Improving the Mechanical Properties of Irradiation ......The overall goal was to develop a method...

Improving the Mechanical Properties of Irradiation Sterilized Bone Allografts

by

Brianne Burton

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science

Institute of Biomaterials and Biomedical Engineering University of Toronto

© Copyright by Brianne Burton 2013

ii

Improving the Mechanical Properties of Irradiation Sterilized Bone

Allografts

Brianne Burton

Master of Applied Science

Institute of Biomaterials and Biomedical Engineering

University of Toronto

2013

Abstract

Bone allografts are used in orthopaedic reconstruction of defects resulting from trauma, bone

cancer or infection. Gamma-irradiation sterilization is a safety measure; however it damages the

tissue, specifically the organic component, and embrittles bone. This research investigated the

effect of ribose pre-treatment on the mechanical properties of ribose-modified irradiated bone.

The overall goal was to develop a method for improving toughness of irradiation-sterilized bone

by modifying collagen with a ribose treatment prior to/during irradiation. Bulk mechanical

properties and fracture properties were evaluated. Collagen characterization techniques were

used to further understand the collagen alterations and suggest toughening mechanisms. We have

shown it is possible to recover some of the mechanical properties of γ-irradiated bone as well as

collagen thermal stability and connectivity using our ribose pre-treatment. We propose that the

recovery of collagen connectivity leads to functionally significant recovery of toughness, fracture

toughness and strength.

iii

Acknowledgments

Firstly, I would like to thank my supervisors, Dr. Thomas Willett and Dr. Marc Grynpas for their

guidance and encouragement throughout this process. I have truly grown as a researcher from the

start of this project, mostly due to their thoughtful questions, suggestions, and expert advice.

Tom, this project could not have been possible without your enthusiasm and helpful

encouragement along the way. I would like to thank my committee members, Dr. Zhirui Wang

and Dr. Eli Sone for their input and engaging discussions.

This work could not have been possible without the help of several people. I would like to

especially thank Anne Gaspar, David Josey, David Lee, and Jindra Tupy for their technical

assistance and problem-solving skills in the lab.

Finally, I would like to thank my friends and family for their love and support through this

difficult and rewarding process, especially my dad, who inspired my love of science at a young

age, and my mom, who has shown me the true meaning of dedication and hard work.

iv

Table of Contents

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Tables ............................................................................................................................... viii

List of Figures ................................................................................................................................ ix

List of Appendices ........................................................................................................................ xii

Chapter 1 ......................................................................................................................................... 1

1 Introduction ................................................................................................................................ 1

1.1 Motivating Problem ............................................................................................................ 1

1.2 Clinical use of Bone Allografts and Irradiation Sterilization ............................................. 1

1.3 Structure of Bone ................................................................................................................ 2

1.3.1 Overall Structure ..................................................................................................... 2

1.3.2 Collagen Structure .................................................................................................. 5

1.3.3 Collagen Crosslinks ................................................................................................ 6

1.4 Bone Material Properties .................................................................................................... 8

1.4.1 The Role of Collagen in Bone Toughness .............................................................. 8

1.4.2 Fracture Toughness Mechanisms in Bone .............................................................. 8

1.5 Effects of Irradiation ......................................................................................................... 11

1.5.1 Effects on Collagen Structure ............................................................................... 11

1.5.2 Mechanical Properties of Irradiated Bone ............................................................ 12

1.6 A Potential Solution for Irradiated Allografts .................................................................. 15

1.7 Objectives and Hypothesis ................................................................................................ 17

1.8 Experimental Approach .................................................................................................... 18

v

1.8.1 Tissue Model – Bovine Cortical Bone .................................................................. 18

1.8.2 Design of Experiments .......................................................................................... 19

1.8.3 Methods for Characterizing Bone Collagen ......................................................... 19

Chapter 2 ....................................................................................................................................... 24

2 Bone Embrittlement and Collagen Modification Due to High Dose Gamma-Irradiation

Sterilization .............................................................................................................................. 24

2.1 Introduction ....................................................................................................................... 24

2.2 Methods ............................................................................................................................. 25

2.2.1 Sample Preparation ............................................................................................... 25

2.2.2 Irradiation .............................................................................................................. 25

2.2.3 Mechanical Testing ............................................................................................... 26

2.2.4 Collagen Characterization Methods ...................................................................... 29

2.2.5 Statistics ................................................................................................................ 34

2.3 Results ............................................................................................................................... 34

2.3.1 Mechanical Properties ........................................................................................... 34

2.3.2 Collagen Characterization ..................................................................................... 34

2.4 Discussion and Conclusions ............................................................................................. 41

Chapter 3 ....................................................................................................................................... 44

3 Ribose Pre-Treatment to Improve Bone Mechanical Properties .............................................. 44

3.1 Introduction ....................................................................................................................... 44

3.2 Methods ............................................................................................................................. 45


3.2.2 Treatment .............................................................................................................. 47

3.2.3 Irradiation .............................................................................................................. 48

3.2.4 Three-point Bending ............................................................................................. 48

3.2.5 Dual Energy X-Ray Absorptiometry .................................................................... 49

vi


3.3 Statistical Analysis ............................................................................................................ 51

3.4 Results ............................................................................................................................... 51




Chapter 4 ....................................................................................................................................... 66

4 Comparing Ribose to other Crosslinking Agents ..................................................................... 66

4.1 Introduction ....................................................................................................................... 66

4.2 Methods ............................................................................................................................. 68


4.2.2 Treatment .............................................................................................................. 68

4.2.3 Irradiation .............................................................................................................. 69

4.2.4 Mechanical Testing ............................................................................................... 69

4.2.5 Dual Energy X-Ray Absorptiometry .................................................................... 70



4.4 Results ............................................................................................................................... 71




Chapter 5 ....................................................................................................................................... 86

5 Fracture Testing of Irradiated and Ribose-Treated Bone ......................................................... 86

5.1 Introduction ....................................................................................................................... 86

5.2 Methods ............................................................................................................................. 88


vii

5.2.2 Treatment and Irradiation ..................................................................................... 88

5.2.3 Fracture Testing .................................................................................................... 88

5.2.4 Imaging the Fracture Surface ................................................................................ 89

5.2.5 Calculating Fracture Toughness ........................................................................... 92


5.4 Results ............................................................................................................................... 96

5.5 Discussion and Conclusions ........................................................................................... 100

Chapter 6 ..................................................................................................................................... 102

6 Discussion, Conclusions, and Future Work ........................................................................... 102

6.1 Discussion ....................................................................................................................... 102

6.2 Conclusions ..................................................................................................................... 116

6.3 Future Work .................................................................................................................... 116

References ................................................................................................................................... 119

Appendices .................................................................................................................................. 127

viii

List of Tables

Table 2.1 Mechanical properties from three-point bending 36

Table 2.2 Crosslinking quantification from HPLC 38

Table 2.3 Data from differential scanning calorimetry 40

Table 2.4 Data from hydrothermal isometric tension testing 40

Table 3.1 Treatment conditions prior to irradiation 48

Table 3.2 Mechanical properties from three-point bending 54

Table 3.3 Bone mineral densities of bovine bone beams measured with DEXA 55

Table 3.4 Average percent soluble matrix after digestion with pepsin 55

Table 3.5 Pentosidine content in irradiated and ribose pre-treated bovine bone 57

Table 3.6 Data from differential scanning calorimetry: ribose pre-treatment 59

Table 3.7 Data from hydrothermal isometric tension testing: ribose pre-treatment 60

Table 4.1 Treatment conditions prior to irradiation for Ribose, Glucose, Fructose,

and Ascorbate 69

Table 4.2 Mechanical properties from three-point bending comparing

different agents 74

Table 4.3 Bone mineral densities of bovine bone beams measured with DEXA 75

Table 4.4 Average percent soluble matrix after pepsin digestion for ribose and

Glucose 75

Table 4.5 Pentosidine content in ribose and glucose treated bone matrix

after irradiation 77

Table 4.6 Data from differential scanning calorimetry: ribose vs. glucose 79

Table 4.7 Data from hydrothermal isometric tension testing: ribose vs. glucose 80

Table 5.1 J-integral and Ki of bovine cortical bone 98

Table 5.2 Stable tearing measurements based on SEM images for fracture

samples tested in SENB fracture 98

ix

List of Figures

Figure 1.1 X-ray image of a bone allograft implanted into the femur of a patient 3

Figure 1.2 The hierarchical structure of bone (From Launey et al. 2006) 4

Figure 1.3 The structure of collagen (Nyman et al. 2005) 5

Figure 1.4 The pathway of non-enzymatic crosslinking (From Saito et al. 2009) 7

Figure 1.5 Fracture toughness mechanisms in bone (From Ritchie 2009) 10

Figure 1.6 Effects of gamma-irradiation on collagen. A proposed mechanism

of damage to bone collagen caused by gamma-irradiation. 14

Figure 1.7 Experimental procedure for treating, irradiating, and testing bovine

bone samples 19

Figure 2.1 Bone beams cut from bovine metatarsal bone 27

Figure 2.2 Test set-up for three-point bending 28

Figure 2.3 A typical heat flow vs. temperature endotherm obtained from

differential scanning calorimetry (left). A load vs. temperature

curve with measured properties from a hydrothermal isometric

tension test (right). 33

Figure 2.4 SDS-PAGE gel and density profile from one matched pair

demonstrating the different banding pattern of non-irradiated

and irradiated bovine bone matrix. 37

Figure 2.5 Comparison of non-irradiated and irradiated DSC endotherms (left)

and HIT load curves (right) for one matched pair. 39

Figure 3.1 Samples obtained from bovine tibia 46

Figure 3.2 SDS-PAGE gel and density profile from one matched set of

demineralized bovine bone 56

Figure 3.3 Representative chromatograms from HPLC. IS = internal

standard Pent = Pentosidine. Ribose pre-treated samples show

peaks corresponding to pentosidine and other glycation products. 57

Figure 3.4 Representative curves for differential scanning calorimetry (left)

and hydrothermal isometric tension (right) for a matched set of

demineralized bovine bone samples. 58

Figure 4.1 SDS-PAGE gel and stain density profile for one matched set

of demineralized bone samples, comparing ribose and glucose

pre-treatment to non-irradiated and irradiated controls. Ribose

treated collagen was less susceptible to pepsin digestion and

therefore not represented on the gels. 76

Figure 4.2 Representative chromatographs from HPLC analysis of one

matched set. IS = Internal Standard. Pent = Pentosidine. High T Ribose

(green curve) is the only curve with a peak corresponding to pentosidine

and other glycation products. 77

Figure 4.3 Representative curve s from DSC (left) and HIT (right) for one set

of matched specimens comparing ribose and glucose pre-treatments. 78

x

Figure 5.1 An optical micrograph showing microcracks formed in human bone

tissue after fatigue. (From Nicolella et al. 2011 ) 86

Figure 5.2 Test set-up for a single-edge notched beam fracture test. The ~2 mm

notch shown is cut with a diamond wire saw (diameter = 300 um) and

sharpened by hand with a razor blade (a). An SEM image of a fractured

sample, looking at the fracture surface (b). 90

Figure 5.3 Load vs. displacement curve for a notched beam tested in three-point

bending 91

Figure 5.4 Calculating the crack length with Image J software. On the left is an

SEM image of the fracture surface of one fractured beam. On the right

is a screen shot of the imaging software with lines that were drawn and

measured to estimate the sample dimensions. 96

Figure 5.5 On the left, an example of the load vs. displacement curves for one

matched set of single-edged notched beams tested in three-point

bending. On the right, a graph representing the average J-integral

values for each group. The J-integral was evaluated using the maximum

load and the crack length at instability. 97

Figure 6.1 Work to fracture vs. maximum isometric stress for all groups tested in

three-point bending. The averages of the normalized values are shown

here and the error bars represent the standard error of the mean. 104

Figure 6.2 Work to fracture vs. slope at half maximum of the HIT load curve for

all groups tested in three-point bending. The averages of the normalized

values are shown here and the error bars represent the standard error of

the mean. 105

Figure 6.3 Work to fracture vs. temperature of denaturation (HIT) for all groups

tested in three-point bending. The averages of the normalized values

are shown here and the error bars represent the standard error of the

mean. 106

Figure 6.4 Work to fracture vs. temperature of denaturation onset (measured in

DSC) for all groups tested in three-point bending. The averages of the

normalized values are shown here and the error bars represent the

standard error of the mean. 107

Figure 6.5 Work to fracture vs. enthalpy (measured in DSC) for all groups tested

in three-point bending. The averages of the normalized values are

shown here and the error bars represent the standard error of the mean. 108

Figure 6.6 A simple schematic of the initial predicted for the relationship between

toughness and connectivity (a) and two simplified curves from actual

testing results (b and c). The gap between normal bone and the other

groups (modified collagen) suggest additional factors other than

thermal stability and connectivity contribute to the toughness in

normal, healthy bone. 111

Figure 6.7 Fracture toughness mechanisms from macro to nano scale in bone.

(From Barth et al 2010) 114

xi

xii

List of Appendices

Appendix 1: Force vs. displacement graphs (four examples) for 3-point bending experiments

described in Chapter 3

Appendix 2: Force vs. displacement graphs (four examples) for 3-point bending experiments

described in Chapter 4

Appendix 3: Details and equations regarding the calculation of fracture toughness measurements

from SENB fracture tests.

1

Chapter 1

1 Introduction

1.1 Motivating Problem

An allograft is a transplant of donor tissue to a recipient patient. Bone allografts are used in

orthopaedic reconstruction that is often necessary for trauma cases and people suffering

skeletal defects due to bone cancer or infection. In the United States, there are over 1.5

million allograft transplants each year [1] and around seventy-thousand each year in Canada

[2]. Of these transplants each year, roughly 450,000 are bone allografts [3]. In order to ensure

there is no pathogen transfer from donor to recipient, bone tissue is often sterilized with

gamma-irradiation, which eliminates bacteria, virus, or fungus that may cause infection. This

sterilization is necessary for the safety of the patient but it damages the collagen in bone,

which is responsible for toughness and resistance to fracture [4]. Compromising the collagen

component alters the mechanical properties of the allograft making it more brittle and easier

to fracture. It has been found that when implanted, allografts are approximately twice as

likely to fracture as non-irradiated autografts [5]. Thus, there is a great need for a method of

sterilization that maintains the mechanical toughness of the tissue while still using gamma-

irradiation.

1.2 Clinical use of Bone Allografts and Irradiation Sterilization

When a patient requires reconstruction of a defect, the best performing graft would be an

autograft, or a transplant of their own tissue from another source in their body. In terms of

bone graft performance, an autograft has been considered the gold standard [6, 7] as there is

no immunological rejection and it provides appropriate mechanical stability and osteogenic

cells stimulate bone incorporation [6, 8]. They may be the first choice for repairing small

defects [9] but, despite the advantages, autografts are often not feasible due to limited

availability for large grafts and donor site morbidity. The use of allografts (tissue from a

donor) is popular due to the greater availability of shapes and sizes without sacrificing host

structures. Figure 1.1 shows an x-ray image of a large structural allograft implanted into the

femur of a patient. The major concern of allograft use is disease transfer and it has become

2

more common for tissue banks to require allograft sterilization. Unfortunately, the process of

gamma-irradiation sterilization has an effect on the mechanical performance of the graft. One

clinical study found that while patients with irradiation-sterilized allograft implants had no

occurrence of infection, they were twice as likely to fracture as non-irradiated allografts [5].

Many research studies have been devoted to understanding the properties of bone and how

these are affected by irradiation. Allografts can be load-bearing and are usually under

mechanical stresses. According to a study by Enneking et al. [10], revitalization of the

allograft is only about 20% at 5 years post implantation. Wheeler et al. [11] demonstrated an

increase in microcrack density in retrieved allografts that were implanted from 0 up to 10

years. They suggest this increase is due to the slow rate of turnover in allograft bone.

Therefore, long-term durability of these grafts is required.

Despite the known damage to tissue caused by gamma-irradiation, it is a widely-used method

by tissue banks because of the superior sterilization capabilities and ease of use. Irradiation is

highly effective at killing pathogens. Ionizing radiation, such as gamma irradiation, kills

pathogens by damaging the DNA and RNA directly by the gamma rays and also indirectly

through highly reactive free radicals created by the radiolysis of water [12]. Gamma-

irradiation has good penetrability into matter, it does not require the use of heat (which could

also alter the tissue), and it can be performed while tissue is inside its packaging to avoid

contamination during re-packing [12]. Because of these qualities, irradiation is often required

by tissue banks as a means of sterilization. Thus a method of rescuing damage caused to

collagen in this process is required in the field of allograft use.

1.3 Structure of Bone

1.3.1 Overall Structure

Bone is a unique material with a complex hierarchical structure. There are two classifications

of bone: cancellous (or trabecular) and cortical bone. Cancellous bone is highly porous, with

bone marrow occupying the voids, and can be found in the ends of long bones and in

vertebrae. Cortical bone is compact, provides structure and protection to the body and organs,

and can be found in the diaphysis of long bones and layered on the exterior of flat bones and

vertebrae. Bone is a composite material made up of tough collagen fibers and mineral

3

crystals. Figure 1.2 provides an illustration of the structure of cortical bone from nanoscale to

macroscale [13]. Type I collagen makes up 90% of the organic matrix of bone [14]. Three

polypeptide chains are tightly bonded in a triple-helix to form a tropocollagen molecule.

Tropocollagen molecules assemble together in a staggered pattern that leaves small gaps in

between each molecule. Mineral crystals are interspersed in these gaps forming mineralized

collagen fibrils. A stacked array of mineralized fibrils forms collagen fibers that are arranged

in patterns and layered in a lamellar structure to form the bulk of cortical bone. A cylindrical

structure called an osteon is formed by concentric lamellar layers surrounding haversian

canals, which are pores allowing for vasculature, cells, and nerves to penetrate into the

otherwise compact structure of cortical bone [13, 15].

Figure 1.1: X-ray image of a bone allograft implanted

into the femur of a patient. Image courtesy of Dr. Peter

Ferguson [101] (Mount Sinai Hospital, Toronto ON

Canada).

4

Figure 1.2: The hierarchical structure of bone (From Launey et al. 2010 [13])

5

1.3.2 Collagen Structure

The amino acid sequence of the peptide chains is a characteristic Gly – X – Y pattern where

Gly is Glycine; the smallest residue that packs neatly into the center of the helix. X and Y are

most often proline and hydroxyproline. This sequence gives collagen its higher order triple-

helical structure. Intramolecular hydrogen bonding of the hydroxyproline residue provides

thermal stability to the helix [16]. Nyman et al. [17] showed that drying bone, even at low

temperatures, will greatly reduce work to fracture (a measure of toughness) due to loss of

stabilizing hydrogen bonding. Water stabilizes collagen with both inter- and intramolecular

bonding [17], and is an important component in both collagen structure and in the properties

of bone [18, 19]. The triple helical section of the molecule is roughly 300 nm long and 1.5

nm in diameter, forming a long rod-like structure [20]. There are two short non-triple helical

regions at each end, called telopeptide regions. These regions do not exhibit the Gly-X-Y

pattern and are remainders after post-translational proteolytic enzymes cleave off larger

propeptides, allowing collagen molecules to assemble together (see Figure 1.3). Collagen

molecules stack together side-by-side with an overlap of about one-quarter of their length,

with a gap region between the end of one molecule and the head of the next [15]. They are

linked together in a microfibril array by crosslinks between molecules.

Figure 1.3: The structure of collagen (From Nyman et al. 2005 [17])

6

1.3.3 Collagen Crosslinks

Hydroxylysine and lysine, other abundant amino acids in type I collagen, serve as sites for

enzymatic crosslinks that form between neighboring molecules. Lysyl and hydroxylysyl

residues become aldehydes via the enzyme lysyl oxidase. The staggered pattern of stacked

collagen molecules allows for covalent bonds to form between two molecules (a non-helical

telopeptide end to a site on the helix). These crosslinks first form between two molecules

(immature divalent) and can turn into stable trivalent crosslinks, classified as pyridinolines or

pyrroles, between three neighboring sites [21]. A unique feature of bone collagen is the fact

that there are roughly equal numbers of pyrroles and pyridinolines. There may be a

functional purpose for this phenomenon however at this point the reason remains unclear.

