Biocompatibility of Bone Allograft Toughened with a Novel ... · Massive bone defects, such as...
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Biocompatibility of Bone Allograft Toughened with a Novel Sterilization Method for
Critically-Sized Segmental Defects: An In Vivo Rabbit Study
by
Si Hyeong Park, MD
A thesis submitted in conformity with the requirements for the degree of
Master of Applied Science (MASc)
Institute of Biomaterials and Biomedical Engineering (IBBME)
University of Toronto
© Copyright by Si Hyeong Park, MD (2017)
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Biocompatibility of Bone Allograft Toughened with a Novel Sterilization Method for
Critically-Sized Segmental Defects: An In Vivo Rabbit Study
Si Hyeong Park, MD
Master of Applied Science (MASc)
Institute of Biomaterials and Biomedical Engineering (IBBME)
University of Toronto
2017
ABSTRACT
Purpose: Bone allografts often undergo γ-irradiation to decrease infection risk. This
consequently degrades bone collagen and makes the bone brittle. Our laboratory found pre-
treatment with ribose could protect the bone. This thesis aimed to determine if ribose-treated γ-
irradiated allografts were biocompatible in vivo.
Materials and Methods: Using the New Zealand White rabbit (NZWr), we first aimed to
establish a proper in vivo model to test the graft using intramedullary wire fixation (Study 1), and
then tested the biocompatibility of ribose-treated grafts (compared to untreated and
conventionally-irradiated grafts) (Study 2). Healing/union was assessed with radiographs, μCT,
histomorphometry, backscatter electron microscopy, and torsion testing.
Results: Intramedullary fixation achieved stable reconstructions. All grafts achieved bony union.
No differences were found in radiographic and biomechanical parameters tested. However,
irradiated grafts had less bone volume (p=0.000) but greater bone forming properties (p<0.002).
Conclusion: Ribose-pretreated grafts were able to achieve bony union with host bone, and may
be protected against graft resorption.
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ACKNOWLEDGEMENTS
I would like to thank my supervisors, Dr. Marc Grynpas and Dr. Thomas Willett, for their
guidance and support through this thesis project. I would further extend my thanks to Miss Lucia
Zhang, Dr. Christopher Kim, Dr. Adeline Ng, Dr. Tarik Attia, Dr. Adele Changoor, Miss Megan
Stajkowski, and Miss Julia Pasquale, for their help and assistance with the various experiments
and analyses related to my research.
I would also like to acknowledge Dr. Peter Ferguson and Dr. Margarete Akens for their
clinical and surgical expertise. I would like to thank Ms. Dezan Rego and the staff at the Mount
Sinai Hospital, Surgical Skills Laboratory for use of the surgical instruments. Many thanks are
also extended to Dr. Kate Banks, Mr. Rainer de Guzman, and Miss Arin Dunning for assistance
with the surgical procedures and the care of the research subjects.
Finally, I would like to extend thanks to my committee members: Dr. Rita Kandel, Dr.
Jay Wunder, Dr. Paul Kuzyk, Dr. Thomas Willett, and Dr. Marc Grynpas.
