Improving the Mechanical Properties of Irradiation ......The overall goal was to develop a method...
Transcript of 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
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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.
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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.
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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
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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
3.2.1 Sample Preparation ............................................................................................... 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
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3.2.6 Collagen Characterization ..................................................................................... 50
3.3 Statistical Analysis ............................................................................................................ 51
3.4 Results ............................................................................................................................... 51
3.4.1 Mechanical Properties ........................................................................................... 51
3.4.2 Collagen Characterization ..................................................................................... 52
3.5 Discussion and Conclusions ............................................................................................. 61
Chapter 4 ....................................................................................................................................... 66
4 Comparing Ribose to other Crosslinking Agents ..................................................................... 66
4.1 Introduction ....................................................................................................................... 66
4.2 Methods ............................................................................................................................. 68
4.2.1 Sample Preparation ............................................................................................... 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
4.2.6 Collagen Characterization ..................................................................................... 70
4.3 Statistical Analysis ............................................................................................................ 71
4.4 Results ............................................................................................................................... 71
4.4.1 Mechanical Properties ........................................................................................... 71
4.4.2 Collagen Characterization ..................................................................................... 72
4.5 Discussion and Conclusions ............................................................................................. 82
Chapter 5 ....................................................................................................................................... 86
5 Fracture Testing of Irradiated and Ribose-Treated Bone ......................................................... 86
5.1 Introduction ....................................................................................................................... 86
5.2 Methods ............................................................................................................................. 88
5.2.1 Sample Preparation ............................................................................................... 88
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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
5.3 Statistical Analysis ............................................................................................................ 95
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
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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
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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
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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.
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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
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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
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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).
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Figure 1.2: The hierarchical structure of bone (From Launey et al. 2010 [13])
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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 intra- molecular
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])
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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-
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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])
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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
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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])
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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
3.2.1 Sample Preparation
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).
3.2.6 Collagen Characterization
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
3.4.1 Mechanical Properties
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.
3.4.2 Collagen Characterization
Collagen Fragmentation and Crosslinks
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.
Collagen Connectivity
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
3.5 Discussion and Conclusions
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
4.2.1 Sample Preparation
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.
4.2.6 Collagen Characterization
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
4.3 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.
4.4 Results
4.4.1 Mechanical Properties
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).
4.4.2 Collagen Characterization
Collagen Fragmentation and Crosslinks
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.
Collagen Connectivity
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
4.5 Discussion and Conclusions
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
5.2.1 Sample Preparation
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.
5.3 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.
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
5.5 Discussion and Conclusions
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)
High T Ribose E3 (60)
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
High T Ribose E2 (55)
High T Ribose E3 (60)
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
High T Ribose E2 (55)
High T Ribose E3 (60)
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
High T Ribose E2 (55)
High T Ribose E3 (60)
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
High T Ribose E2 (55)
High T Ribose E3 (60)
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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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)
Set 8 Force vs. Displacment in 3-point bending
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)
Set 9 Force vs. Displacment in 3-point bending
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)
Set 16 Force vs. Displacment in 3-point bending
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)
Set 15 Force vs. Displacment in 3-point bending
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.