[21]. Pyridinolines have been used as a biomarker in urine (indicator of bone remodeling)

because they fluoresce and can be easily quantified using high performance liquid

chromatography. Pyrrole crosslinks are more difficult to characterize [21]. Enzymatic

crosslinking in bone collagen is an important microstructural feature that contributes greatly

to the mechanical integrity of bone as a material [22, 23, 20].

Collagen is also susceptible to the formation of non-enzymatic crosslinks. In vivo, oxidation

of a free reducing sugar (such as glucose) leads to a more reactive compound [24] which

reacts with a free ε-amine group of a lysine, arginine, or hydroxylysine in the collagen helix

to form a Schiff base. The Schiff base spontaneously undergoes Amadori rearrangement, and

the resulting product can further react with another amino acid to form a highly stable

crosslink between two collagen molecules [25, 26, 27]. Figure 1.4 depicts a schematic of the

pathway of non-enzymatic crosslinking from Saito et al. [28]. While enzymatic crosslinks are

specific to the telopeptide regions of the collagen molecule, these glyco-oxidation crosslinks

(GOCs) are thought to be non-specific and distributed all throughout the structure, mostly

forming helix-helix crosslinks [28]. Because of this, a high level of GOCs in normal tissue

has been shown to stiffen the collagen network and lead to embrittlement of tissue (skin,

vasculature, tendons, and bone) [28, 20]. According to Avery and Bailey [26], glycation of a

collagen fiber will lead to an increase in both tensile strength and temperature of

denaturation, as well as an increase in stiffness. These all indicate a high level of

crosslinking, and in particular the stiffening decreases the ductility of tissues [23]. Glyco-

7

oxidation crosslinks are also often referred to as Advanced Glycation Endproducts (AGEs).

There are many different forms of AGEs, some of which are known (such as pentosidine,

glucosepane, and non-crosslink formations like carboxyl methyl lysine) while many more

remain to be characterized, making overall AGE quantification difficult. However, there is

one crosslink, pentosidine, which has been well characterized and can be detected using a

high performance liquid chromatography technique. Pentosidine is accepted as a marker for

AGEs.

Figure 1.4: The pathway of non-enzymatic crosslinking (From Saito et al. 2009 [28])

8

1.4 Bone Material Properties

1.4.1 The Role of Collagen in Bone Toughness

While mineral plays an important role in bone stiffness and yield strength, the post-yield

toughness of bone relies mostly on intact and functional collagen network. In fact, many

disorders affecting collagen have devastating effects on the mechanical properties of bone.

For example, Osteogenisis Imperfecta is a genetic mutation that inhibits osteoblasts from

forming proper collagen. Glycine is replaced by a larger amino acid, which alters the packing

of the three polypeptide chains in the triple helix and therefore disrupts the packing of

collagen molecules [29]. This leads to an increase in bone fragility and a decrease in bone

mass [17, 20]. Lathyrism, a disease effecting lysyl oxidase and therefore the ability to form

collagen crosslinks, results in a decrease in bone strength and toughness. Oxlund et al. [22]

demonstrated the importance of collagen crosslinks for the mechanical integrity of bone. A

reduction in pyridinolines in rats treated with a lathyrtic agent was associated with an

increase in susceptibility to enzyme digestion and a decrease in stiffness, bending strength,

and deflection at failure. Wang et al. [30] studied the effect of two forms of collagen

modification on properties of demineralized human cadaveric femur bone: unwinding the

helix (heat denaturation) and enzymatic cleavage of the peptides. Both unwinding and

cleavage increased the percent denatured collagen and decreased the mechanical integrity

(lower ultimate strength, stiffness, work to fracture, and strain at failure). There was no

change in crosslinking, suggesting that the integrity of collagen molecules is just as important

as the connectivity of the network. All factors that influence the connectivity of the collagen

network, including the structure of the triple helices, stabilizing hydrogen bonding, crosslink

quality and quantity, and interaction between collagen and mineral seem to play an important

role in the strength and toughness of the bone tissue.

1.4.2 Fracture Toughness Mechanisms in Bone

There are several important mechanisms provided by the unique structural organization of

matrix and mineral that influences the toughness of bone. Toughness is defined as the ability

of a material to absorb energy before fracturing. Fracture toughness is similar but distinct, as

it is the ability of a material containing a crack to resist fracture. Fracture toughness

9

measurements indicate the energetic cost of crack growth. Bone is a complex material and

does not necessarily have traditional “plasticity” like, for example, a metal. Plasticity is

permanent deformation under loading, which dissipates energy before failure of a material.

Most of the plastic behavior in bone is due to the formation of microdamage. Microcracking

in bone is often referred to as a mechanism of plastic deformation. A study by Zioupos et al

[31] suggests that toughness in bone comes from the natural ability of bone to form and

accommodate microdamage. They found that when cortical bone samples were tested at high

strain rates there was no time to form microcracks before fracture. There was a correlation

between lower amounts of microdamage and low post-yield toughness and strain. Smaller

scale toughening mechanisms may include microcracking of mineralized fibrils, inter and

intra-fibrillar sliding, and molecular uncoiling of the triple helix [13, 32]. Most of the

resistance to crack propagation, or fracture toughness, relies on deflection of cracks from the

crack path via the highly anisotropic structure and properties of the material. Since osteons,

which are cylindrical structures formed by concentric layers of lamellae (see Figure 1.2), are

much stronger than the cement lines in between them, cracks are deflected to the cement

lines when they encounter an osteon because cracks take the path of least resistance [13]. Un-

cracked ligament bridging is an unbroken area that forms in between the main crack tip and

smaller cracks ahead (see Figure 1.5). This region is able to withstand greater loads and thus

increases the fracture toughness [32]. Crack bridging by collagen fibers, where fibers span

the cracked region, requires higher loads and energy to further open the crack [32].

Fracture toughness in bone can be linked to the properties of the collagen phase in bone. It

has been shown that fracture properties decrease in aging [33, 34, 35]. Aging is also

associated with a deterioration of the collagen [33, 36], which suggests that there is a

relationship between the properties of collagen and the fracture toughness in bone. Barth et

al. [37] demonstrated that modification of collagen by high-dose x-ray irradiation of human

femur specimens dramatically lowered the measure of fracture toughness (Kjc) by a factor of

five.

10

Figure 1.5: Fracture toughness mechanisms in bone (From Ritchie [32])

11

1.5 Effects of Irradiation

1.5.1 Effects on Collagen Structure

Irradiation disrupts the collagen network in bone by causing a breakdown in the peptide

backbone. It is proposed that cleavage of peptides is caused directly by the gamma rays [4,

12] and by damaging free radicals. The majority of damage is thought to be caused by the

radiolysis of water molecules which creates free radicals that attack collagen molecules and

change their chemical structure [4, 3]. It has been shown that bone treated with a chemical

free radical scavenger during the irradiation process decreases the deleterious effect of

irradiation on the collagen properties [3, 38, and 39], which upholds the theory that free

radical damage of collagen molecules is a major mechanism of radiation damage in bone.

It is important to note that the effects of irradiation on collagen are not fully known,

especially when it comes to crosslinking. One study showed no difference in mature

enzymatic pyridinoline crosslink content between control and irradiated bone; however the

effect on immature crosslinks was not studied [40]. Irradiation of collagen in gels or solution

has been shown to create crosslinks via modification of side chains making them more

reactive [41, 42]. It seems that the type of tissue and the conditions under which irradiation is

taking place (in presence of water, frozen, etc.) has a major influence on the type of

modifications [12, 41, and 43]. In the case of bone allografts, the major concern is cleavage

of the peptide chains.

Several studies have shown evidence of the fragmentation of collagen (in many types of

tissue) as a result of irradiation. Increased solubility, for example leaching of collagen

fragments from irradiated skin samples into solution [44], and increased susceptibility to

enzyme digestion indicate a less stable collagen structure [12, 42, 43, 44, 45]. Gouk et al.

[45] found that irradiated skin (human cadaveric) was more susceptible to digestion by

trypsin, an enzyme that cleaves only denatured collagen at lysine and arginine sites. An

increase in exposed lysine and arginine suggests collagen denaturation due to chain scission,

perhaps. They also showed a decline in mechanical strength and a lowering of the

denaturation temperature, further suggesting irradiation-induced damage. Dzeidzic-

Goclawska et al. [12] showed both an increase in solubility of irradiated rat bone collagen

12

and an increased rate of resorption when irradiated bone was implanted into the abdominal

muscles of rats. Akkus et al. [3] demonstrated the broken-down structure of collagen from

irradiated human femur bone using gel electrophoresis. Gels showed there were fewer intact

alpha-chains and more low-molecular weight fragments present in the supernatant of pepsin-

digested irradiated samples. Figure 1.6 is a simplified schematic of the proposed mechanism

of damage caused by irradiation. This cleavage of collagen chains is thought to be the major

reason for the changes in the mechanical properties of irradiated tissue.

1.5.2 Mechanical Properties of Irradiated Bone

The mechanical properties of bone suffer greatly from the effects of irradiation. It has been

widely demonstrated that the mechanical properties of irradiated bone are significantly

inferior to those of non-irradiated bone [3, 4, 12, 38, 39, 43, 46]. Irradiation of bone tissue

has been shown to mostly affect the post-yield properties. This includes post-yield strain,

toughness and fracture toughness; properties attributed to the collagen component of bone

[3]. Bone that has been irradiated loses toughening mechanisms making it a brittle material

with little ability to absorb energy. If you consider the stress-strain curve from a mechanical

loading test, the area under the curve reflects the ability of the material to absorb energy

before fracture. Prior to the yield point (the point at which irreversible behavior starts), the

mechanical behaviour is considered elastic. Elastic energy to fracture depends on the

stiffness of the material, which is mostly controlled by the mineral component. Toughening

mechanisms and plasticity contribute to energy absorbed during the non-linear post-yield

portion of the curve. After irradiation, collagen molecules become fragmented and weak,

causing a loss of connectivity of the collagen network. In theory, collagen is unable to

support mechanisms of pseudo-plasticity and resistance to crack propagation. Post-yield

toughness seems to be directly related to the integrity of collagen [30].

In comparing non-irradiated controls and irradiated specimens from human femurs, Currey et

al. [46] showed that bending strength, work to fracture, and impact energy were significantly

lower in the irradiated specimens. Akkus et al. [3] demonstrated a striking decrease in work

to fracture (70% of non-irradiated bone), post-yield energy (87%), and fatigue resistance

(87%) in bone samples irradiated at 36.4 kGy. The Akkus group also compared the fracture

surface of irradiated bone to control bone to demonstrate the difference in fracture

13

mechanisms. The native control exhibited a tortuous fracture surface attributed to crack

deflection and bridging. Irradiated samples exhibited a flat surface, indicating the absence of

these mechanisms. This suggested loss of fracture toughness mechanisms. This, together with

evidence of damage to collagen structure as a result of irradiation, provides support to the

theory that collagen integrity influences bone toughness.

An important correspondence between the degree of damage to collagen and the dose of

irradiation has also been established in previous research [37, 46, and 47]. In other words,

their investigation has shown degradation of material properties as the irradiation dose

increases. Currey et al. [46] demonstrated this relationship by comparing bending strength

and work to fracture of human cortical bone allografts at irradiation doses of 0, 17, 29.5, and

94 kGy. Both strength and work-to-fracture showed a decreasing trend in properties with

increasing dose. Because of this relationship, there is not a standard dose amount and tissue

banks usually use a moderate dose of ~20-30 kGy in an effort to maximize the sterilization

while minimizing the effect on mechanical integrity [47]. It would be ideal to use higher

doses because certain viruses of concern such as HIV require doses as high as 36 kGy to

eliminate risk of infection [12, 46, 47] but even at moderate doses (20-30 kGy) the material

integrity is significantly affected. An extra measure of irradiation sterility would certainly

increase the safety of clinical allograft use.

14

15

1.6 A Potential Solution for Irradiated Allografts

As stated above, free radical scavenging is one method that has been previously pursued as a

solution to irradiation damage. Due to concerns about carcinogenic effects and compromising

the sterility of the graft (free radical scavengers protect the pathogens); this method would

not be successful in clinical use [3]. Crosslinking collagen prior to irradiation has also been

pursued, although to the best of our knowledge only for tendons and never in bone because

of the generally excepted idea that crosslinking would further embrittle bone. Ng et al. [48]

tested the effects of genipin pre-soaking on irradiated bovine and human patella tendons.

Genipin is thought to generate crosslinks between adjacent collagen microfibrils. The results

showed radioprotection of the elastic modulus in the bovine model. No significant results

were found in the human model, most likely due to biological variation in donors. Dunn and

his colleagues have published several studies on crosslinking before and during irradiation

and radioprotection of collagen and tendons [38, 39, and 49]. In one study, collagen films

were prepared with glucose and irradiated at 25 kGy, which maintained strength [49]. In

further studies, rabbit tendons soaked in glucose and irradiated at 50 kGy maintained strength

and modulus. The addition of free radical scavengers (ascorbate, mannitol, or riboflavin)

helped maintain higher strength and modulus [38, 39].

To the best of our knowledge, crosslinking has not been attempted in bone tissue as a method

of maintaining tissue toughness while still using gamma irradiation to sterilize the tissue. The

mechanism of damage to irradiated bone is widely accepted as the cleavage of peptide chains

in the collagen molecule. This leads to a weakened molecule, and thus a weakened collagen

network. Collagen is thought to loose connectivity due to this damage. One way to increase

the connectivity of the network is to introduce additional crosslinks. The idea is that these

crosslinks would compensate for the cleavage by “tying” the loose pieces together to

maintain a continuous structure. Since we know that collagen plays an integral role in the

overall toughness of bone, creating a more connected network could toughen the otherwise

brittle irradiated bone. The use of a glyco-oxidation crosslinker such as ribose to induce

crosslinks is one option. Irradiation causes oxidation which might have two effects; first, it

may accelerate glycation of the sugar, and secondly it may accelerate crosslink formation

(pentosidine formation is oxidation dependent) which would reduce the time barrier for the

16

reaction. This accelerates the glycation step and furthers progress to crosslink formation,

greatly reducing the time for these crosslinks to form. AGEs can form in vitro by incubation

of proteins with sugars such as glucose or ribose. In vivo, AGE formation is associated with

aging or diseases (such as diabetes) as it is thought to cause hyper-crosslinking and therefore

embrittlement of bone. For native bone, it is thought to be undesirable to introduce more

crosslinks into the already tough, continuous, structure. For our purposes, irradiation causes

collagen to become fragmented, weak polymeric chains that could gain toughness and

strength through the addition of new crosslinks.

17

1.7 Objectives and Hypothesis

The overall objective of this thesis was to develop a technique for improving irradiation-

sterilized allograft performance, specifically by improving toughness of irradiation-sterilized

bone. It was critical to assess the effect of irradiation on the mechanical properties and

collagen characteristics. Since it is known that irradiation damages collagen by cleaving the

backbone polypeptide chains, our novel idea was to introduce new crosslinks to restore the

connectivity of the collagen network. It is thought that crosslink formation by pre-soaking

with ribose (a glyco-oxidation crosslinker) that is subsequently driven by irradiation may

alter the compromised collagen phase of bone material and make it tougher. This would

improve the integrity and clinical performance of allograft bone. To our knowledge, this was

investigated for the first time in this project. The study can be divided into four main

objectives:

1) Evaluate changes in bone as a result of irradiation and understand what collagen

damage can tell us about the embrittlement of bone.

2) Evaluate ribose treatment as a method to improve the toughness of irradiated bone

3) Study changes in bone collagen as a result of ribose pre-treatment + irradiation to

gain insight into collagen alterations

4) Evaluate the effect of ribose pre-treatment on fracture toughness of bovine bone

Hypothesis: We expect that ribose pre-treatment will improve the mechanical properties of

irradiated bone, more specifically there will be an increase in measures of toughness (work to

fracture and fracture toughness). It is anticipated that due to crosslinks created through the

glyco-oxidation reaction between ribose and the amino acid side chains of collagen

molecules, the connectivity of the collagen network will improve. Four experiments, each

described in a separate chapter of this thesis, aim to address these objectives.

18

1.8 Experimental Approach

1.8.1 Tissue Model – Bovine Cortical Bone

In order to evaluate the material properties of bone under various treatment conditions, the

experimental set-up calls for a large number of cortical bone samples. Cortical bone is the

more dense form of bone that provides structure and protection to the body and organs. Since

we are concerned with the mechanical properties of large structural allografts, cortical

sections of long bones are of interest in the study. In order to eliminate the influence of whole

bone shape and size and to simplify the testing procedure, small uniform beams of cortical

bone were tested. Due to the difficulty in obtaining large amounts of human bone, bovine

bone was used. The experiments in this study were conducted using cortical bone from the

tibia and metatarsal of steers aged 2 years old. Bovine bone has been used by other

investigators to evaluate the mechanical properties of bone [50, 51, 52, 53]. The tibia and

metatarsal bones of steers are large and the cortical wall is very thick, allowing for many

sample beams to be cut from just one bone. Steers are fully grown at 2 years, meaning they

have a much faster growth rate than humans (who are fully grown around 16 years of age

[50]). Bovine bone differs from human bone in several ways. Humans exhibit secondary

osteonal bone, where pre-existing bone is resorbed and replaced with cylindrical structures

called osteons that consist of concentric layers of lamellae around a Haversian canal

(containing blood vessels and nerves). Cattle, on the other hand, grow much faster and

larger. Because of this difference in growth rate, they exhibit a different form of bone called

plexiform bone. Plexiform bone is characterized by sheets of lamellar bone and sheets of

blood vessel networks, with highly mineralized non-lamellar bone in the interstitial spaces

[15]. Bovine plexiform bone is remodeled by osteons as the cow ages, past this initial stage

of fast growth. Plexiform bone is more mineralized than secondary osteonal bone, and

therefore it is stiffer [53]. Because of these differences, bovine bone serves as a good model

for the initial study of these treatments however further validation in human bone will be

required in the future.

19

1.8.2 Design of Experiments

In each of the experiments in this study, small rectangular beams (see each chapter for

specific dimensions) cut from the cortical wall of bovine bone was subjected to various

conditions prior to irradiation. Except for the controls, all samples were treated and then

irradiated. Irradiated samples were “treated” simply by incubating in PBS to control for the

incubation conditions of the samples treated in ribose solutions. The general experimental

procedure for each experiment is demonstrated in Figure 1.7. Note that fracture testing was

only performed in one experiment (see Chapter 5), while three-point bending was used as the

mechanical test in all others.

1.8.3 Methods for Characterizing Bone Collagen

One aim of this study was to characterize the material properties of bone and more

specifically bone collagen in native, irradiated, and ribose-treated bone to assess the

modifications caused by these processes. There are several established techniques to

investigate the structure, thermal stability, and thermomechanical properties of the organic

matrix of bone.

Figure 1.7: Experimental procedure for treating, irradiating, and testing bovine bone samples

20

Collagen structure: SDS-PAGE and HPLC

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique often

used to obtain the molecular weight distribution of a protein sample. It has been used by

previous investigators to assess the fragmentation of collagen due to irradiation damage [3,

42]. Protein samples are mixed with SDS, which is a powerful detergent with a hydrophobic

end and a highly charged end. The hydrophobic end of SDS interacts with the amino acid

side groups and destroys any tertiary protein structure. The result is a linear protein coated in

highly charged SDS, and within the sample there is a constant mass to charge ratio. These

proteins are loaded into the wells on a polyacrylamide gel and once an electric field is

applied, the proteins move through the gel as a function of their molecular weight. At the end

of the run, the proteins are stained and the MW distribution of the sample can be imaged and

analyzed. In a pure sample, where all the proteins are the same, there would be one band at

the MW of that particular protein. A heterogeneous sample with a continuous distribution of

molecular weights would stain throughout the lane, creating a smearing effect.

