Raman spectroscopy investigation of organic matrix orientation and ...

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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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

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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]

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[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.

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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.

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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/

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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

Raman spectroscopy investigation of organic matrix orientation and ...

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