -helix and relative values (XX/ZZ=0.537)
and (YY/ZZ=0.537).
Here, we have evaluated the Amide I band response at different polarization angle of the
incident light of alpha helix and collagen-like peptide structures (by transfer