Collagen has four distinct bands, one for three alpha chains crosslinked together (gamma

band), one for two alpha chains crosslinked together (beta band), and two bands for the two

alpha chains, one for each type (alpha-1 and alpha-2). Fragmented collagen will exhibit a

decrease in stain density at the bands and an increase in smearing below the alpha bands [3,

42]. It is expected that irradiation damage will have abnormal banding patterns and damage

can be easily identified as a smear based on previous work by Akkus et al. [3]. In preparation

for SDS-PAGE, the bone sample is demineralized and digested with pepsin. Pepsin cleaves

peptide bonds at hydrophobic amino acids (such as alanine and tyrosine) which are mostly

found in the telopeptide regions of collagen. Enzymatic crosslinks are found in this region, so

cleaving here will remove intermolecular crosslinks [54] and free individual collagen

molecules from the crosslinked network. The bone samples are run on the gel against a

reference of purified rat tail tendon collagen in order to compare bone samples to a relatively

clean collagen profile.

21

There are three types of collagen crosslinks with fluorescent properties considered in this

study, including two mature enzymatic crosslinks (hydroxylysyl pyridinoline and lysyl

pyridinoline) and a nonenzymatic glycation crosslink (pentosidine). Although other glyco-

oxidation products are thought to possibly have fluorescent properties (such as imidazole),

pentosidine is the only well-characterized fluorescent glyco-oxidation crosslink [55]. The

fluorescent properties of pyridinolines allow them to be used as a biomarker in urine to

indicate increased bone turnover in people with osteoporosis [21, 56]. The crosslinks in a

given sample are quantified using a High Performance Liquid Chromatography (HPLC)

technique that can also be used to quantify the amount of crosslinks present in a

demineralized bone sample. Hydrolyzed bone samples are introduced into a chromatography

column in a constantly running flow of solvent. Inside the column, the chromatographic

material achieves separation of different analytes which will have a known elution time. The

analytes „stick‟ inside the column until high pressures and harsh solvents carry them out of

the column. A fluorescence detector located downstream of the column measures

fluorescence peaks, with separate peaks corresponding to the two mature enzymatic

crosslinks and pentosidine. When compared to a peak of a standard with known

concentrations of these crosslinks, the concentration of crosslinks can be quantified for each

sample. Since enzymatic crosslinks are an important structural feature of bone collagen that

influences the mechanical properties of bone, it is important to identify if irradiation has an

effect on them. Pentosidine is a marker of glyco-oxidation crosslinking and will indicate if

we are introducing crosslinks with our ribose pre-treatment of irradiated bone.

Thermal Properties of Collagen: Differential Scanning Calorimetry and Hydrothermal

Isometric Tension Testing

Heating of collagen will result its denaturation, or the loss of secondary and tertiary protein

structure (i.e. melting). The temperature at which this occurs is called the temperature of

denaturation. The temperature of denaturation reflects the stability of the native structure of

the molecule, in other words the forces keeping the triple helix together. Collagen molecules

are metastable at 370C [57] but gain considerable stability from fibril formation (temperature

of denaturation is increased to ~650C) [58]. Once enough kinetic energy is added during

heating, the molecular motion increases to a point at which the triple helical structure is lost

22

to a more thermodynamically favorable configuration (a random coil). Things that effect

denaturation temperature include protein folding, hydrogen bonding, intermolecular

interactions, and crosslinking [59]. The overall heat used to completely denature the sample

is called the enthalpy, or ΔH.

In differential scanning calorimetry, a sample in an air-tight sealed pan and an empty

reference pan are heated at a constant rate. The heat flow into both pans is measured, and the

difference in heat flow is recorded. During a phase transition, in this case denaturation of the

collagen, more heat will be needed by the sample than the reference pan. A resulting peak in

the measured heat flow vs. temperature curve can be analyzed to study the thermal stability

of the sample [60]. A lowering of the expected temperature of denaturation would indicate

collagen degradation [44, 60, 61]. Enthalpy, or the total heat required to melt the collagen,

can be obtained as the area under the denaturation peak. A change in enthalpy indicates

degradation or modification of the collagen molecules [60, 62].

Hydrothermal Isometric Tension (HIT) testing has not been used to study demineralized bone

collagen very frequently, although it can provide a lot of insight into the nature of irradiation

damage. It takes advantage of an interesting property of collagen: when heated slowly it will

contract. Heating collagen to the required temperature causes a transition from crystalline

chains to random amorphous coils [63] and thus shrinkage in length. In an HIT test, a small

collagen specimen is held in isometric constraint (constant length) while temperature is

slowly increased. This way, the contraction is inhibited and the sample creates a contractile

force. At a certain temperature, the collagen is driven to denature and an increase in tension

is recorded. For bone, this temperature (sometimes called shrinkage temperature) has been

shown to decrease with age in both rats and humans [36]. Since collagen stability has also

been shown to decrease with age [36, 63] it has been suggested that a decrease in collagen

stability will lower this shrinkage temperature. After the initial onset of contraction, there is a

period of increasing tension until the sample reaches its maximum force and ruptures. The

behavior during this test gives a measure of the connectivity of the collagen network.

Connectivity can be defined as the integrity and density of collagen crosslinks and the

stability of the network. In other words, how well collagen is connected together in a network

and the stability that this conformation provides. The thermomechanical properties measured

23

in this study reflect the thermal stability of collagen molecules as well as the quality and

quantity of intermolecular crosslinks.

24

Chapter 2

2 Bone Embrittlement and Collagen Modification Due to

High Dose Gamma-Irradiation Sterilization

2.1 Introduction

Gamma-irradiation is a widely-used method by tissue banks because of concerns for the

possibility of pathogen transfer and the superior sterilization capabilities of this technique

[64, 65]. Gamma irradiation kills pathogens by damaging the DNA and RNA directly by the

gamma rays and also indirectly through highly reactive free radicals created by the radiolysis

of water [12]. Because of the effectiveness and ease of use, irradiation is often favored by

tissue banks as a means of sterilization over other methods, such as chemical or heat

sterilization [64]. Preparation of allograft tissue under aseptic conditions and screening

procedures are currently part of the tissue harvest process, but some incidence of infection

occurs even with these precautionary measures in place (reports range from 7% to 53%

incidence of infection from the use of cadaveric allograft bone, according to Ngyuyen et al.

[65]). Terminal sterilization limits the risk of infection; unfortunately, the same process that

kills pathogens is thought to damage the collagen of tissues, especially in connective tissues

such as tendon and bone. Thus a method of rescuing damage caused to collagen in this

process would greatly benefit the field of allograft technology.

Many investigators have reported embrittlement of bone due to gamma irradiation, yet the

underlying mechanisms are not well understood. Since collagen is a major structural part of

bone and lends itself to toughening mechanisms, the study of changes to collagen could shed

light on the loss of toughness seen in irradiated bone. It has become abundantly clear that the

changes in collagen due to gamma-irradiation play a major role in the loss of toughness seen

in bone allografts. The experiments in this chapter aim to evaluate both the integrity of

irradiated bone as a whole and the thermal and structural properties of irradiated collagen in

order to further understand collagen alterations and investigate embrittlement mechanisms.

25

The objective of this study was to evaluate changes in bone as a result of irradiation and

understand what collagen damage can tell us about the embrittlement of bone. The

hypothesis was that by looking at both the bulk mechanical properties and some properties

that reflect the state of collagen, we would be able to suggest a relationship between the

embrittlement of bone and the damaging effects to the structure and thermal behavior of

collagen.

2.2 Methods

2.2.1 Sample Preparation

Ten metatarsal bones from steer (aged 2 years old) were obtained immediately after slaughter

from a local abattoir and dissected after approximately 24 hours of refrigeration at 4oC. The

bone was stripped of all soft tissue such as muscle and fat. The posterior section of the bone

was then isolated into a block (approximately 100 mm x 25 mm x 4mm) using a band saw.

Later, each posterior block was cut into rectangular beams with the length along the

longitudinal dimension and the thickness in the radial direction using an Isomet 1000

diamond wafer saw (Buehler Canada, Whitby, ON, Canada). See Figure 2.1. The dimensions

of each beam were 80 mm x 6 mm x 3 mm (l x w x t). Two beams from each bone block

were taken as a matched pair. One beam from each pair was randomly assigned either the

control or irradiated group. All samples were wrapped in saline soaked gauze, stored

individual in empty 15 mL centrifuge tubes, and frozen at -200C.

2.2.2 Irradiation

Irradiation was performed with the help of Allograft Technologies at Mount Sinai Hospital

(Toronto, ON, Canada). All samples were kept frozen inside their tubes and packed in the

center of a box surrounded with dry ice. The box was sent to Steris Isomedix (Whitby, ON,

Canada) and irradiated at ~30 kGy (according to dosimeters located inside and on the outside

of the box) with a Cobalt-60 gamma irradiation source. The box was returned within 24

hours of irradiation and samples were transferred into the freezer until testing.

26

2.2.3 Mechanical Testing

Three-point bending tests were performed in order to evaluate bulk mechanical properties of

the test beams. All specimens were thawed in their saline-soaked gauze wraps at room

temperature (overnight) and rehydrated for 2 minutes in PBS before testing. Measurements

of thickness and width were taken immediately before testing using a Mitutoyo digital

micrometer (Mitutoyo Canada Inc., Mississauga ON, Canada). Three-point bending tests to

failure methods were based on ASTM D790 [66]. A beam was held in a fixture by two

circular supports (diameter 6.35 mm) separated by a span of 65.0 mm (span to thickness ratio

> 20:1). The beams were oriented so that the periosteal side of the bone beam is facing down

(this face was in tension during loading). A cross-head (diameter 6.35 mm) was lowered onto

the center of the test beam. The loading rate was adjusted on a beam-by-beam basis in order

to achieve a strain rate of 1% strain per minute at the tensile surface. See Figure 2.2 for test

set-up. The applied load was measured using a calibrated load cell with a data acquisition

rate of 60 Hz. The cross-head displacement, time, and load were recorded using data

acquisition software. The three-point bending tests were conducted using a Test Resources

100LE2 mechanical testing device with custom made fixtures (Test Resources, Shakopee,

Minnesota, USA).

From the load and displacement data, a stress-strain curve was created using the following

equation to obtain engineering stress:

Where σ is the engineering stress (MPa), t is beam thickness (mm), P is applied load

measured by the load cell (N), s is the span separating the centers of the two supports, I is the

second moment of area (mm4). I = wt

3/12 where w is the width of the beam.

Engineering strain, ε, was calculated as:

27

Where d is the displacement of the crosshead (mm), t is beam thickness (mm), and s is the

span separating the supports (mm).

See Figure 2.2 for a graphical representation of a typical stress-strain curve. The following

parameters were determined from the stress-strain curve: elastic modulus (E), yield stress

(σy), yield strain (εy), ultimate stress (σu), work to fracture (WFx), and failure strain (εf). The

yield point was calculated as the intersection of the curve and a 0.2% strain offset line (see

Figure 2.2). Elastic modulus is calculated as the slope of the elastic (linear) portion of the

curve prior to the yield point. Work to fracture is an estimate of the energy required to break

the beam and can be calculated as the area under the load displacement curve divided by two

times the cross-sectional area. Fail strain is the strain at failure of the beam and represents the

amount of deflection before fracture.

1 2

Posterior

Anterior

Figure 2.1: Bone beams cut from bovine metatarsal bone

28

29

2.2.4 Collagen Characterization Methods

2.2.4.1 SDS-PAGE

SDS-PAGE was performed on demineralized samples to assess the amount of fragmentation

in the collagen chains of irradiated bone. A decrease in the distinct banding pattern (collagen

polypeptide chains) and/or smearing in low molecular weight regions would indicate

collagen fragmentation. A roughly 15 mm x 6 mm x 3 mm portion of the fractured bone

beams was removed using a Buehler Isomet 1000 diamond wafer saw; located away from the

fracture site. These portions were demineralized in 0.5M ethylenediaminetetraacetic acid

(EDTA) for 4 weeks at 40C. Beams were initially considered demineralized when they were

easily cut with razor blade, then tested radiographically to be confident of full

demineralization. Samples were dried, defatted, and ground to a fine powder using a 6750

Freezer Mill (SPEX CertiPrep, Metuchen NJ, USA) to yield approximately 100 mg of

demineralized bone powder. 10 mg of powder was digested with pepsin from porcine gastric

mucosa (lyophilized powder, Sigma Aldrich) in 0.5M acetic acid for 24hrs at room

temperature (1 mg of pepsin for every 10 mg of bone powder). Pepsin cleaves peptide bonds

in the end regions of the collagen molecule, where the enzymatic crosslinks are located.

Cleaving these regions will remove the crosslinks to liberate individual tropocollagen

molecules from the matrix.

Following digestion, the samples were neutralized with 5N NaOH and centrifuged for 30

minutes at 4000 rpm. The supernatant was separated from the insoluble pellet. Four

milliliters of supernatant was transferred into a dialysis-filter centrifuge tube (Amicon Ultra-

15 Centrifugal Filter Device, 10kDa MW cut-off) and centrifuged for 45 minutes at

4000rpm. 50 uL of the concentrate was taken to be used in a colorimetric hydroxyproline

assay to determine the concentration of collagen in the sample (assuming bone collagen is

14% hydroxyproline by mass [67]).

Based on the concentration of each sample, a volume containing 35 ug of collagen was

prepared for SDS-PAGE based on a protocol previously used in our lab [68]. Laemmli

buffer containing 62.5 mM Tris-HCl, 25% glycerol, 2% SDS, 0.01% Bromophenol Blue (pH

30

6.8, from BioRad Laboratories Inc., Mississauga, ON, Canada) plus 10% β-mercaptoethanol

was added to samples in a 1:1 sample to buffer ratio. Samples were boiled for 5 minutes,

cooled, and quickly centrifuged. Samples were loaded into each lane of a 4-20% gradient

Criterion TGX pre-cast polyacrylamide gel (BioRad). A BioRad Precision Plus Protein Dual

Colour Standard (5 uL) and acid-soluble rat tail tendon collagen standard (7.5 uL, Sigma

Aldrich) were run on each gel to act as a molecular weight standard and a pure collagen

control. Gels were run at 200V for 40 minutes, then stained with Commassie Blue (BioRad)

for 1 hour followed by de-staining overnight in a methanol/acetic acid de-staining solution

(1:1 ratio, diluted to 10% each in de-ionized water). De-stained gels were scanned onto a

computer (Canon imageRunner 2525) so that the images could be analyzed with Image J

analysis software to compare alpha, beta, and gamma chain content (band intensity) as well

as the extent of fragmentation (smearing) below the alpha bands.

2.2.4.2 High Performance Liquid Chromatography

HPLC was performed to quantify crosslinks using a previously published protocol from our

lab by Willett et al. [69]. Another portion of each beam (approximately 100 mg) away from

the fracture site was removed and hydrolysed using 6 N HCl at 110 °C for 18 hours. The

samples were diluted and added to a sample buffer containing 10% acetonitrile, 1% HFBA

and water plus an internal standard (pyridoxine). Mature enzymatic crosslinks (lysyl-

pyridinoline (L-Pyr), hydroxylysyl-pyridinoline (H-Pyr)), pentosidine (Pent) and

hydroxyproline (OH-Pro) contents were measured using HPLC methods [69]. The crosslinks

were quantified using a slight modification of a previously published method [70] using

standards of pentosidine and lysyl-pyridinoline (PolyPeptide Group, Strasbourg, France).

Hydroxyproline was quantified using a slight modification of a previously published

method [71] using both hydroxyproline and amino acid standards (Sigma-Aldrich). The

columns were Agilent Zorbax Eclipse XDB-C18 Reversed-Phase C18 HPLC columns

(150 × 4.6 mm, 5 um particle size, 80 Å pore size, endcapped; Agilent Technologies,

Mississauga, ON, Canada). After a sample was run, an elution profile (fluorescence vs.

elution time) for the run time was obtained and the areas under the peaks were determined

and compared to a standard curve in order to get a concentration of each particular crosslink.

These concentrations were normalized to the amount of collagen in the sample, estimated by

measuring hydroxyproline.

31

2.2.4.3 Thermal and Thermomechanical Analysis of Bone Collagen

Differential Scanning Calorimetry

Additional portions of the fractured bone beams located away from the fracture site were

demineralized in EDTA as described above (see section 2.2.4.1) for thermal and

thermomechanical analysis of the organic matrix. A 5 mm x 6 mm x 3 mm demineralized

portion was used for differential scanning calorimetry. Small discs were cut out of the sample

using a 3mm-diameter cylindrical biopsy punch. These discs were halved in thickness so that

they fit flat into hermetically sealed aluminum pans (TA Instruments, New Castle, DE, USA)

in order to ensure an even heat flux. The test method was based on previously published

methods from our lab [68, 69]. Using a TA Instruments Q-2000 DSC with refrigerated

cooling system (TA Instruments, New Castle, DE, USA), the samples were slowly heated

from 250C to 85

0C at a ramp of 5

0 per minute. Temperature and heat flow were calibrated

using an Indium standard. The heat flow was measured as a function of temperature.

The endotherm recorded during collagen denaturation was analyzed using TA Universal

Analysis software. Figure 2.3 shows an example DSC endotherm along with the parameters

calculated during analysis. The temperature at the start of the denaturation peak is TONSET,

calculated as the temperature at the intersection of the steepest tangent line and the

temperature axis. The temperature at the maximum heat flow is labeled TPEAK. The area

under the curve represents the amount of heat absorbed during denaturation, ΔH. The width

of the curve at half the maximum (FWHM) is a measure of the heterogeneity of the thermal

stabilities within a sample [44]. TONSET and ΔH are a function of the molecular structure of

the collagen [61]. Lowering of the expected melting point could indicate collagen

degradation. A change in enthalpy indicates degradation/denaturation or modification of the

heat labile bonding in the collagen molecules. After testing, samples are freeze-dried,

weighed, hydrolyzed and assayed for hydroxyproline content in order to normalize the data

to the amount of collagen in each pan.

Hydrothermal Isometric Tension Testing

32

The remaining portions of demineralized beams were halved with a surgical scalpel to

produce 2 mm x 2 mm x 15 mm samples for hydrothermal isometric tension tests. Testing

was done at the Tissue Mechanics Laboratory at Dalhousie University (Halifax NS, Canada).

The samples were clamped at each end and held in isometric constraint while submerged in a

water bath in a six-specimen HIT device (updated from the version published in [72]). The

temperature was increased at a fixed rate of 10C per minute from room temperature to 90

0C.

At the point when collagen starts to denature (Td), there is a driving force to increase

conformational entropy and the collagen helices start to become amorphous. Without

constraint, the amorphous coils would decrease in length. Because the specimen is held at a

fixed length, tension increases until a maximum isometric force is reached and the specimen

fails. The denaturation temperature (Td), maximum isometric force (MIF), and temperature at

maximum isometric force (TMIF) are labeled on a representative HIT curve in Figure 2.3. The

slope of the curve is a function of the collagen network connectivity. It reflects the crosslink

density (more crosslinks lead to a steeper slope [72]) and the size of the parts between

crosslinks [63]. The slope of the curve was calculated in excel by graphing a linear trend line

that included the temperature and load at half of the maximum force and one degree above

and below this point. The slope of this line was considered the slope at half maximum. The

thermomechanical strength is measured in MIF and TMIF and therefore these parameters

reflect the connectivity of the collagen network [61]. More crosslinking usually leads to

higher values for the slope, MIF, and TMIF [72]. The temperature of denaturation indicates the

initiation of contraction and therefore reflects the thermal stability of collagen molecules

[63]. Maximum Isometric Stress (MIS) is the MIF normalized to the geometry of the sample.

It is calculated as the MIF divided by the cross-sectional area of the specimen.

33

34

2.2.5 Statistics

Paired T-tests were used to test differences between the non-irradiated control and irradiated

groups at the 95% confidence level (p < 0.05).

2.3 Results

2.3.1 Mechanical Properties

Analysis of 3-point bending data revealed significantly lower ultimate stress, yield stress, fail

strain, elastic modulus, and work-to-fracture in irradiated bone, while no difference was

detected in yield strain (refer to Table 2.1). Irradiated samples also showed a 17% loss in

yield stress and a 20% loss in ultimate stress (p = 0.002, p ≤ 0.001). The most notable

differences were the over 50% loss of work-to-fracture (p ≤ 0.001); a measure of the bone‟s

toughness and the 36% loss in failure strain (p ≤ 0.001). There is a decrease in the yield

stress but no significant difference in yield strain. The large loss of toughness is mostly due

to the reduction in strain-to-failure seen in the irradiated samples.