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TABLE OF CONTENTS
Page Number
ACKNOWLEDGEMENTS……………………………………………………………... iii
TABLE OF CONTENTS………………………………………………………………... iv
ABBREVIATION OF TERMS…………………………………………………………. viii
LIST OF TABLES………………………………………………………………………. ix
LIST OF FIGURES……………………………………………………………………... x
LIST OF APPENDICES………………………………………………………………... xiii
1. INTRODUCTION………………………………………………………………….. 1
1.1 Allograft Bone Healing…………………………………………………….... 2
1.2 Irradiation of Bone Allografts………………………………………………. 2
1.3 Mechanism of Degradation…………………………………………………. 4
1.4 Ribose Pre-Treatment……………………………………………………….. 6
1.5 In-Vivo Testing………………………………………………………………. 8
1.6 Thesis Hypotheses and Objectives…………………………………………... 9
2. MATERIALS AND METHODS (Common to Both Studies)…………………… 11
2.1 New Zealand White Rabbit………………………………………………….. 11
2.2 Donor Bone Allograft Preparation………………………………………….. 11
2.3 Surgical Procedure………………………………………………………….. 13
2.4 Post-Operative Care………………………………………………………… 17
2.5 Data Collection……………………………………………………………… 18
2.6 Radiographic Analysis……………………………………………………… 18
2.7 μCT Analysis………………………………………………………………... 20
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TABLE OF CONTENTS
Page Number
2.8 Histomorphometric Analysis………………………………………………... 20
3. STUDY 1……………………………………………………………………………. 22
3.1 Study Design………………………………………………………………… 22
3.2 Methods (Study 1)…………………………………………………………… 22
3.2.1 Radiographic Analysis……………………………………………… 22
3.2.2 Data Collection……………………………………………………... 23
3.2.3 μCT Analysis………………………………………………………... 23
3.2.4 Histomorphometric Analysis………………………………………... 23
3.2.5 Statistical Analysis………………………………………………….. 24
3.3 RESULTS (STUDY 1)……………………………………………………… 24
3.3.1 Radiographic Analysis……………………………………………… 24
3.3.2 μCT Analysis………………………………………………………... 27
3.3.3 Static Bone Histomorphometry……………………………………... 28
3.3.4 Complications………………………………………………………. 30
4. STUDY 2…………………………………………………………………………….. 31
4.1 Study Design………………………………………………………………… 31
4.2 Methods (Study 2)…………………………………………………………… 32
4.2.1 Radiographic Analysis……………………………………………… 32
4.2.2 Data Collection……………………………………………………... 32
4.2.3 μCT Analysis………………………………………………………... 33
4.2.4 Static Bone Histomorphometry……………………………………... 36
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TABLE OF CONTENTS
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4.2.5 Dynamic Bone Histomorphometry………………………………….. 38
4.2.6 Backscatter Electron Microscopy…………………………………... 40
4.2.7 Torsion Testing Set-Up Protocol…………………………………… 42
4.2.8 Torsion Testing Data Analysis……………………………………… 45
4.2.8.1 Structural Properties………………………………….... 46
4.2.8.2 Material Properties……………………………………. 46
4.2.9 Statistical Analysis………………………………………………….. 49
4.3 RESULTS (STUDY 2)………………………………………………………. 50
4.3.1 Radiographic Analysis……………………………………………… 50
4.3.2 μCT Analysis………………………………………………………... 52
4.3.3 Static Bone Histomorphometry……………………………………... 54
4.3.4 Dynamic Bone Histomorphometry………………………………….. 56
4.3.5 Backscatter Electron Microscopy…………………………………... 59
4.3.6 Histomorphometric Analysis Within the Grafts…………………….. 61
4.3.7 Biomechanical Testing……………………………………………… 63
4.3.8 Complications……………………………………………………….. 65
5. DISCUSSION……………………………………………………………………….. 67
5.1 STUDY 1…………………………………………………………………... 67
5.2 STUDY 2…………………………………………………………………... 70
6. CONCLUSION…………………………………………………………………….... 77
7. FUTURE DIRECTIONS…………………………………………………………… 78
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TABLE OF CONTENTS
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8. REFERENCES……………………………………………………………………… 80
9. APPENDIX………………………………………………………………………….. 91
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ABBREVIATION OF TERMS
AGE Advanced Glycation Endproduct
ANOVA Analysis of Variance
APL Abductor Pollicis Longus
BSE Backscatter Electron Microscopy
BS Bone Surface
BV Bone Volume
C Control
CSD Critically Sized Defect
CT Computed Tomography
ECR Extensor Carpi Radialis
EDC Extensor Digitorum Communis
FCR Flexor Carpi Radialis
I Conventionally Irradiated
IM Intramedullary
ISO International Organization for Standardization
kGy Kilogray
μCT Micro-Computed Tomography
MAR Mineral Apposition Rate
Mrad Millirad
MS Mineralizing Surface