2.3.2 Collagen Characterization

Collagen Fragmentation and Crosslinks

Banding at the molecular weight of the two types of alpha chains from the triple-helical

collagen molecule, alpha-1 and alpha-2, are clearly visible in the control sample lanes on

SDS-PAGE gels, as shown in Figure 2.4. Beta bands (two alpha chains together) and gamma

bands (three alpha chains together) are also present in the control sample. Irradiated samples

show less dense alpha bands and lack beta and gamma bands. There is more “smearing” in

the lower regions of the lane. There was no significant difference between non-irradiated and

irradiated bone for any of the three measured collagen crosslinks including: pentosidine (an

oxidation-dependent AGE), deoxypyridinoline, and pyridinoline (mature enzymatic

crosslinks). See Table 2.2.

Thermal Stability

35

The denaturation behaviour was significantly altered in irradiated bone collagen, as

illustrated in a comparison of control and irradiated DSC curves in Figure 2.5. Irradiated

samples on average showed a 140C decrease in TONSET and an 11

0C decrease in TPEAK when

compared to non-irradiated controls. This corresponds to a 22% decrease in onset

temperature and an 18% decrease in peak temperature (p<0.001 for both). There was an

increase of 300% (p<0.001) in the average enthalpy and an increase of 600% (p<0.001) in

the FWHM for the irradiated group. See Table 2.3. The overall shape of the peak is changed,

in that there is a broadening of the peak corresponding to an increase in both full width at

half maximum (FWHM) of the curve and overall enthalpy.

Collagen Connectivity

The thermomechanical behaviour of irradiated bone collagen was also dramatically different

from that of non-irradiated controls (see Figure 2.5 and Table 2.4). On average, the

temperature of denaturation during HIT testing for irradiated bone was 100C lower than the

control (p ≤ 0.001), which is a 20% decrease. The temperature at maximum isometric force

was also lowered by 20% from around 850C to 67

0C on average for irradiated bovine bone

collagen (p ≤ 0.001). Both the slope of the load vs. temperature curve and the maximum

isometric force were significantly lower in irradiated samples, losing 31% of the slope at half

maximum and 50% MIF when compared to matched controls (p = 0.008 and p ≤ 0.001

respectively).

36

Tab

le 2

.1: M

ech

anic

al p

rop

erti

es f

rom

th

ree

-po

int

ben

din

g. n

= 7

.

37

38

Tab

le 2

.2: C

ross

linki

ng

Qu

anti

fica

tio

n f

rom

HP

LC. n

= 7

39

40

Tab

le 2

.3: D

ata

fro

m d

iffe

ren

tial

sca

nn

ing

calo

rim

etry

. n =

7

Tab

le 2

.4: D

ata

fro

m h

ydro

ther

mal

iso

met

ric

ten

sio

n t

esti

ng.

n =

7

41

2.4 Discussion and Conclusions

The loss of work to fracture and failure strain confirms previous findings that irradiated bone

is more brittle and easier to fracture than a matched non-irradiated control. As shown in

previous studies [3, 4, 12, 38, 39, 43, 46], it appears that irradiation has a bigger effect on

post-yield properties of tissues. Bone gets its toughness from various mechanisms that

dissipate energy. The ability to form microdamage in the form of small diffuse microcracks

[17, 63] absorbs energy through the creation of new surfaces. Microdamage formation cannot

account for all post-yield toughness in bone, however, according to a study by Fondrk et al.

[73]. Their work suggested that collagen-dependent mechanisms such as crack fiber-bridging

and molecular sliding contribute to toughness as well. Losing the ability to form

microdamage prior to fracture will embrittle bone [17]. In the case of irradiated bone, it

would be interesting to further investigate the microdamage formation. The results from this

experiment suggest that there is a lower capacity for energy dissipation before fracture,

which might equate to less formation of microdamage prior to failure [12, 31, 46]

The loss of alpha bands seen in SDS-PAGE suggests peptide fragmentation and smearing

suggests heterogeneous fragments at lower molecular weights. This could mean that

irradiation leads to cleavage of the peptides that make up the collagen molecule, creating

weaker molecules and therefore a weaker network of collagen, which is supported by our

HIT data because the connectivity of the collagen network is decreased with no apparent

change in the crosslinks (that were measured). It could be possible that other collagen

crosslinks are affected, such as immature divalent enzymatic crosslinks not considered in this

study. Further analysis of the effect of irradiation on these crosslinks is required. However,

previous studies showed that irradiation caused an increase in collagen solubility and an

increase in the rate of enzyme digestion for other animal and tissue models which also

supports the theory that irradiated collagen is fragmented [12, 42, 43].

In DSC, increased temperature leads to thermally induced denaturation resulting from an

increase in the kinetic energy of the molecules that eventually causes collagen to melt.

Melting occurs through loss of stabilizing hydrogen bonds and the semi-crystalline structure

of the collagen fibrils that leads to the unwinding of the triple helical molecules [41, 74]. On

42

average, the irradiated bovine bone collagen showed a decrease in denaturation onset and

peak temperature, which reflect a decrease in thermal stability.

A change in enthalpy indicates modification of the collagen molecules. Interestingly, the

increase in enthalpy means it takes more energy to melt irradiated collagen, although it

happens at a lower temperature. Irradiation-induced scission of peptide chains may lead to

the creation of new reactive sites that form new heat labile bonds with surrounding

molecules. Gamma rays can also modify amino acid side chains (without chain scission),

which could alter the interaction of amino acid side chains with bound water. Although there

are possibly new bonds forming, the initiation of melting occurs at a lower temperature,

indicating that while the overall energetic cost has increased, the thermal stability of

irradiated bone collagen is much lower than normal bone collagen. If we consider Gibbs free

energy equation, ΔG = ΔH – TΔS, we can assume that ΔG for irradiated and control are

equal to zero during the phase change at the denaturation temperature. That means that

TONSET (or Td, Tpeak) = ΔH/ΔS and we would expect to see an increase in temperature with an

increase in enthalpy. In this case, we see a large increase in enthalpy but a decrease in

temperature for irradiated collagen. There must also be a large gain of entropy to account for

the decrease in temperature, which is an interesting finding that will require further

investigation.

In HIT, Td is the temperature when load starts to increase due to the driving force of the

unwinding alpha helices to decrease in length [61]. Irradiated samples showed a decrease in

HIT denaturation temperature as well as a decrease in the maximum isometric force (MIF)

reached at failure. MIF and the temperature at MIF reflect the connectivity of the collagen in

the test specimen [72]. The significant decrease in MIF and temperature at MIF for irradiated

bovine bone collagen indicate a loss of connectivity of the collagen network. According to

the crosslink quantification, there was no decrease in two of the mature enzymatic crosslinks

and no change in glyco-oxidation crosslinks. A loss of connectivity and fragmentation of

collagen chains suggests the weakened collagen molecules as the major mechanism for loss

of toughness, similar to the effects of aging on bone properties [Zioupos 1999].

43

Although this experiment provided a lot of information, there are several important

limitations to note. Only three types of crosslinks have been measured here and there are two

other types of crosslinks (mature enzymatic crosslinks called pyrroles and immature

enzymatic crosslinks) that are thought to play a major role in the mechanical and thermal

properties of bone collagen. While we did not see any changes in the measured crosslinks, it

is still unclear if irradiation has an effect on immature crosslinks, as some studies have

suggested [75]. Another limitation is the material used: bovine metatarsal cortical bone.

There was an unexpected significant lowering of the modulus in irradiated bone. Although

irradiation is suspected to mostly effect the post-yield properties of bone, in this study the

pre-yield properties were also slightly decreased. Modulus, or material-level stiffness, is

expected to be mainly influenced by the mineral. It is possible that these young bones are not

as mineralized as, for example, the tibia or femur. BMD from tibia bones of 2-year old steers

was 12% higher than metatarsal bone used in another experiment in this lab (unpublished

data). A different bone would be more ideal for future studies, which is why we have now

switched to tibia in all further experiments.

We can conclude that irradiation damages collagen and this leads to inferior mechanical

properties. Both toughness and strength were decreased in the irradiated model. At the

collagen level, thermomechanical measures of connectivity also suggest that the damage to

collagen weakens the collagen network. This data along with the evidence of degradation of

collagen structure, suggests that a weakened collagen network is unable to support

toughening energy dissipation mechanisms, such as microdamage formation, that give bone

its post-yield ductility and toughness.

44

Chapter 3

3 Ribose Pre-Treatment to Improve Bone Mechanical

Properties

3.1 Introduction

It is clear that irradiated bone has inferior mechanical qualities to non-irradiated bone [3, 4,

12, 46, 75]; however, the use of gamma-irradiation is popular amongst tissue banks in order

to implant biologically safe allografts. Evidence from Chapter 2 suggests irradiation damage

to collagen, specifically fragmentation of collagen molecules, could play a role in the

decrease in mechanical properties. There are several studies that have shown evidence of the

fragmentation of collagen as a result of irradiation. Examples of this evidence include

increased susceptibly to enzyme digestion, increased solubility in solution, decreased thermal

stability, the presence of voids in irradiated soft tissue structure, and increased rate of

resorption of irradiated bone by osteoclasts [12, 42, 44, 45]. Crosslinking collagen prior to

gamma irradiation in a tendon model has been pursued as an attempt to overcome the

deleterious effects of fragmentation [38, 39, 47, 48]. Using crosslinking agents such as

genipin and glucose, some studies showed radioprotection of the elastic modulus in a bovine

tendon model and the strength, modulus, and toughness in a rabbit tendons model [38, 39]. In

irradiated bone, it may be beneficial to create new links between the collagen molecules to

compensate for the loss in connectivity. We suspect that the addition of new crosslinks with

ribose, a glyco-oxidation crosslinker, could strengthen weakened chains leading to a tougher

material.

In this study, bovine bone beams were evaluated in 3-point bending for mechanical

properties, radiographically for bone mineral density, and with several collagen

characterization techniques. The objective of this study was to evaluate ribose treatment as a

method to improve the toughness of irradiated bone. This was achieved by comparing the

performance of ribose pre-treated and irradiated bone to that of non-irradiated bone and

irradiated bone with no treatment. Because this was the first investigation of this kind, a

45

variety of ribose groups with slightly different treatment procedures were tested. Secondary

purposes of this portion of the study were to 1) discover optimal treatment conditions and 2)

compare collagen characteristics and potentially link them to the mechanical outcomes. Our

hypothesis was that ribose pre-treatment of bone would have a positive effect on the

mechanical properties of bone, particularly the measures of toughness, and that there would

also be an improvement in the collagen stability and connectivity.

3.2 Methods


Eight tibia bones from steers (aged 2 years old) were obtained immediately after slaughter

from a local abattoir and kept frozen (-200C) for 3-10 days until dissection. Frozen bones

were thawed stripped of all soft tissue (muscle and fat). The periosteum was scraped from the

bone surface using a surgical scalpel. Using a band saw, bones were cut into blocks

approximately 70 mm x 25 mm x 6 mm with three blocks from each bone: distal anterior,

distal posterior, and proximal as shown in Figure 3.1. The location and animal number was

noted and blocks were stored frozen until further processing. A total of 16 blocks from 8

tibias were used. Only distal anterior and distal posterior blocks were processed further

because the proximal blocks had irregular shapes making it difficult to ensure the correct

orientation and consistent microstructure in each of the specimens. Later, each block was cut

into rectangular beams with the length along the longitudinal dimension and the thickness in

the radial direction with an Isomet 1000 diamond wafer saw (Buehler Canada, Whitby, ON,

Canada). Beams had the dimensions of 60 mm x 4 mm x 2 mm (l x w x t). The endosteal

side of the beam is marked with a permanent marker to keep track of orientation. The beams

from each block (10-20 per block) were kept together as a matched set. The beams were

randomly assigned to be a non-irradiated control, irradiated control, or one of the ribose test

groups including: low concentration ribose treatment, medium concentration ribose

treatment, high concentration ribose treatment, and medium concentration + high temperature

ribose treatment (see Table 3.1 for concentrations and conditions).

46

47

Each set of beams was kept matched with controls from the same animal and block. A set

includes one non-irradiated control, one irradiated control, and one of each of the test

specimens. By matching controls to each test sample from same location on the same animal,

we reduce any differences resulting from comparison of one animal to another. Bone is

extremely heterogeneous in microstructure, which is why the beams from each group were

also matched with beams as closely together as possible in the same plane. Further analysis,

however, showed no significant difference in non-irradiated beams from different locations.

Matching each treatment group to a control from the same animal and location will account

for a variation in statistics and provide more power to statistical comparisons between

groups.

A non-destructive screening test was performed prior to further testing to screen for beams

with major defects (i.e. large blood vessel) that would affect the mechanical performance. All

beams from one set were soaked in PBS for 30 minutes. Beams were loaded endosteal side-

up into a custom three-point bend fixture in an Instron E1000 mechanical testing machine

with a 100N load cell. The beams were loaded up to 100 MPA which is known to be well

below the yield point so that any deformation was elastic (non-permanent). The flexure

modulus was calculated as the slope of the stress-strain curve. Beams with moduli two or

more standard deviations away from the group mean were not used in effort to start with

uniform groups and eliminate any pre-treatment differences. After the test, the beam was

unloaded and wrapped in saline soaked gauze. At the end of all sample preparation methods,

there were 16 matched sets containing 6 beams each. All samples were wrapped in saline

soaked gauze, stored individual in empty 15 mL centrifuge tubes, and frozen at -200C.

3.2.2 Treatment

Sixteen matched sets of cortical bone beams were used. Each set contained one of each of the

following six groups: Non-Irradiated, Irradiated, Ribose 1 (0.6M ribose solution), Ribose 2

(1.8M ribose solution), Ribose 3 (3.0M ribose solution), and High T Ribose (1.8M ribose

solution at 550C). Control bones were untreated and left in the freezer (-20

0C) until

mechanical testing. The Irradiated group was incubated in PBS for 24 hours (prior to

irradiation) at 370C to control for the incubation conditions of the test groups. Solutions were

prepared by dissolving powdered D-Ribose (Sigma Aldrich) into PBS to the appropriate

48

concentration and adjusting the pH to 7.4 with dilute HCL or NaOH as needed. Samples

(aside from the controls) were placed in tubes with 45 mL of their respective solutions and

left to incubate for 24 hours. High T Ribose was the only group to be incubated in a water

bath at an elevated temperature of 550C (all other groups were incubated in a warm room at

370C). See Table 3.1 for a list of the treatment conditions. The three different concentrations

of ribose in solution were tested to measure mechanical performance depending on

concentration. In addition, the high temperature (550C) incubation condition was tested as an

attempt to increase the diffusion of ribose into the bone and possibly increase crosslinking

action. Following incubation, all samples were wrapped in saline soaked gauze, stored

individually in empty 15mL centrifuge tubes, and frozen at -200C.

Sample Name Treatment Incubation solution Conditions: time/temp NonIrrad None None Frozen until testing Irrad Irradiation PBS 24hrs/37

0

Ribose 1 Ribose conc. Low PBS + 0.6M D-Ribose 24hrs/37

0

Ribose 2 Ribose conc. Med PBS + 1.8M D-Ribose 24hrs/37

0

Ribose 3 Ribose conc. High PBS + 3M D-Ribose 24hrs/37

0

High T Ribose Ribose high temp PBS + 1.8M D-Ribose 24hrs/55

0

3.2.3 Irradiation

Irradiation was performed with the help of Allograft Technologies at Mount Sinai Hospital in

the same manner as described in section 2.2.2. Briefly, all samples were packed in the center

of a box surrounded with dry ice and sent to Steris Isomedix (Whitby, ON) where it was

irradiated at ~30kGy from a Cobalt-60 gamma irradiation source. The box was received

within 24 hours of irradiation and samples were transferred into the freezer until testing.

3.2.4 Three-point Bending

Following Irradiation, bone beams were thawed at room temperature and polished by hand to

a 1-um finish. Immediately after polishing, beams were placed in 15 mL PBS to soak for 4

hours (at room temperature) prior to testing in order to rehydrate the sample. Three-point

Table 3.1: Treatment conditions prior to irradiation

49

bending tests to failure methods were based on the ASTM D790. Measurements of thickness

and width were taken immediately before testing using a micrometer and entered into the

computer test method before testing each beam. The beam was held by two circular supports

(diameter 6.35 mm) separated by a span of 40 mm (span to thickness ratio > 20:1). The

beams were oriented so that the periosteal side of the bone beam was facing down (this face

will be in tension during loading). A crosshead (diameter 6.35 mm) was lowered onto the

center of the test beam at a constant loading rate of 1.04 mm/min based on the following

equation from ASTM D790 [66]:

Where R is the loading rate, Z is the strain rate (equal to 0.005), L is the span (equal to 50

mm) and d is the beam thickness.

The applied load was measured using a calibrated 100N load cell. The position of the

crosshead, time, and load was recorded using Instron Bluehill data acquisition software. The

three-point bending tests were conducted using an Instron E1000 mechanical testing device

with custom made fixtures. From the load and displacement data, a stress-strain curve was

created from which various mechanical properties are calculated. Refer to section 2.2.3 for

calculations. The following parameters were determined from the stress-strain curve: elastic

modulus (E), yield stress (σy), yield strain (εy), ultimate stress (σu), work to fracture (WFx),

and failure strain (εf). Yield point was taken as the intersection of the curve and the 0.05%

strain offset line (determined experimentally).

3.2.5 Dual Energy X-Ray Absorptiometry

Dual Energy X-Ray Absorptiometry (DEXA) was performed on half of the beam from each

sample after failure in three-point bending. Samples were scanned one at a time on a polymer

tray with positioning markers in the same orientation to avoid variations based on placement

in the machine. The bone mineral density (BMD) of each specimen was measured using a

PIXImus dual energy x-ray absorptiometer. A measure of bone mineral content was divided

over the projected area of the sample surface. Measurements of thickness of the samples

were taken (using digital vernier calipers) at three locations and then the bone mineral

50

content was divided by average thickness to obtain an estimate for the volumetric bone

mineral density (bone mineral content/area x thickness).


Of the groups tested in 3-point bending, four were selected for collagen characterization

(Non-Irradiated control, Irradiated, 1.8M Ribose at 370C, and 1.8M Ribose at 55

0C). These

groups were of interest because the High T Ribose group had the best recovery of toughness

and the 1.8M Ribose group was chosen as a comparison since it was treated with the same

concentration of ribose but at a temperature of 550C instead of 37

0C. Portions of the fractured

beams located away from the fracture site were taken for collagen characterization. These

portions of the bone beams were demineralized in EDTA for 3 weeks at room temperature,

and then prepared for each of the collagen characterization methods. The methods used in

section 2.2.5 were repeated with these samples. Following is a brief overview of each

method:

When investigating collagen fragmentation with SDS-PAGE, samples were demineralized

and ground into a fine powder, then digested with pepsin to liberate individual collagen triple

helices. The digested solution was centrifuged to separate the soluble proteins in the

supernatant and the insoluble fraction in the pellet. The soluble supernatant was filtered,

mixed with Laemmli sample buffer containing SDS, and run on a polyacrylamide gel where

proteins are separated based on molecular weight. The uniformity of the gamma, beta, and

alpha chains of collagen results in distinct bands and heterogeneous fragments will appear as

a smear. In this study, some of the insoluble pellets were freeze-dried and weighed. This

weight was compared to the starting the weight, and the percent matrix solubilized was

calculated (dividing the difference between the starting and pellet weight by the starting

weight) in order to determine susceptibility to pepsin digestion.

Pentosidine crosslinks were quantified using HPLC in order to determine if ribose pre-

treatment was in fact crosslinking collagen. The concentration of pentosidine in the sample

was normalized to the amount of collagen in the sample using a colorimetric assay for

hydroxyproline. In order to measure thermal stability, differential scanning calorimetry was

used. Demineralized bone was heated slowly and the heat flow was measured, which records

a peak during the denaturation that can provide information about the thermal stability of the

51

helix. Hydrothermal isometric tension testing was used to evaluate thermomechanical

properties and give a measure of collagen connectivity. Strips of demineralized collagen

were slowly heated and the increase in tension created by the driving force for amorphous

coils to shrink was recorded. From this curve, several parameters are calculated that reflect

the connectivity of the material. Refer to section 2.2.5 for specific details on these methods.

3.3 Statistical Analysis

The data in this chapter are all presented as mean ± standard deviation, with a p value of less

than 0.05 considered statistically significant. Statistical analysis was performed using SPSS

v18 (SPSS, Chicago, IL, USA). One-way repeated measures ANOVA (RM ANOVA) was

used to detect differences between the means of each group. Repeated measures ANOVA

considers each sample within its matched set, which controls for inter-animal variance. A

Holms-Sidak post-hoc analysis was used for multiple comparisons between groups when

significance was detected using RM ANOVA. The adjusted p-values are reported when

discussing a comparison between two groups.

3.4 Results


The results of three-point bending showed irradiation and ribose treatments had an effect on

ultimate stress, failure strain, and work to fracture while modulus, yield stress, and bone

mineral density were unaffected. See Table 3.2 for mechanical properties and Table 3.3 for

bone mineral density data. On average, Irradiated samples had 20% lower ultimate stress (p =

0.003), 62% loss of work-to-fracture (p ≤ 0.001), and 45% loss of failure strain (p ≤ 0.001)

compared to the non-irradiated control. The 0.6M Ribose treatment was essentially

ineffective, with no parameters significantly different from the Irradiated group. It was also

the only group with significantly lower yield strain (p = 0.019, compared to Non-Irradiated).

1.8M Ribose had 11% recovery of ultimate stress, 13% recovery of work to fracture, and

15% recovery of failure strain although none of these parameters were detected as

significantly different from the Irradiated group. Similarly, 3M Ribose samples demonstrated

recovery of these parameters but they were not significantly different from the Irradiated

52

group. The most notable result was the effect of the High T Ribose treatment. There was a

47% recovery of work-to-fracture, 70% recovery of ultimate stress (p=0.004) and 43%

recovery of failure strain (p = 0.006). 1.8M Ribose and High T Ribose had the same solution

concentration of ribose (1.8M) but different incubation temperatures (370C and 55

0C,

respectively). High T Ribose demonstrated a larger recovery of work-to-fracture (p=0.055

when compared to 1.8M Ribose), which suggests high temperature incubation increases the

positive effect of the treatment on the mechanical properties.



The molecular weight distributions on SDS-PAGE gels showed that rat-tail tendon collagen

and the non-irradiated control bone had four distinct bands: one for gamma (three alpha

chains), beta (two alpha chains), and one for each of the two types of alpha chains. The gels

showed less defined alpha bands, absence of the gamma and beta bands, and smearing in

both the irradiated and ribose-treated groups. Figure 3.3 is a scanned gel image and density

profiles for one of the 16 sets of our samples. Irradiated bone collagen shows lower density

alpha and beta chain banding and more smearing in the region lower than alpha bands.

Similar to the Irradiated sample, the 1.8M Ribose samples also demonstrated less defined

banding and smearing below the alpha chains. High T Ribose-treated bone collagen showed

almost no alpha banding and an overall lower density stain in the lane than all other groups.

In addition, the High T Ribose insoluble pellets were, on average, much denser than control

and irradiated groups. See Table 3.4 for measures of solubility based on the difference

between the starting weight and the weight of the freeze-dried pellet after the soluble

compartment was removed by centrifugation. The High T Ribose samples were only 11%

solubilized while the Non-Irradiated controls were 25% soluble and the Irradiated samples

were 35% soluble. High T Ribose was significantly lower than Irradiated (p = 0.007) but not

statistically different from Non-Irradiated (probably due to high variance).

Pentosidine was not detected in Non-Irradiated and Irradiated samples using HPLC. Figure

3.3 shows representative HPLC elution profiles for one set of samples. There was a

significant amount of pentosidine crosslinks measured in 1.8M Ribose treated samples and,

53

on average, approximately 1.5 times that amount of pentosidine in the High T Ribose

samples; however, a significant difference was not detected between the two ribose groups

(p = 0.342). See Table 3.5.

Thermal Stability

Irradiated bovine bone demonstrated significantly lower denaturation and peak temperatures

in differential scanning calorimetry tests (p ≤ 0.001 and p = 0.001 respectively). When

treated with 1.8M Ribose prior to irradiation, the demineralized bone collagen recovered

25% of denaturation and peak temperatures. The High T Ribose group demonstrated 70%

recovery of onset and peak temperatures. Figure 3.4a displays the behaviour of the different

test groups with DSC curves from one matched set of demineralized bovine bone samples.

Table 3.6 presents data for DSC on the control, irradiated, 1.8M Ribose, and High T Ribose

groups. There were no significant differences in measures of enthalpy; however this is likely

due to high variation in the data.


Bovine bone collagen subjected to irradiation had significantly different HIT curves when

compared to normal bone. Figure 3.4b shows typical load curves from HIT testing for one

matched set of bone collagen specimens. Irradiated bone denaturation temperature and

temperature at MIF were both reduced by ~20% (p ≤ 0.001) while the slope of the curve and

MIS were both reduced by ~47% (p ≤ 0.001). The average Td, slope at half max, TMIF and

MIS for the 1.8M Ribose group were slightly higher than that of the Irradiated group,

however the only significant difference detected was a 30% recovery of TMIF (p = 0.016

compared to Irradiated). The High T Ribose group, on the other hand, recovered 74% of the

slope, 90% of TMIF and 100% of Maximum Isometric Stress (p values indicate no significant

difference detected between Control and High T Ribose). Table 3.7 shows data for HIT tests

for control, irradiated control, ribose treated, and high temperature ribose treated bovine bone

collagen.

54

E =

flex

ura

l mo

du

lus,

σy =

yie

ld s

tres

s, ε

y = y

ield

str

ain

, σu =

ult

imat

e st

ress

, WFx

= w

ork

to

fra

ctu

re, ε

f = f

ailu

re s

trai

n

a Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o N

on

-Irr

adia

ted

(ad

just

ed p

< 0

.05

)

b S

tati

stic

ally

sig

nif

ican

t d

iffe

ren

ce d

etec

ted

co

mp

ared

to

Irra

dia

ted

(ad

just

ed p

< 0

.05

)

c Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o H

igh

T R

ibo

se (

adju

sted

p <

0.0

5)

x* in

dic

ates

p v

alu

es le

ss t

han

or

equ

al t

o 0

.10

bu

t gr

eate

r th

an 0

.05

(fo

r ex

amp

le c

* m

ean

s 0

.05

< p

val

ue

< 0

.10

fo

r

com

par

iso

n t

o H

igh

T R

ibo

se)

Tab

le 3

.2: M

ech

anic

al p

rop

erti

es o

f b

ovi

ne

bo

ne

bea

ms

test

ed in

th

ree

-po

int

ben

din

g. n

= 1

4

55

56

57

Figure 3.3: Representative chromatrograms from HPLC. IS = internal standard Pent = Pentosidine. Ribose pre-treated samples show peaks corresponding to pentosidine and other glycation products.

IS

Pent

58

59

Tab

le 3

.6: D

ata

fro

m d

iffe

ren

tial

sca

nn

ing

calo

rim

etry

. n =

8

T ON

SET =

tem

per

atu

re o

f d

enat

ura

tio

n, T

PEA

K =

tem

per

atu

re a

t p

eak

hea

t fl

ow

, FW

HM

= f

ull

wid

th a

t h

alf

max

imu

m

a Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o N

on

-Irr

adia

ted

(ad

just

ed p

< 0

.05

)

b S

tati

stic

ally

sig

nif

ican

t d

iffe

ren

ce d

etec

ted

co

mp

ared

to

Irra

dia

ted

(ad

just

ed p

< 0

.05

)

c Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o R

ibo

se (

adju

sted

p <

0.0

5)

d S

tati

stic

ally

sig

nif

ican

t d

iffe

ren

ce d

etec

ted

co

mp

ared

to

Hig

h T

Rib

ose

(ad

just

ed p

< 0

.05

)

60

Tab

le 3

.7: D

ata

fro

m h

ydro

ther

mal

iso

met

ric

ten

sio

n t

esti

ng.

n =

13

T d =

tem

per

atu

re o

f d

enat

ura

tio

n, M

IS =

max

imu

m is

om

etri

c st

ress

, T

MIF

= te

mp

erat

ure

at

max

imu

m is

om

etri

c fo

rce

a Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o N

on

-Irr

adia

ted

(ad

just

ed p

< 0

.05

)

b S

tati

stic

ally

sig

nif

ican

t d

iffe

ren

ce d

etec

ted

co

mp

ared

to

Irra

dia

ted

(ad

just

ed p

< 0

.05

)

c Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o R

ibo

se (

adju

sted

p <

0.0

5)

d S

tati

stic

ally

sig

nif

ican

t d

iffe

ren

ce d

etec

ted

co

mp

ared

to

Hig

h T

Rib

ose

(ad

just

ed p

< 0

.05

)

61


There is abundant evidence of the major deleterious effect of irradiation on the mechanical

properties of bone. Recovery of a significant amount of mechanical performance was

achieved by incubating bone in ribose at 550C for 24 hours prior to irradiation. The ribose

pre-treatment also had an effect on the collagen properties, suggesting that for irradiated bone

there may be a link between the recovery of toughness and the modifications in collagen

induced by glyco-oxidation crosslinking.

The modulus and yield stress were not affected by irradiation of bone or any of the pre-

treatments (RM ANOVA p = 0.123 and p = 0.279). These properties are considered to be

mainly influenced by the mineral in bone, since they reflect the stiffness and the transition

point from pre-yield behaviour to post-yield behaviour, respectively. Pre-yield behaviour of

bone means behaviour that is elastic, in other words recoverable deformation. After the yield

point, permanent deformation occurs. Permanent changes in the micro and nano structure of

bone, for example microdamage formation and collagen fiber deformation, absorb energy

until failure. Although not exclusively, it is thought that collagen has more of a role in the

post-yield properties that in the pre-yield properties [17, 32, 76]. It therefore makes sense that

the pre-yield properties are less affected by irradiation and ribose pre-treatment, since they

modify collagen and are thought to have less of an effect on the mineral. This argument is

strengthened by the bone mineral density measurements, which show no significant

difference between irradiated, ribose pre-treated, and control groups (RM ANOVA p =

0.817).

With that said, it would be expected that the yield strain would also be similar between all

groups. However, the 0.6M Ribose group had a significantly lower yield strain than the Non-

irradiated and High T Ribose groups (p = 0.019 and p = 0.042). It was not detectably

different from the Irradiated group. It is important to remember that while pre-yield behavior

is mostly controlled by the mineral component, the composite nature of the material means

these there may be some small effects on these properties due to modifications in the

material, which is probably the case here.

62

While incubation with three different concentrations of ribose at 370C prior to irradiation

demonstrated some recovery of ultimate stress, work to fracture, and failure strain, a

significant difference from the Irradiated group was not detected for these treatment groups.

The treatment with the highest recovery of these parameters, High T Ribose, had a

concentration of 1.8M ribose and was incubated at 550C. This group had a large recovery of

work-to-fracture, a measure of toughness, and also the largest recovery of ultimate stress and

failure strain. It is unclear whether this is due to increased diffusion of ribose into the bone or

due to increase crosslinking reaction as a result of the higher incubation temperature;

however, in either case, it is clear that the high temperature incubation is required for optimal

performance of the bone material.

In vivo, crosslinks formed by the reaction of simple sugars and the collagen in bone (and

other tissues) form over time. In this study, the acceleration of crosslinking reaction leads to

the addition of many new glyco-oxidation crosslinks, as suggested by the increased levels of

pentosidine in the 1.8M Ribose and High T Ribose samples shown in Table 3.5. Pentosidine

is one of many glyco-oxidation crosslinks, so it can be used as a marker or indicator of

crosslinks formed [26, 28]. Pentosidine levels in the High T Ribose group were higher than

that of the 1.8M Ribose group. The HPLC data is important because it confirms that our

treatment is introducing new crosslinks, but it is not a quantification of total crosslinking.

Another thing we do not know is where these crosslinks are located within the matrix. We

can speculate that they are intra- and intermolecular collagen crosslinks, because the group

with highest increase in pentosidine (High T Ribose) also has an increase in the slope and

maximum load reached in HIT testing, an indication of crosslinking density but also that

these crosslinks are acting to hold the collagen network together by linking molecules. On

the other hand, 1.8M Ribose had a substantial amount of pentosidine detected (77.5 ± 28.0

mmol pentosidine per mole of collagen) but a smaller increase in connectivity (37% recovery

of MIS for 1.8M Ribose vs. 100% recovery of MIS for High T Ribose). This suggests that

somehow, the pentosidine crosslinks induced with the 1.8M Ribose treatment were not as

successful in improving connectivity. Whether this is simply due to a lower number of

crosslinks (a little over half the pentosidine content of High the T Ribose group) or some

other aspect of the crosslinking action affected by the high temperature incubation remains to

be determined.

63

Fragmentation of collagen molecules due to irradiation is suggested by the smearing and loss

of gamma, beta, and alpha bands seen on SDS-PAGE gels and the increase in soluble

fraction. It is possible that ribose treatment and/or irradiation can modify the solubility of the

collagen, making it either more or less soluble to pepsin-digestion. The solubility data (see

Table 3.4) suggests that the irradiated matrix is more soluble to pepsin digestion than the

control (35% vs. 25%) which could mean the structure of the molecules is less stable. This

agrees with DSC data showing a lowering of the denaturation onset temperature, another

indication of unstable molecules. The average solubility of the High T Ribose group is lower

than both Irradiated and Non-Irradiated matrix, with only 10% soluble after pepsin digestion.

This means it is possible that most of the modified collagen remains in the pellet and is not

represented on the gel. Collagen with increased levels of glyco-oxidation crosslinking has

been shown to be less soluble to pepsin digestion [28], so this data further supports the

crosslink quantification and HIT data suggesting increased crosslinking due to the high

temperature ribose treatment.

In DSC, the denaturation temperature can be used as an indicator of thermal stability because

it is the temperature at which the kinetic energy of the molecules eventually causes collagen

to start melting. A higher denaturation temperature indicates a more energetically stable

conformation. Collagen denaturation involves the loss of stabilizing hydrogen bonds and the

fibril structure that causes to triple helix to unwind and become amorphous [16, 74]. Peak

temperature, the temperature at maximum heat flow into the sample, also reflects thermal

stability. On average, the irradiated bovine collagen showed a decrease in denaturation onset

and peak temperature which reflects a decrease in thermal stability. 1.8M Ribose treatment

was able to recover some of the onset denaturation and peak temperatures (increased from

Irradiated by about 50C and 4

0C, respectively) but only the increase in denaturation

temperature was significant (p = 0.032). The High T Ribose treatment had significant

recovery of both temperatures, indicating that the crosslinking action (evidenced by

pentosidine data) may have caused an increase in stability by creating stable links between

molecules. This seems to have happened to a lesser extent in the lower temperature

incubation, and had nearly full recovery of these temperatures in the high temperature

incubation.

64

There was a similar trend in the HIT tests. Irradiated collagen showed a dramatic decrease in

temperature of denaturation, slope of the curve at half maximum, maximum isometric stress,

and temperature at maximum isometric stress. 1.8M Ribose showed little to no recovery, and

the High T Ribose group had significantly higher temperature of denaturation, slope of the

curve at half maximum, maximum isometric stress, and temperature at maximum isometric

stress. A higher slope and maximum isometric force suggests there is an increase in collagen

connectivity. This is not simply a function of the number of crosslinks, but reflects the extent

to which the collagen is connected, so the links but also the integrity of the molecules that are

linked together. Lee et al. [72] showed that chemically induced crosslinks increase the slope

and maximum force well above that of the control in bovine pericardia. This increase in HIT

properties also related to a stiffening of the material, so an increase in HIT properties does

not necessarily mean a tougher material. In our case, we do see a recovery of work-to-

fracture which suggests that increased connectivity in irradiated bone collagen will lead to an

increase in toughness. This is most likely due to the fact that irradiated collagen is broken

and in order to maintain mechanical integrity, new links must be made to hold the material

together under loading.

It is important to note that if our theory is correct, the High T Ribose treated bone recovers

toughness but not native collagen structure. In other words, there may be a limit to the

recovery of toughness using this treatment method because increasing the number of

connections between fragments does not return collagen to its normal, native state. Native

collagen exists in long molecules connected together in a network, with links in the end

regions of the molecules. The reason that glycation is thought to embrittle bone is because it

creates crosslinks between the helices, and restricts motion and stretching of the molecules.

In our case, stitching fragments back together with non-site specific crosslinks results in a

network that is more connected than irradiated collagen, but perhaps not as ductile as native

collagen networks.

An interesting discovery is that ribose incubation at a high temperature resulted in better

recovery of toughness, connectivity, and thermal stability. The initial reason for testing high

temperature incubation was to increase the diffusion of ribose into the bone. Something

interesting to note, however, was an increased browning of samples incubated at 550C over

samples incubated at 370C. Browning is an indicator of the Maillard reaction [55] although it

65

is not clear whether full crosslinks have formed or just the precursor products that would

eventually form crosslinks. Either way, it suggests that perhaps there are reactions happening

prior to irradiation. It seems feasible that the increased temperature would increase the

number of reactions and therefore create more crosslinks, which could be the reason that

there was more recovery of connectivity, thermal stability, and toughness in the High T

Ribose group.

While we have evidence supporting the fact that crosslinks lead to connectivity which leads

to an increase in toughness, we cannot be sure there is not more to the story. DSC data

indicates that the thermal stability of the molecules is also recovered in the High T Ribose

treated samples. It has been shown that increased crosslinking increases the temperature of

denaturation [69, 77], so our data could suggest that the crosslinking from ribose treatment

and fragmentation from irradiation, in a sense, cancel each other out. The result is a DSC

endotherm very similar to that of native collagen, however more information is needed on the

structure of pre-treated and irradiated ribose in order to more fully understand our results.

We can conclude from this experiment that it is possible to recover toughness in irradiated

bone using a pre-treatment with ribose. Incubating the bone in ribose solution at 550C

recovered more work to fracture, ultimate stress, and fail strain than the same solution at

370C. High temperature ribose treatment also had the effect of increasing measures of

thermal stability and collagen connectivity (measured in DSC and HIT). The crosslinks

created during treatment made the collagen more resistant to pepsin digestion and therefore

an accurate molecular weight distribution cannot be obtained using our SDS-PAGE method.

Overall, this suggests that the degradation of collagen due to irradiation can be rescued with

modification via glyco-oxidation crosslinking to increase the connectivity and stability of the

collagen network, which in turn results in tougher bone.

66

Chapter 4

4 Comparing Ribose to other Crosslinking Agents

4.1 Introduction

The collagen component plays an important role in the mechanical properties of bone,

especially the toughness [13, 17, 32]. Modifications to collagen will have an effect on the

overall properties of bone, as we have seen in the case of irradiation embrittlement (Chapters

2 and 3). Others have suggested that the mechanical integrity of normal, healthy bone is

negatively affected by glyco-oxidation crosslinking in pathologies such as aging and diabetes

[26, 28, 25]. Non-enzymatic glyco-oxidation crosslinks are thought to be distributed all

throughout the collagen structure, including at helix-to-helix locations. A high level of glyco-

oxidation crosslinks in normal tissue may lead to a stiffening of tissues [24, 55, 78]. Several

studies have demonstrated the embrittlement of bone due to induced glycation [69, 79, 80].

In the case of irradiated bone, an increase in the level of crosslinking may not have a negative

effect on the mechanical properties because the scission of the peptide backbone due to

irradiation results in a broken structure with low levels of connectivity in the collagen

network. As shown in Chapter 3, incubation of bone samples in a ribose solution at an

elevated temperature prior to irradiation demonstrated recovery of work-to-fracture

compared to an irradiated control.

The objectives of the experiments in this study are to:

1) Evaluate ribose, glucose, fructose, and ascorbate treatments as a method to improve

the toughness of irradiated bone and identify which is the optimal treatment

2) Study changes in bone collagen as a result of ribose and glucose pre-treatment plus

irradiation in order to gain insight into collagen alterations

An important part of this study was to compare ribose to glucose, a glyco-oxidation

crosslinking agent that has been used by other investigators to crosslink tendons prior to

irradiation [49] and is essentially a competitor (from an IP perspective). Pre-treatment of

67

bovine bone with ribose, glucose, fructose, and ascorbate was tested using the most

successful incubation condition from previous experiments (see Chapter 3). Glucose and

fructose are similar to ribose (all are ring-structured simple sugars in the aldose family) and

ascorbate (vitamin-C) is a free radical scavenger with some ability to glycate. All

crosslinking agents were tested for mechanical property recovery using three-point bending.

For the control, irradiated, high temperature ribose and high temperature glucose groups,

differential scanning calorimetry, hydrothermal isometric tension testing, high performance

liquid chromatography and SDS-PAGE were performed on demineralized specimens.

Glucose and fructose are similar to each other in structure, and because no positive results (or

any differences from the glucose group) were detected in mechanical testing, the fructose

group was not processed for further testing. Glucose has been used in literature as a

crosslinking agent for tendons, so the mechanism of collagen modification (or lack thereof) is

of interest. Ascorbate, as mentioned, is not ideal for use in a sterilization process because it

protects the pathogens (free radical damage is the mechanism of pathogen elimination) thus it

was not included in the collagen characterization.

It is our hypothesis that ribose treatment will be superior to all other agents in the recovery of

mechanical properties (such as ultimate stress and work to fracture). As a smaller molecule,

ribose should be able to diffuse more thoroughly into the bone material. More importantly,

the reaction requires sugars in an open chain form. Because ribose is more often found in this

form, it has faster glycation kinetics than glucose [81] and should be a more effective

crosslinking agent. We also anticipate that ribose treatment will have better recovery of the

collagen thermal and thermomechanical properties over glucose treatment. We will explore

the relationship between the properties of modified collagen to the recovery of the bulk

mechanical properties of bone, in an effort to explain the differences in behavior of these two

different treatments.

68

4.2 Methods


A total of 10 tibias from steers (aged 2 years old) were used for this experiment. Samples

were prepared as described in section 3.2.1. Briefly, tibias were obtained immediately after

slaughter and frozen (-200C) for 3-10 days. Then they were thawed, cleaned and cut into

bone blocks. Two blocks from the distal portion of each tibia were used, allowing for 20 sets

(see Figure 3.1). Beam dimensions were 60 mm x 4 mm x 2 mm (l x w x t). After screening

for any outlier samples with a non-destructive measurement of the modulus (see section

3.2.1), there were at least seven (7) beams per set. Each beam was wrapped in saline-soaked

gauze and stored frozen until further processing.

4.2.2 Treatment

Before treatment, all samples (aside from controls) were thawed at room temperature. All

four agents (ribose, glucose, fructose, and ascorbate) were purchased in powder form from

Sigma Aldrich. The agents were dissolved in PBS to a concentration of 1.8M and pH was

adjusted to 7.4 with dilute HCL or NaOH as needed. A concentration of 1.8M solution was

successful in previous experiment (Chapter 3). Each set contained seven groups: Non-

Irradiated, Irradiated, High T Irradiated, High T Ribose, High T Glucose, High T Fructose,

and High T Ascorbate. The “High T” indicates the sample was incubated at 600C. The agent

was different for each group, with no agent in the High T Irradiated group. The beams were

incubated in 45 mL of their respective solution. See Table 4.1 for a list of treatment

conditions.

69

Group Treatment Incubation solution Conditions: time/temp

NonIrrad None None Frozen until testing Irrad Irradiation PBS 24hrs/37

0

High T Irrad Irrad + high temp incubation PBS 24hrs/600

High T Ribose Ribose + high temp

incubation PBS + 1.8M Ribose 24hrs/60

0

High T Glucose Glucose+ high temp

incubation PBS + 1.8M Glucose 24hrs/60

0

High T

Fructose Fructose+ high temp

incubation PBS + 1.8M Fructose 24hrs/60

0

High T

Ascorbate Ascorbate+ high temp

incubation PBS + 1.8M

Ascorbate 24hrs/60

0

4.2.3 Irradiation

Irradiation was performed with the help of Allograft Technologies at Mount Sinai Hospital in

the same manner as described in section 2.2.2. Briefly, all samples were packed in the center

of a box surrounded with dry ice and sent to Steris Isomedix (Whitby, ON) where it was

irradiated at ~30kGy from a Cobalt-60 gamma irradiation source. The box was received

within 24 hours of irradiation and samples were transferred into the freezer until testing.

4.2.4 Mechanical Testing

Three-point bending to failure was performed on all test samples to evaluate bulk mechanical

properties. The method described in section 3.2.4 was repeated for this set of samples.

Briefly, bone beams were thawed at room temperature and polished by hand to a 1-um finish.

Immediately after polishing, beams were placed in 15 mL PBS to soak for 4 hours (at room

temperature) prior to testing in order to rehydrate the sample. Beams were placed into the

machine (periosteal side down) and loaded at a constant crosshead displacement rate of 1.04

mm/min based on beam dimensions and strain rate of 0.005 %/sec (based on [66], see section

3.2.4 for calculations). The position of the crosshead, time, and load was recorded. The

three-point bending tests were conducted using an Instron E1000 mechanical testing device

Table 4.1: Treatment conditions prior to irradiation for Ribose, Glucose, Fructose, and Ascorbate

70

with custom made fixtures. From the load and displacement data, a stress-strain curve was

created from which various mechanical properties are calculated. Refer to section 2.2.3 for

calculations. The following parameters were determined from the stress-strain curve: elastic

modulus (E), yield stress (σy), yield strain (εy), ultimate stress (σu), work to fracture (WFx),

and failure strain (εf). Yield point was taken as the intersection of the curve and the 0.05%

strain offset line (determined experimentally).

4.2.5 Dual Energy X-Ray Absorptiometry

Dual Energy X-Ray Absorptiometry (DEXA) was performed on one-half of each fractured

sample after failure in three-point bending. Samples were scanned radiographically one at a

time and in the same orientation to avoid differences based on placement in the device. The

protocol described in section 3.2.5 was repeated for these samples; refer to that section for

details on the methods. The volumetric bone mineral density was calculated and averaged for

each test group.


Following mechanical testing, four of the groups were chosen for collagen characterization:

Non-Irradiated controls, the Irradiated group, the High T Ribose treatment group and the

High T Glucose treatment group, as discussed in the introduction.

Portions of the fractured beams located away from the fracture site were taken for collagen

characterization. These portions of the bone beams were demineralized in EDTA for 3 weeks

at room temperature, and then prepared for further procedures. The methods of collagen

characterization used in this experiment were previously described in section 2.2.5 and a

brief overview was provided in section 3.2.5. They included investigating collagen

fragmentation with SDS-PAGE, solubility to pepsin digestion, measuring thermal stability in

differential scanning calorimetry, measuring thermomechanical properties with hydrothermal

isometric tension testing, and quantifying pentosidine crosslinks with HPLC. Please refer to

section 2.2.5 for a detailed description of these methods or section 3.2.5 for an overview.

71








significance was detected using RM ANOVA. The adjusted p-values are reported when


4.4 Results


Three point bend tests to failure demonstrated the embrittlement of bone due to irradiation

and recovery of toughness with the use of high temperature ribose pre-treatment. Controlling

for the high temperature incubation had no protective effect; all measured parameters were

nearly equal and no significant difference was detected between the Irradiated and High T

Irradiated groups. From now on we will refer to these groups together simply as the

“Irradiated” group. Table 4.2 lists the results for modulus, yield stress, yield strain, ultimate

stress, work to fracture, and failure strain. Modulus, yield stress, and bone mineral density

(see table 4.3) were not affected by irradiation or any of the treatments. Interestingly, the

yield strain of the High T Ribose group was slightly higher than all other groups. It was

significantly higher than the Irradiated, High T Glucose, and High T Fructose groups but not

detectably different from Non-Irradiated controls.

The Irradiated group lost 15% ultimate stress, 56% work-to-fracture, and 43% failure strain

(p ≤ 0.001 for all three parameters when comparing to Non-Irradiated controls). The High T

Ribose treatment was superior when compared to glucose, fructose and ascorbate in terms of

recovery of mechanical properties. When comparing the High T Ribose group to the

Irradiated group, there was a 57% recovery of work-to-fracture (p < 0.001), a 50% recovery

of fail strain (p < 0.001), and 100% recovery of ultimate stress (p=0.001). High T Glucose

72

resulted in some mild improvement of work to fracture, ultimate stress, and failure strain,

although none of these parameters were detected as significantly different from the Irradiated

group. High T Fructose treatment was also ineffective in recovery of mechanical properties;

none of the calculated parameters were significantly different from the High T Glucose

group. Ascorbate demonstrated protection (as expected because it is a free radical scavenger)

however not as much recovery as the ribose treatment group (98% recovery of ultimate

stress, p = 0.001, and 33% recovery of work to fracture, p = 0.004).



SDS-PAGE gels demonstrate the effects of irradiation and crosslinking treatments on the

pepsin-soluble organic matrix after demineralization. The gamma, beta and alpha bands are

less dense for irradiated samples and more smearing is apparent. High T Glucose samples

had similar density profiles to the irradiated group, with a loss of gamma and beta bands, less

dense alpha bands, and more smearing at low molecular weights. Figure 4.1 shows a typical

gel and stain density profile for one set of matched specimens. The High T Ribose treated

samples show no evidence of banding (gamma, beta or alpha) and little staining in the lane at

all, suggesting that perhaps the modified collagen remains in the pellet. To investigate this,

some pellets were dried and weighed, then compared to the starting amount of bone powder.

It was found that High T Ribose insoluble pellets were, on average, much denser than

control, irradiated, and High T Glucose groups. On average, the High T Ribose samples were

only 9% solubilized while the Non-Irradiated controls were 25% soluble, Irradiated samples

were 31% soluble, and High T Glucose samples were 31% soluble. Significance, however,

was only detected between High T Ribose and High T Glucose groups (p = 0.043).

Pentosidine was not detected in Non-Irradiated, Irradiated, or High T Glucose samples using

HPLC. See Figure 4.2 and Table 4.5 for representative HPLC chromatograms from one set

and pentosidine crosslink quantification data for each group. The only group with

pentosidine measured was the High T Ribose group, with an average of 45 ± 4.7 mmol

73

pentosidine per mol of collagen. It is important to note that while glucose is capable of

forming pentosidine, pentosidine is only one of the many crosslinks possibly formed due to

the glucose treatment. It is likely that other crosslinks or adducts were formed as a result of

the reaction between glucose and collagen, however, these crosslinks were not quantified

with HPLC.

Thermal Stability

Following the trend of previous experiments, the irradiated bovine bone collagen showed a

significant loss in denaturation and peak temperatures and an increase in enthalpy (p ≤ 0.001

for all). The High T Ribose group demonstrated 100% recovery of onset and peak

temperatures. In fact, denaturation temperature, peak temperature, enthalpy, and full width at

half maximum (FWHM) were not significantly different between non-irradiated control and

high temperature ribose treated groups. Figure 4.2 shows example DSC curves for the non-

irradiated, irradiated, and high temperature ribose groups from one matched set of specimens.

The high temperature Glucose group demonstrated moderate recovery of TONSET and FWHM

(p = 0.001, p ≤ 0.001). Table 4.6 presents DSC results for control, irradiated, high

temperature Ribose and high temperature Glucose groups.


HIT testing revealed a loss of thermomechanical parameters for irradiated samples and

recovery with the use of ribose and glucose pre-treatments. The temperature of denaturation

and temperature at maximum isometric force were decreased by 100C and 20

0C (p ≤ 0.001

for both) due to irradiation, which corresponds to a ~20% loss for both temperatures. On

average, the irradiated samples showed a loss of 55% of the maximum isometric stress and

35% of the slope at half maximum when compared to non-irradiated controls (p ≤ 0.001 for

both). The High T Ribose group had 54% recovery of denaturation temperature, 93%

recovery of temperature at MIF, 100% recovery of slope, and 100% recovery of Maximum

Isometric Stress (p ≤ 0.001 for all). See Table 4.7 for a list of measurements. Figure 4.2

demonstrates the recovery of these thermomechanical measurements with example curves

from one matched set of specimens. The High T Glucose group also demonstrated some

recovery of the slope of the curve (72%, p = 0.016), TMIF (39%, p ≤ 0.001) and MIS (83%, p

74

≤ 0.001) however the temperature of denaturation was not significantly different from the

irradiated group.

75

Tab

le 4

.2: M

ech

anic

al p

rop

erti

es f

rom

th

ree

-po

int

ben

din

g co

mp

arin

g d

iffe

ren

t ag

ents

E =

flex

ura

l mo

du

lus,

σy =

yie

ld s

tres

s, ε

y = y

ield

str

ain

, σu =

ult

imat

e st

ress

, WFx

= w

ork

to

fra

ctu

re, ε

f = f

ailu

re s

trai

n

a Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o N

on

-Irr

adia

ted

(ad

just

ed p

< 0

.05

)

b S

tati

stic

ally

sig

nif

ican

t d

iffe

ren

ce d

etec

ted

co

mp

ared

to

Irra

dia

ted

(ad

just

ed p

< 0

.05

)

c Sta

tist

ical

ly s

ign

ific

ant

dif

fere

nce

det

ecte

d c

om

par

ed t

o H

igh

T R

ibo

se (

adju

sted

p <

0.0

5)

Irra

d a

nd

Irra

d (

hig

h T

) w

ere

no

t si

gnif

ican

tly

dif

fere

nt

fro

m e

ach

oth

er f

or

any

me

asu

red

pro

per

ty

Hig

h T

Glu

cose

an

d H

igh

T F

ruct

ose

wer

e n

ot

sign

ific

antl

y d

iffe

ren

t fr

om

eac

h o

ther

fo

r an

y m

easu

red

pro

per

ty

76

77

78

79

80

81

82


In this study, High T Ribose incubation is superior in recovering mechanical properties over

glucose and fructose treatments. Ribose (five carbons) has a shorter chain length than glucose

and fructose (six carbons) and therefore it is suspected that faster glycation kinetics [81] lead

to a more crosslinked network. Ascorbate was the only other treatment that demonstrated

promising recovery of mechanical properties; however free radical scavenging is not an ideal

approach, as it would also protect pathogens during irradiation [3] making the sterilization

process ineffective.

The increase in incubation temperature from 550C (Chapter 3) to 60

0C (Chapter 4) increased

the recovery of most mechanical and thermal properties, for example 47% (for High T

Ribose at 550C) versus 57% (for High T Ribose at 60

0C) recovery of work to fracture. It is

possible that the increase in temperature increased the amount of ribose diffusion into the

bone; however it is also possible that the crosslinking reaction happens or begins to happen

during the heat incubation meaning there are more crosslinks in the higher temperature

treated samples. The reason for testing 600C incubation was to possibly increase effects but

also to expand the range of temperatures tested in the interest of patenting this procedure.

600C, however, is considered the upper limit to avoid causing any collagen denaturation

during the incubation.

It appears that SDS-PAGE may not be an appropriate method to study glyco-oxidation

crosslinked (GOC) collagen because it is possible that the GOC collagen is less soluble

following pepsin digestion than native collagen [26, 82]. Pepsin cleaves peptide bonds

between hydrophobic amino acids, which are located mostly in the telopeptide region of

collagen. It is used to digest collagen for SDS-PAGE because native enzymatic crosslinks are

located in the telopeptide region, so cleavage at these regions would remove the crosslinks

holding the collagen together, separating individual triple helix molecules for further study.

Glyco-oxidation crosslinks are non-site specific, so they would form all over including from

helix to helix. Pepsin cleavage would not be sufficient in digesting this network, thus more of

the GOC collagen remains insoluble. More work investigating the solubility and

susceptibility to enzyme digestion of glycated bone collagen is required, however, one

83

conclusion we can gain from the data is that the structure of collagen is in fact modified by

ribose incubation, although it is still not entirely clear how. It is possible that both Irradiated

and High T Glucose samples were more prone to pepsin digestion than non-irradiated

controls. This would indicate a decrease in molecular stability, and possibly some

denaturation induced by irradiation damage since denatured collagen is more susceptible to

enzyme digestion [30, 44]. To verify this, future investigation should include digestion by

trypsin, a serine protease that has been used by others to measure enzyme solubility of

irradiation damaged collagen [44, 45].

With the use of the High T Ribose pre-treatment, the DSC endotherm is comparable to that

of normal, native bone. This includes Tonset, Tpeak, enthalpy, and FWHM, none of which were

detectably different from Non-Irradiated controls (Table 4.5). If we assume that two

modifications of collagen occur: 1) irradiation creates cleavage sites and 2) ribose treatment

creates new crosslinks, then the structure of treated collagen is very different from that of

native collagen. If this is true, what mechanism returns the enthalpy value back to normal? If

we assume that the enthalpy of irradiated collagen is increased because irradiation creates

new reactive sites, then crosslinking would not explain why these sites disappear in the High

T Ribose group. This perhaps suggests that there is partial protection of some molecules from

irradiation damage, which limits the amount of new bond sites formed and keeps the

enthalpy from expanding. It could also mean that there is another reason for the increase in

enthalpy in irradiated collagen.

High T Ribose demonstrated better recovery of thermomechanical properties in HIT than the

High T Glucose treatment, however, High T Glucose did demonstrate some recovery of

measures of connectivity (72% recovery of slope at half maximum, p = 0.016, and 83%

recovery of Maximum Isometric Stress, p ≤ 0.001). One important question raised in this

study is: why does glucose treatment recover measures of collagen connectivity but not bone

toughness? It is clear that connectivity is not the only factor influencing toughness of the

material. Glucose recovers connectivity but does not recover thermal stability, indicated by

the low temperature of denaturation in DSC. This suggests that the stability of the collagen

molecule may be just as important as the connectivity of the network in producing a tough

bone-derived material.

84

Another difference in the glucose treated samples is the type of crosslinking. The increase in

slope and maximum isometric stress in HIT testing suggests that there is an increase in

connectivity via crosslinking, yet HPLC did not detect pentosidine in the High T Glucose

samples. Pentosidine was detected in High T Ribose samples (45 mmol pentosidine/mol

collagen). Pentosidine is one form of glyco-oxidation crosslinking; it is a product of the

reaction between ribose and amino acids in collagen polypeptides. Glucose reactions can

form pentosidine [55] but in the current experiment it was not a major product of the reaction

between glucose and bone matrix. The new crosslink may be some other form of non-

fluorescent crosslink that remains unknown without further investigation, or perhaps has yet

to be identified. It is possible that this crosslink does not have the quality of protecting the

stability that ribose crosslinking has, since ribose treatment recovers temperature of

denaturation in differential scanning calorimetry and glucose treatment does not.

A possible reason for this is that because the ribose molecule is smaller than glucose, the

length of the pentosidine crosslink is shorter and keeps the structure together in a tighter

formation. This could cause irradiation-induced denaturation to have less of an effect than on

the glucose-treated collagen. If we consider both the HIT and DSC curves in Figure 4.2, it is

important to remember that HIT measures the tension in the sample and this measurement is

influenced by crosslinks. This means the glucose-induced crosslinks could have an effect on

measurements post-denaturation in HIT, but may have no protective effect on the

denaturation and post-denaturation measurements in DSC since this only measures heat flow

required to denature the collagen molecules.

We can conclude that high temperature incubation with ribose is more successful at

recovering toughness than two other similar sugars: glucose and fructose. The explanation is

not simple; in other words more collagen connectivity does not necessarily equate to higher

toughness. There seems to be another important aspect of collagen structure that is modified

or protected by high temperature ribose treatment. One possibility is that ribose crosslinking

protects the native structure of the triple helix, which increases molecular stability and

contributes to the increase in toughness and strength. There are toughening mechanisms at

the molecular scale, such as molecular stretching, sliding between molecules, and helical

unwinding that dissipate energy before fracture and could explain why molecular stability is

85

important for overall tissue toughness [37]. These mechanisms will be further discussed in

Chapter 6. It is also possible that the mineral-matrix interactions are modified by irradiation,

and perhaps also somehow rescued by ribose treatment. Further investigation is required to

more completely understand the toughening mechanism.

86

Chapter 5

5 Fracture Testing of Irradiated and Ribose-Treated

Bone

5.1 Introduction

In order to truly understand the failure process of bone specimens, it is important to use

fracture testing. Fracture testing measures the resistance to fracture in the presence of an

existing crack, so it can be viewed as a measure of defect tolerance. Defect tolerance is an

especially important property of bone because microcracks are forming in our bones from

daily loading cycles. In vivo, bone is constantly remodeled in order to replace the damaged

bone with new bone. Allografts are not remodeled as often as normal bone [10, 83], so it is

especially important that allograft bone is resistant to crack propagation because

microdamage will build up without being replaced. Figure 5.1 demonstrates an optical

micrograph of human cortical bone that has been cyclically loaded in fatigue [84]. Note the

microcracks that have formed throughout the specimen, especially at the osteon borders and

extending from Haversian canals.

There are many mechanisms that contribute to the resistance to fracture in cortical bone.

They rely on the complex hierarchical composite structure of bone [13, 85]. On the macro to

micro scale, these mechanisms include crack deflection at the osteons, collagen fibril

bridging, and microcracking ahead of the crack tip [13, 86]. At the micro to nano scale,

microcracking of individual fibrils, fibrillar sliding, and possibly even collagen molecular

sliding, stretching, and unwinding act as energy dissipating mechanisms [37]. The

importance of stable collagen to the fracture properties of bone has been stressed in previous

investigations [32, 37, 33, 87].

The first objective of this study was to evaluate the effect of irradiation on the fracture

properties of bovine bone and evaluate high temperature ribose treatment as a method of

improving these properties in irradiated bone. We hypothesized that irradiation would have

deleterious effects on the fracture properties of bone, as it does with bulk mechanical

87

properties. We also anticipate that the ribose pre-treatment, which has been shown to

improve the stability and connectivity of the collagen component of bone, will improve the

fracture toughness.

88

5.2 Methods


Five tibias from steers (aged 2 years old) were used for this experiment. Samples were

prepared in a similar manner to the method described in section 3.2.1. Briefly, tibias were

obtained immediately after slaughter and frozen (-200C) for 3-10 days. Then they were

thawed, cleaned and cut into bone blocks. Two blocks from the distal portion of each tibia

were used, allowing for ten sets but only nine were used. The blocks were machined into a

set of three beams with dimensions of 60 mm x 4 mm x 4 mm (l x w x t) using a

metallurgical saw (Buehler Isomet 1000) with a custom built fence followed by hand

grinding and polishing. One beam was assigned to be a non-irradiated control, one beam was

irradiated, and one beam was pre-treated with ribose and then irradiated. Each beam was

wrapped in saline-soaked gauze and stored frozen until further processing.

5.2.2 Treatment and Irradiation

The samples (aside from control, which were left frozen until testing) were thawed at room

temperature and placed in individual 50 mL tubes. D-Ribose in powdered form was dissolved

in PBS to a concentration of 1.8M and pH was adjusted to 7.4 with dilute HCL or NaOH as

needed. This concentration was selected to match the three-point bending experiments

described in Chapter 4. There were three groups in this experiment: Non-Irradiated,

Irradiated, and High T Ribose. The Irradiated beams were incubated in 45 mL of PBS and the

High T Ribose beams were incubated in 45 mL of 1.8M Ribose solution. Both the Irradiated

samples and the High T Ribose samples were incubated in a water bath at 600C for 24 hours.

After incubation they were removed from their solution, wrapped in saline soaked gauze, and

frozen down for irradiation. Both the Irradiated and the High T Ribose group were irradiated

at 30 kGy from a Cobalt-60 source at Steris Isomedix (Whitby, ON Canada).

5.2.3 Fracture Testing

Based on the ASTM fracture testing standards a single-edge notched beam (SENB) loaded in

3-point bending was used [75, 88, 89]. A machined notch was cut at mid-length into one face

(periosteal to endosteal direction) of the sample with a 300 um-diameter diamond wire saw.

89

The notch was further sharpened by hand by sliding a razor blade back and forth across the

machined notch tip with 1 um diamond paste [75, 86]. The resulting machined notch and

razor cut was a ~2 mm long singular crack, as shown in the inset SEM image in Figure

5.2(a). To encourage crack propagation down the center of the sample, side grooves with a

depth of 0.5 mm were cut on the two side faces (lined up with the notch). Mode I loading

was used, which requires that the direction of crack propagation is perpendicular to the

direction of crack extension [90]. The beams were placed notch-side down into a 3-point

bending jig, making sure that the notch was lined up with the center-line of the loading

crosshead (6.35 mm diameter). The two supports (also 6.35mm in diameter) were spaced 40

mm apart. Figure 5.1a is a schematic representation of the testing set-up. Figure 5.1b is an

SEM image of the fracture surface with the notch, razor notch, and side-grooves labeled to

illustrate the samples. Beams were loaded at a rate of 0.5 mm/min using an Instron E100

testing machine. Instron Bluehill data acquisition software was used to produce a load vs.

displacement curve. The beams were loaded until there was a 10% drop in load, so that the

point at maximum load was captured during the test but the samples were not fully fractured.

Samples were mostly fractured (almost all the way through) at this point, so the two halves

were separated carefully by hand by rapid snapping in order to expose the fracture surface for

imaging with SEM (Figure 5.2b). A load vs. displacement curve like the one in Figure 5.3

was created with the data from a test.

5.2.4 Imaging the Fracture Surface

The fracture surface was imaged using scanning electron microscopy (SEM) methods after

failure in three-point bending. A portion of bone containing the fracture surface was removed

from the rest of the sample with a Buehler Isomet 100 wafer saw, leaving at least 5 mm

between the cut and the fracture surface to avoid damage. Methods previously published in

the lab were used for preparation and imaging [69, 91, 92]. The fracture sample portions

were soaked in 3% hydrogen peroxide for 48 hours, rinsed with distilled water, defatted in a

50:50 solution of methanol–chloroform (24 hours) then placed in 100% methanol for 1 hour

and dried overnight in a desiccator. Samples were mounted on SEM stages such that the

direction of propagation of the crack was parallel to the stage surface. They were then affixed

to specimen stubs using conductive carbon cement (Leit-C Plast, Plano GMBH, Wetzlar,

90

Germany). The mounted samples were sputter coated with gold for 125 s with a Denton

Vacuum Desk II sputter coater (Moorestown, NJ, USA). Imaging was conducted with a

scanning electron microscope (XL30 ESEM; Philips USA). Beam conditions were set at 20

kV accelerating voltage and a spot size of 4.

91

92

5.2.5 Calculating Fracture Toughness

Two important fracture toughness values calculated from test data were K and J. K is a

parameter that describes the intensity of the triaxial stress at the crack tip, and is called the

stress intensity factor. See Appendix 1.1 for the three equations describing stress at the crack

tip, which are all dependent on K. When the stresses and strains reach a certain value, the

crack will start to propagate and K will have reached a critical value called Kc [93]. Kc is also

known as the fracture toughness, an intrinsic material property independent of specimen

geometry [90]. The following equation is used to calculate K from a load vs. displacement

curve:

(

)

√

Where P=load, a=crack length, W=width of the specimen, B=thickness of the specimen, and

f(a/W) is a known function based on the geometry of the specimen (see Appendix 1.2). The

load used in the calculation of Kc must be the critical load that initiates crack propagation. If

93

we consider the load vs. displacement curve, the non-linear portion of the curve from yield

point to maximum load represents plastic deformation, including but not exclusively crack

propagation. Thus, an estimation of the critical load, PQ, is usually taken by using the

intersection of the 95% secant line and the load-displacement curve [90]. The critical load

and initial crack length are used to calculate Kc, the critical stress intensity required to

initiate crack growth. After this point, there is stable crack propagation while the load and

displacement continue to increase non-linearly. The maximum load marks instability point;

the point after which the crack is unstable and fast fracture occurs. The beam is considered

failed after the instability point is reached as there is no longer a building resistance to crack

progression. In a material such as bone, which is not as homogeneous in microstructure as a

metal, it is difficult to know an appropriate estimation of the critical load. For this study, we

can also evaluate fracture toughness using Ki, or K calculated at the maximum load

(instability point).

The second value to be calculated from load-displacement curves for the fracture tests is J. J

is a mathematical representation of the energy release rate during crack propagation in a

region of the material containing the crack tip. Under plane-strain conditions, meaning the

following condition is satisfied:

(where B is specimen thickness and σy is

yield strength):

(

)

Where G is the elastic energy released per unit area of a new crack surface forming for an

infinitesimal increment of crack extension [Hertzberg 1995], ν is Poisson‟s ratio (estimated at

0.3) and E is Young‟s Modulus. The above equation only accounts for the elastic energy

release rate, so another term to account for plastic energy release and possible crack

propagation must be added:

94

Where a0 is the initial crack length, B is the thickness of the specimen, W is the width and Apl

is the area under the plastic portion of the load-displacement curve. Jc is the J-integral

evaluated at the critical load with the initial crack length. This value is a criterion for crack

propagation. If we evaluate J-integral at the maximum load (Pmax) it will give the energy

released at the point of instability. This is called Jtotal and is calculated using the maximum

load and initial crack length. Even though using the initial crack length assumes there was no

crack propagation up to this point, it is assumed that some of the energy goes towards crack

propagation as well as plastic energy. We also chose to evaluate J and K at Pmax with

estimation for crack growth (instead of using initial crack length) to give a measure of each

value at the instability point, Ji and Ki and because previous work has shown that irradiation

greatly effects the crack growth between crack initiation and instability (R-curve) [75].

Estimating the crack length

Prior to the instability point at the maximum load, it is assumed that there may be some

stable crack propagation. The surface appearance between stable and fast fracture is

noticeably different [51]. Using SEM images of the fracture surface, it is possible to visually

distinguish stable tearing from unstable tearing. Measurement of the stable tearing region on

SEM images can give an estimate of the crack growth at the maximum load. This crack

length can then be used in the calculation of Ki (and subsequently in Ji) instead of just

assuming the crack at maximum load is still the initial notch length.

Preliminary experiments were performed to explore this idea and more accurately estimate

crack growth based on SEM images. Several specimens were loaded to Pmax and

subsequently stained with alizarin red. The stain marked the edge of the crack, and when

samples were then cut and imaged on the pre-fracture surface, it was possible to see the crack

propagation leading up to instability (maximum load). The stain measurements from

microscope images were comparable to separate measurements from SEM images using

visual roughness as the measure of crack propagation. Information from this experiment

confirmed that roughness of the stable tearing region is a good estimate of stable crack

growth; however it is important to note that the specimens in the current experiment were not

95

stained for crack growth, only measured on SEM images with the preliminary results as a

reference for what constitutes as stable tearing.

Figure 5.4 demonstrates the method for measuring crack propagation prior to instability. An

SEM image of the entire fracture surface of each specimen was taken and analyzed with

ImageJ analysis software. Lines were normalized to the length bar scale on the image so that

accurate measurements could be taken. Five vertical and five horizontal lines were drawn

across the entire sample to get average measurements of the width and thickness. The depth

of the side grooves, notch, and razor notch were also measured at five points. The edge of the

stable tearing region was traced and five measurements across the fracture surface were

taken. The average of these measurements was called as for the stable crack propagation. The

crack length was calculated as follows:

Where Ac is the crack length at instability, a0 is the machined notch, ar is the razor notch and

as is the stable crack propagation. Ac was used in the calculation of Ki, specifically in the

f(a/w) function (see Appendix 1 for formula). Using this estimation for the crack length and

based on the load vs. displacement curves for each sample, Ji and Ki were calculated for Non-

Irradiated, Irradiated, and High T Ribose groups.








significance was detected using RM ANOVA. The adjusted p values are reported when


96

5.4 Results

Irradiation had a negative effect on the fracture properties of bovine cortical bone. Kc, the

critical stress intensity factor, did not differ statistically between groups. The J-integral

values were lowered in the irradiated group by 44% and 49% for Jc and Jtotal (p = 0.006 and p

= 0.001, respectively). Both the J-integral at instability (Ji) and fracture toughness (Ki) were

lowered due to irradiation. Figure 5.5 shows example load vs. displacement curves for one

matched set of fracture beams. There was a loss of 61% for Ji (p = 0.005) and a loss of 42%

for Ki (p = 0.048) when comparing the Irradiated group to the Non-Irradiated controls. High

T Ribose treatment resulted in a 30% recovery of Ji and 43% recovery of Ki although this

was not detectably significant (p=0.093 and p = 0.080 for comparison of Irradiated to High T

Ribose). Table 5.1 presents the average Kc, Jc, Jtotal, Ji and Ki values for each group. Table

5.2 presents the average measurements for as, the stable tearing region found using SEM

image analysis, for each group. The stable tearing region for the Non-Irradiated group was

significantly longer than that of the Irradiated group (p = 0.020) but the High T Ribose group

was not detectably different from Non-Irradiated or Irradiated groups.

97

98

99

100


Our hypothesis that high temperature ribose pre-treatment could provide recovery of some of

the fracture properties was correct. Although collagen characterization was not performed on

these samples, we can assume that the ribose pre-treatment had similar effects to those

described in Chapters 3 and 4. Irradiation has been shown to damage collagen and we know

that collagen integrity plays a role in the ability of cortical bone to resist fracture [37, 75, 94,

95]. Pre-treatment with ribose has been shown to recover collagen connectivity and thermal

stability. It is now clear that the modifications of irradiated collagen that are induced by high

temperature ribose treatment allow for better fracture resistance than irradiated bone, since Ji

and Ki were higher in the High T Ribose group than the Irradiated group, although there was

only a significant difference detected for Ji (p = 0.047). The measurements for stable crack

growth were not detectably different between groups. A more precise method of

measurement is needed, as some SEM images were easier to interpret than others, leaving

room for human error.

Other studies of the fracture properties of bone have suggested that collagen plays a major

role in the mechanisms that prevent crack propagation. For example, Fantner et al. [95]

compared fracture mechanisms of healthy and heat-denatured bovine vertebral bone. They

demonstrated that normal bone exhibited failure by delamination, and collagen fibrils

bridging cracks were visible in SEM images. Baking bone denatured the collagen and caused

a change in the failure mechanisms to one of random fracture with no visible collagen

bridging [95]. Zioupos et al. [63] demonstrated that work to fracture, Kc, and J-integral all

decreased with age. While the mechanism is not known, it is suspected that collagen becomes

less connected with aging [63]. Barth et al. [37] found that in single-notched bend specimens

of human femoral bone there was a dose-dependent decrease in fracture properties (K0 at

crack initiation, Kjc at failure, and crack-growth toughness) with increasing irradiation dose.

All of these studies present a degradation model of collagen (heat denaturation, aging, and

irradiation) that leads to a loss of fracture toughness. Our modification of collagen (high

temperature ribose treatment plus irradiation) has better fracture properties than irradiation

alone, but does not return them to the level of normal, healthy bone.

101

One limitation of this study is the method for measuring crack propagation at instability.

Estimation of the crack length using visual evaluation of SEM images is not ideal because

there is a lot of room for human error. It was found to be difficult to distinguish stable tearing

regions from fast fracture regions for some samples. For this initial study, it was sufficient

especially because we were comparing between groups and all analysis was performed by the

same person. It would be beneficial to further investigate staining and imaging methods to

better quantify both the crack initiation point and the stable crack propagation prior to failure.

We can conclude that high temperature ribose pre-treatment demonstrates recovery of some

of the fracture toughness of irradiated bovine cortical bone. This recovery is most likely due

to the fact that irradiated bone collagen alone is weakened by cleavages of the peptide bond,

and high temperature ribose treatment prior to irradiation induces glyco-oxidation

crosslinking that stabilizes the organic network.

102

Chapter 6

6 Discussion, Conclusions, and Future Work

What we know from the results of the experiments in this study is, first, that irradiation

damages collagen and decreases toughness, and secondly that our treatment recovers some

work to fracture by restoring/protecting collagen integrity and connectivity. Our high

temperature pre-treatment with ribose has been successful in improving the mechanical

properties of irradiation sterilized bone. This chapter will discuss all experiments in this

thesis as well as draw conclusions and recommendations for future work.

6.1 Discussion

We have gained some insights into the mechanisms of embrittlement due to collagen, mainly

that decreases in collagen connectivity and thermal stability are concomitant with

embrittlement after irradiation. Our best performing high temperature ribose treated group is

successful in recuperating the thermal stability and thermomechanical measures. Zioupos et

al. [63] studied the fracture properties of human bone and as well as the thermal properties of

collagen in aging. They found that collagen became less stable with age, and the temperature

of denaturation of collagen has a positive correlation with fracture toughness. They

performed HIT testing on demineralized collagen and their most interesting finding was that

an increase in the rate of load contraction in HIT (which indicates more crosslinking) had a

positive correlation to work to fracture. Like this study, our results suggest that increasing

connectivity in an irradiation model results in an increase in toughness.

Looking at relationships between toughness and collagen properties

It is important to note that while our best performing ribose treatment was able to recover

100% of the connectivity measures in HIT testing, there was only partial recovery of the

work to fracture. Similarly for glucose treatment, there was a recovery of connectivity but not

much recovery of toughness. Clearly, our initial theory for the relationship between bone

toughness and collagen connectivity is not as simple as we anticipated. In an effort to study

this relationship, a series of summary curves have been constructed from the three-point

103

bending data, HIT data, and DSC data from both of the experiments conducted in Chapters 3

and 4. Figures 6.1, 6.2, 6.3, 6.4, and 6.5 are the average work to fracture vs. the average

maximum isometric stress (HIT), slope at half maximum (HIT), temperature of denaturation

(HIT), denaturation onset temperature (DSC) and enthalpy (DSC) of each group from the two

experiments. The groups from chapter 3 are labeled „E2‟ and the groups from chapter 4 are

labeled „E3‟. For example, the irradiated group from chapter 3 is labeled „Irrad E2‟. The

normalized averages were calculated by dividing each sample measurement by the

measurement for its matched non-irradiated control and taking the average of these

normalized values. The error bars represent the standard error of the mean, calculated by

dividing the standard deviation by the square root of the sample size (16 for Chapter 3, 20 for

Chapter 4).

These curves reveal that recovering toughness is not as simple as tying the collagen

fragments back together with new crosslinks. Considering the relationships between work to

fracture and MIS, work to fracture and slope, and work to fracture and Tonset (Figures 6.1,

6.2, and 6.4), it seems that the non-irradiated control group stands out from the other groups.

Excluding the non-irradiated control, there is a non-linear increase in toughness with an

increase in MIS. This would suggest that there is a positive correlation between the

connectivity (measures of collagen crosslink density and network stability) and the toughness

in bone. However, the non-irradiated bone has the highest measure of toughness yet it does

not have the highest measure of MIS. The glucose group has an MIS average close to that of

the non-irradiated control, but has almost 40% lower work to fracture. In examining the

position of glucose on the work to Fracture vs. Tonset curve, however it is clear that there was

minimal recovery of the thermal stability. This indicates that there is something aside from

the connectivity of the network that contributes to toughness; perhaps the stability and

integrity of the molecules themselves is also important.

Although the onset of denaturation in DSC (Tonset) and the temperature of denaturation are

thought to be similar [96], there was a large difference between the Tonset and Td averages for

the High T Ribose groups in both experiments (see Tables 3.6 & 3.7, and Tables 4.5 & 4.6).

In general for the ribose treated groups, Td was lower than Tonset, for example in Chapter 4

Td was 550C and Tonset was 61

0C. If both temperatures reflect the temperature at which

104

collagen starts to melt, they should be closer together. The difference may be a function of

the shape of the HIT load curve for High T Ribose. In Figure 4.2 the High T Ribose curve

has a steadily increasing baseline, while the other groups exhibit a relatively sharp transition

at Td. This made estimating Td for the High T Ribose group not as straight forward as the

other groups. Td is estimated as the temperature at which a „steady climb in force‟ begins,

according to the method of Lee et al. [72]. More specifically, Td was calculated by estimating

a straight line for the baseline at the beginning of the test and visually determining the point

of deviation from this line. The less sharp transition for High T Ribose samples made this

point perhaps artificially lower than it actually was. The more interesting question, however,

is why does ribose treatment cause this behaviour in HIT tests? It is possible that there is a

heterogeneous fraction of collagen material in these modified samples that becomes engaged

in tension over a temperature range, instead of a sharp transition temperature that we see in

the homogeneous non-irradiated controls. This could be due to a heterogeneous placement of

crosslinks, making some collagen portions very short, or there could be some amount of

amorphous collagen present in the sample as a result of irradiation.

The relationship between enthalpy (energetic cost of thermally melting collagen) and

toughness is unclear (Figure 6.5). It seems all ribose pre-treated groups return the enthalpy

back to that of the non-irradiated, but not all recover work to fracture. By what mechanism is

the enthalpy returned to normal in ribose treated groups? Something this might suggest is that

crosslinks create a more thermally stable matrix, however it could also suggest that the

treatment has some protecting effect on collagen from modification during irradiation. If you

take away the comparison to irradiated bone and just look at control vs. ribose treatment plus

irradiation, you could say that the toughness has decreased much like in a glycation model of

bone that received no irradiation [69]. Also, we have reason to believe that instead of just

migrating into the bone, ribose molecules are reacting prior to irradiation because of the

browning reaction during incubation. It is possible that the „tightening‟ of the interaction

between collagen molecules could shield some of the damage due to free radicals.

Rabotygova et al. [97] suggested that the triple-helical structure protects the collagen from

damage by showing that heat-denatured (unwound) collagen was more susceptible to

cleavage via irradiation. Unlike glycated bone from Willett et al. [69], however, we do not

see a significant increase above that of normal bone in the stiffness, strength, and temperature

105

of denaturation suggesting that there is some differences in these two modifications (glycated

and irradiated vs. only glycated) due to the subsequent irradiation of the bone.

y = 0.9388x2 - 1.096x + 0.7466 R² = 0.9528

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Wo

rk t

o F

ract

ure

(n

orm

aliz

ed

to

no

n-i

rrad

iate

d)

Maximum Isometric Stress (normalized to non-irradiated)

Work to Fracture vs. Maximum Isometric Stress (Normalized)

NonIrrad

Irrad E2

Irrad E3

Ribose E2

High T Glucose E3

High T Ribose E2 (55)


Figure 6.1: Work to fracture vs. maximum isometric stress for all groups tested in three-point bending. The averages of the normalized values are shown here and the error bars represent the standard error of the mean.

106

y = 0.6476x + 0.0446 R² = 0.8685

0

0.2

0.4

0.6

0.8

1

1.2

0.2 0.4 0.6 0.8 1 1.2 1.4

Wo

rk t

o F

ract

ure

Work to Fracture vs. Slope (Normalized)

NonIrrad

Irrad E2

Irrad E3

Ribose E2

High T Glucose E3



Figure 6.2: Work to fracture vs. slope at half maximum of the HIT load curve for all groups tested in three-point bending. The averages of the normalized values are shown here and the error bars represent the standard error of the mean.

Slope at half max (normalized to non-irradiated)

107

y = 3.3544x - 2.2404 R² = 0.963

0

0.2

0.4

0.6

0.8

1

1.2

0.7 0.75 0.8 0.85 0.9 0.95 1 1.05

Wo

rk t

o F

ract

ure

Temperature of Denaturation

Work to Fracture vs. Td (Normalized)

NonIrrad

Irrad E2

Irrad E3

Ribose E2

High T Glucose E3



Figure 6.3: Work to fracture vs. temperature of denaturation (HIT) for all groups tested in three-point bending. The averages of the normalized values are shown here and the error bars represent the standard error of the mean.

108

y = 1.5396x - 0.7917 R² = 0.9784

0

0.2

0.4

0.6

0.8

1

1.2

0.5 0.6 0.7 0.8 0.9 1 1.1

Wo

rk t

o F

ract

ure

(n

orm

aliz

ed t

o n

on

-irr

adia

ted

)

Temperature of denaturation onset (DSC) (normalized to non-irradiated)

Work to Fracture vs. Tonset (Normalized)

NonIrrad

Irrad E2

Irrad E3

Ribose E2

High T Glucose E3



Figure 6.4: Work to fracture vs. temperature of denaturation onset (measured in DSC) for all groups tested in three-point bending. The averages of the normalized values are shown here and the error bars represent the standard error of the mean.

109

y = -0.1408x + 0.7793 R² = 0.4427

0

0.2

0.4

0.6

0.8

1

1.2

0.4 0.9 1.4 1.9 2.4 2.9 3.4

Wo

rk t

o F

ract

ure

(n

orm

aliz

ed t

o n

on

-irr

adia

ted

) Work to Fracture vs. Enthalpy (Normalized)

NonIrrad

Irrad E2

Irrad E3

Ribose E2

High T Glucose E3



Figure 6.5: Work to fracture vs. enthalpy (measured in DSC) for all groups tested in three-point bending. The averages of the normalized values are shown here and the error bars represent the standard error of the mean.

Enthalpy (normalized to non-irradiated)

110

Collagen modification and toughness

In a study of collagen in aging in bone, Zioupos et al. [63] showed that collagen becomes, in

their words, less stable due to increasing age. They also demonstrated a loss of toughness and

fracture toughness with increasing age. In terms of collagen properties, they see a decline in

the temperature of denaturation onset in HIT and slope of the load curve in HIT. In terms of

mechanical properties, they see a lowering in work to fracture and fracture toughness

measured in notched beam fracture testing. Similar to our results, their data suggests that

there is a positive correlation in between the collagen network connectivity and work-to-

fracture, and the temperature of denaturation and work-to-fracture. In fact, when they did a

correlation analysis and accounted for overall correlations with age, HIT slope (a measure of

collagen connectivity) was the only property to correlate with J-integral fracture toughness

and the correlation was strong (R2 = 0.86 and p ≤ 0.01)

Willett et al. [69] showed that incubating normal bone in 0.6M ribose for 2 weeks at 370C

leads to ribose-induced glyco-oxidation crosslinking. The resulting bone (note: no irradiation

in this study) demonstrated an elevated temperature of denaturation in DSC, an elevated

MIS, and a lowering of work-to-fracture and failure strain. This model represents an over-

crosslinked modification of native collagen; essentially stiffening the collagen and limiting

the ability for ductility and strain accommodation. The exact mechanisms of embrittlement in

their study are not entirely elucidated.

In Chapter 2 of this study we demonstrated that fragmentation due to irradiation seems to

have an effect on both the thermal and mechanical properties of collagen. There was a

decrease in the onset temperature of denaturation in DSC, the slope of the load curve in

HIT, and the MIS in HIT. This collagen is an under-connected collagen network, where

fragmentation decreases the stability and thermomechanical properties of the matrix as well

as the toughness of bone.

We originally believed that there was an optimal level of connectivity that could be found in

normal, healthy bone. It was assumed that diverting in either direction from the optimum

would cause a decrease in toughness. Figure 6.6a is a schematic of what this relationship

111

between toughness and connectivity might look like. Irradiated collagen and aged collagen

would fall to the left of optimum point, with both lower connectivity and lower toughness.

Over-crosslinked collagen, such as the ribated collagen mentioned above, would fall to the

right of the optimum point, with higher connectivity but lower toughness due to over-

stiffening of the matrix. With our ribose pre-treatment of irradiated bone, we attempted to

increase the connectivity with glyco-oxidation crosslinks and therefore approach the

optimum level of toughness. This ideal relationship is not reflected in our results, which are

shown in a simplified form in Figures 6.6b and 6.6c. A point representing the ribated bone

data from Willett et al. [69] (note: this is ribose treated with no irradiation) is represented by

a black „X‟. There seems to be a positive correlation between connectivity and toughness, but

at a level below the toughness of the normal, healthy control. The irradiated group has both

lower connectivity and lower toughness than the normal bone, but as you increase the

connectivity with glyco-oxidation crosslinking you do not approach the optimum and fall

back down the other side of the peak, but instead continue to rise while passing beneath it.

The ribated bone has the highest measure of connectivity (higher than the control) and the

highest toughness out of the modified groups. This shift downwards suggest that these

modifications of collagen effects more than just connectivity, and that there are other aspects

of the structure of bone that contributes to its toughness. This is important both for future

work on this project and in the study of aging and disease models in bone. Many

investigators attribute most of the loss of toughness in aging or diabetic people to the

addition of glyco-oxidation crosslinks, when in fact our data suggests there is another factor

(or several other factors) that could be negatively affecting the mechanical properties of bone

in these disease models.

112

113

The role of small-scale collagen deformation in bone toughness

Do our results suggest that toughening mechanisms exist at the molecular level? Barth et al.

[37] suggest that collagen plays a major role in toughening due to its micro and nano – scale

toughening mechanisms. These include molecular uncoiling of the collagen molecules,

fibrillar sliding of mineralized collagen fibrils and fibers, and microcracking of mineralized

fibers. Figure 6.5 demonstrates a schematic depiction of these toughening mechanisms from

Barth et al. [37]. They believe that embrittlement due to irradiation is due to the loss of these

molecular-level toughening mechanisms, because larger-scale mechanisms, for example

crack deflection, were still present in x-ray irradiated human bone specimens. If stretching

and sliding of the molecules is essential for ductility and toughness, then the integrity of the

collagen molecules is important to the overall toughness of bone. This could provide an

explanation for the gap in our data, particularly in Figure 6.1 and 6.3, between non-irradiated

bone and all the other groups, which essentially represent different forms of modified

collagen.

While we characterized some aspects of these modifications, the ultra-fine structure of

ribose-treated and irradiated collagen is still unknown. The treatment was shown to induce

glyco-oxidation crosslinks according to HPLC measurements of pentosidine, and the HIT

data suggests an increase in crosslinks over irradiated collagen as well. These crosslinks are

not present in the non-irradiated control, so it is possible that the differences in toughness are

a result of this new formation of collagen. It is possible that the location of the crosslinks in

the collagen matrix have an effect on the nano-scale toughening mechanisms described above

(see Figure 6.7). Although glyco-oxidation crosslinks improve connectivity, they are not

specific to location on the molecules. Enzymatic crosslinks are specific to telopeptide – helix

locations; essentially they are contained at the ends of the molecules. It is proposed that

molecules in this formation can „stretch‟ but they will not slip away from each other. Glyco-

oxidation crosslinks bind the molecules tighter together and form crosslinks throughout the

lattice structure, including helix-to-helix [81]. These non-specific crosslinks essentially

shorten the length of the „stretchy‟ regions which would explain an increase in stiffness, or at

least a decrease in post-yield strain accommodation.

114

The mineral-matrix interface

Another important question to ask is: what about the role of the mineral? Specifically, what is

the effect of irradiation and our treatment on the matrix-mineral interface? When making

connections between the thermal denaturation and thermomechanical behaviour of collagen

and the bulk mechanical properties, one main difference is that the collagen samples are

demineralized so any influence of the mineral is not accounted for in the collagen

characterization tests. It seems reasonable that if irradiation and ribose treatments are

modifying proteins in the collagen triple helix, they could also be affecting the non-

collagenous proteins that may be involved in the interactions of mineral and matrix. Non-

collagenous proteins are thought to play a role in mineralization, initiating crystal formation

in the gap regions between tropocollagen molecules [98]. Siegmund et al. [94] suggests that

intermolecular helix-helix crosslinks decrease the sliding of collagen molecules and instead

of a gradual de-bonding between collagen and mineral, there is a transfer of higher loads to

the matrix-mineral interface which leads to a decrease in strain to failure [94].

Barth et al. [37] suggest that x-ray irradiation actually creates crosslinks (based on a shift in

peaks using UV-Raman spectroscopy) and that this inhibits the sliding mechanisms between

tropocollagen molecules and hydroxyapatite crystals. More specifically, they suggest that an

initial slip at the mineral-matrix interface in normal bone allows for a large amount of gliding

between the tropocollagen and hydroxyapatite molecule, essentially the slip at the interface

frees up a new dissipative sink for energy. Thompson et al. [99] also shows evidence of so-

called „sacrificial bonding‟ although it is not clear whether these bonds are between matrix

and mineral or collagen and other collagen molecules. They suggest that as bone collagen

fibers are loaded, there are bonds that break and release „hidden length‟ in the polymer which

must be stretched further before another bond can be broken, increasing the energy needed

for the structure to fail. This interesting discovery is more evidence suggesting that the

toughness of bone relies on the integrity of individual collagen molecules, the interactions

between the molecules, and the interaction between the mineral-matrix components.

115

Figure 6.7: Fracture toughness mechanisms from the macro- to nano-scale in bone

from Barth et al 2010 *37+.

116

6.2 Conclusions

There were four main objectives of this thesis, as outlined in Chapter 1. In addressing these

objectives, we can make the following conclusions:

1) We can conclude that γ-irradiation of bone decreases strength and toughness. The

evidence of collagen degradation in the form of fragmentation of molecular chains

and lowering of the thermal stability and connectivity suggests collagen damage is a

contributing factor to these changes.

2) In γ-irradiated bone, we have shown it is possible to recover some of the mechanical

properties using a ribose pre-treatment. In particular, we see recovery of strength and

some recovery work to fracture and failure strain.

3) The proposed mechanism is that recovery of collagen connectivity leads to

functionally significant recovery of toughness, fracture toughness and strength.

However, connectivity must be defined as the integrity of molecular structure and the

connections between molecules in the network. It seems likely that some other aspect

of the ultrastructure of bone plays a role in toughness.

4) Fracture properties of γ-irradiated bovine bone, including J-integral and fracture

toughness at instability (Ji and Ki) were partially recovered using a high temperature

ribose pre-treatment.

The improved mechanical properties of this bone-derived material are a promising step

towards a potential solution for poor graft quality and poor clinical outcomes resulting from

graft fracture. Interesting novel data on the relationship between collagen properties and the

bulk mechanical properties of bone also further our understanding of the role of collagen in

the toughness of bone.

6.3 Future Work

In summary, there is a lot that remains unknown about the modifications induced by our

ribose pre-treatment, but we can say that both the bulk mechanical properties and the fracture

toughness are improved over irradiated bone without the treatment. Further investigation into

the structure and properties of this modified form of bone is required. Eventually, work

117

beyond understanding the mechanisms of toughening is required if this bone-derived material

is to be used as an allograft, specifically, the biological implications of the treatment. This

section provides recommendations on future work required for the success of this

technology.

First, it is imperative to determine at what point during the treatment process the glyco-

oxidation crosslinks form. Quantifying pentosidine crosslinks for samples that have been pre-

treated but not irradiated would indicate whether the glyco-oxidation crosslinking happens

before or during irradiation, which could help to understand if there is a protective effect on

the collagen during irradiation due to pre-crosslinking with ribose. It is also necessary to

study the effects of ribose treatment on microdamage and fracture toughness mechanisms of

bone. A recovery of microdamage accumulation or smaller-scale fracture mechanisms could

indicate if ribose-treated bone can support the fracture resistance mechanisms that are

important for the functionality of allograft bone.

The model used in this study, bovine cortical bone, was sufficient as a first step but

validation in human cortical bone is necessary. Human bone exhibits a secondary osteonal

structure, where pre-existing bone is resorbed and replaced with cylindrical osteons that

consist of concentric layers of lamellae around a Haversian canal. Cattle, on the other hand,

have a different form of bone called plexiform bone in which sheets of lamellar bone and

sheets of blood vessel networks alternate, with highly mineralized non-lamellar bone in the

interstitial spaces [15]. Osteonal bone is more anisotropic because the osteons tend to run in

the same direction, which has an effect on the mechanical properties and failure mechanisms.

Because the age of human donors is typically older, the effects of aging on bone must be

considered which could include degradation of collagen [63], increased porosity, and

changes in bone mineral density (effects of changes in bone turnover with age).

The sample dimensions used in this thesis were chosen in order to evaluate bulk mechanical

properties of the material, and thus physiological shape was not considered. In clinical

applications, the allografts receiving this pre-treatment could be various shapes and sizes,

including large tube-shaped long bone segments. Diffusion of ribose into the allografts

becomes a concern, so experiments designed to model the diffusion of ribose into bone

118

material based on the size, shape, and porosity should be completed in order to establish an

appropriate protocol for clinically relevant allograft processing.

From a clinical perspective, the future of the use of these types of allografts requires two

important considerations. First, it must be determined if ribose treatment has an effect on the

destruction of pathogens. If ribose pre-treatment is found to have a protective effect on

collagen, this could also mean it would have a protective effect on pathogens, thus defeating

the purpose of the sterilization procedure. An experiment designed to test the effect of high

temperature ribose pre-treatment on the efficacy of sterilization with γ-irradiation would be

required to move forward with this study. This could be achieved using incubation of known

pathogens with bone material, followed by ribose pre-treatment, irradiation, and then culture

of the tissue and analysis of surviving pathogens

If there is no effect on the sterilization of the bone, it is also important to understand how this

treatment effects the remodeling of bone allografts in vivo. There are indications that

glycation may alter the interaction of cells with collagen [82] thus have an effect on allograft

incorporation. An ideal allograft would be completely remodeled over time, but in the case of

large allografts the current clinical data shows only about 20% revitalization at 5 years post-

implantation, with about 10% at each end of the graft creating a union between the graft and

the patient‟s native bone [10, 83]. Irradiated grafts have been shown to demonstrate union as

well [100]. It is vital not to lose the incorporation of the ends of the allograft into the native

bone because non-union of the graft is considered a failure.

119

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127

Appendices

Appendix 1: Force vs. displacement graphs for 3-point bending experiments described in

Chapter 3. Four sets containing four specimens each (Non-Irradiated, Irradiated, 1.8M

Ribose, and High T Ribose) are shown.

0

10

20

30

40

50

60

0 1 2 3 4 5 6

Forc

e (N

)

Displacment (mm)

Set 15 Force vs Displacment in 3-point bending

NonIrrad

Irrad

Ribose

High T Ribose

0

5

10

15

20

25

30

35

40

45

50

0 0.5 1 1.5 2 2.5 3

Forc

e (N

)

Displacment (mm)

Set 13 Force vs. Displacment in 3-point bending

NonIrrad

Irrad

Ribose

High T Ribose

128

0

10

20

30

40

50

60

0 1 2 3 4 5 6

Forc

e (N

)

Displacment (mm)

Set 16 Force vs. Displacement in 3-point bending

NonIrrad

Irrad

Ribose

High T Ribose

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5

Forc

e (N

)

Displacment (mm)

Set 11 Force vs Displacment in 3-point bending

NonIrrad

Irrad

Ribose

High T Ribose

129

Appendix 2: Force vs. displacement graphs for 3-point bending experiments described in

Chapter 4. Four examples of sets; each set containing a control, irradiated, high T Ribose,

and high T glucose specimen.

0

10

20

30

40

50

60

0 1 2 3 4

Forc

e (N

)

Displacment (mm)


NonIrrad

Irrad

High T Glucose

High T Ribose

0

10

20

30

40

50

60

70

0 1 2 3 4 5

Forc

e (N

)

Displacment (mm)


NonIrrad

Irrad

Glucose

High T Ribose

130

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5

Forc

e (N

)

Displacment (mm)


NonIrrad

Irrad

High T Glucose

High T Ribose

0

10

20

30

40

50

60

0 1 2 3 4 5

Forc

e (N

)

Displacment (mm)


NonIrrad

Irrad

High T Glucose

High T Ribose

131

Appendix 3: Details and equations regarding the calculation of fracture toughness

measurements from SENB fracture tests.

For a singular crack (zero thickness and zero root radius) in mode I loading, there are three equations

that describe the stress, strain, and displacement around the crack. These three equations reflect the

stress state around a crack tip. The equations are written in terms of the polar coordinates (From

Hertzberg [90]):

√ 𝜋𝑟 𝜃

√ 𝜋𝑟 𝜃

𝜏

√ 𝜋𝑟 𝜃

All three equations depend on a variable K, which is a parameter that describes the intensity of stress

at the crack tip, and is called the stress intensity factor. When the stresses and strains reach a certain

value, the crack will start to propagate and K will have reached a critical value called Kc. Kc is the

fracture toughness, an intrinsic material property independent of specimen geometry. The following

equation is used to calculate K from a load displacement curve:

(

)

√

/ 𝜉

𝜁 [𝐶 𝐶 / 𝐶 / 𝐶 / 𝐶 / ]

𝜉 3 / /

𝜁 / /

𝐶 , 𝐶 , 𝐶 0 , 𝐶 3, 𝐶 7

Figure A1: A schematic of an

ideal crack tip, showing polar

coordinates.

132

Where P=load, a=crack length, W=width of the specimen, B=thickness of the specimen, and f(a/W) is

a known function based on the geometry of the specimen.

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