MSK Musculoskeletal
NZWr New Zealand White Rabbit
OS Osteoid Surface
OV Osteoid Volume
R Ribose-Pretreated Irradiated
ROI Region of Interest
SC Subcutaneous
TRAP Tartrate-Resistant Acid Phosphatase
TV Tissue Volume
vBMD Volumetric Bone Mineral Density
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LIST OF TABLES
TABLES Page Number
Table 1. Radiographic scoring system, modified from An and Friedman. 19
Table 2. Radiographic analysis of periosteal reaction. 51
Table 3. Radiographic analysis of cortical remodeling. 51
Table 4. μCT analysis of the volumetric bone mineral density, bone volume
(normalized to tissue volume), and tissue volume, at the osteotomy site ROI. 53
Table 5. Measured growth of bony callus on to the graft. 53
Table 6. Quantitative static bone histomorphometry of the three graft types. 56
Table 7. Structural properties of the three graft groups following torsion
testing. 64
Table 8. Material properties of the three graft groups following torsion
testing. 65
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LIST OF FIGURES
FIGURES Page Number
Figure 1. The Maillard reaction. 7
Figure 2. Longitudinal skin incision along the distal anterior forelimb. 14
Figure 3. Diaphysis of the radius exposed following surgical dissection. 14
Figure 4. Critically-sized bone defect created in the mid-diaphysis of the
radius. 15
Figure 5. Intramedullary fixation of the bone allograft using a 0.8-mm
Kirschner wire. 16
Figure 6. A harvested reconstructed forelimb with Kirschner wire in situ. 24
Figure 7. Post-operative radiographs at 2 weeks of a critically-sized radial
defect reconstructed using bone allograft and fixed with Kirschner wire. 25
Figure 8. Radiographs at 4 weeks post-operative. 26
Figure 9. Radiographs at 8 weeks post-operative. 26
Figure 10. Radiographs at 10 weeks post-operative demonstrate cortical
remodeling occurring. 26
Figure 11. Radiographs at 12 weeks show evidence of bony union and
cortical remodeling. 27
Figure 12. Three-dimensional reconstruction of μCT scan of the graft-host
bone junction at 12 weeks post-operative (sagittal cut shown). 28
Figure 13. Static bone histomorphometry (stained with Goldner’s
Trichrome) of the graft-host bone junction at 12 weeks post-operative. 29
Figure 14. Radiographs at 2 weeks showing graft displacement after
proximal Kirschner wire migration in one NZWr. 30
Figure 15. Study design for testing the biocompatibility of ribose-pretreated
irradiated bone allograft. 31
Figure 16. Summary of all analyses performed on reconstructed samples in
Study 2. 33
Figure 17. Three-dimensional rendered image illustrating new bony growth
onto the graft. 35
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Figure 18. Calculating distance from osteotomy site to furthest leading edge
of callus. 36
Figure 19. A 4 mm ROI (green outline) drawn centered at the osteotomy site
for static bone histomorphmetric analysis. 37
Figure 20. A 2 mm ROI (green outline) drawn within the graft for dynamic
bone histomorphometric analysis. 38
Figure 21. Example of graft resorption (in a ribose-treated irradiated graft). 40
Figure 22. Backscatter electron microscopy image. 41
Figure 23. The distal reconstructed radius bone segment separated from the
ulna. 43
Figure 24. Sandpaper attached to ends 4 mm distal and 4 mm proximal to
the osteotomy site. 44
Figure 25. Reconstructed radius potted in to hex nuts using
polymethylmethacrylate (PMMA). 44
Figure 26. The potted reconstructed radius samples secured into the Instron
ElectroPuls E1000 mechanical testing machine. 45
Figure 27. Measured distance (mm) from the potted static end to the bone
spike at the osteotomy site (ι). 47
Figure 28. Radiographs at 2-, 6-, and 12-weeks, post-surgical reconstruction
of the critically-sized radial defect. 50
Figure 29. Three-dimensional rendering of the μCT scans at the osteotomy
sites. 52
Figure 30. Bony growth of the callus onto the graft. 54
Figure 31. Static bone histomorphometry (stained with Goldner’s
Trichrome) of the graft-host bone junction at 12 weeks post-operative. 55
Figure 32. Dynamic bone histomorphometry of the graft-host bone junction
at 12 weeks post-operative. 57
Figure 33. Graft healing via creeping substitution in the ribose-pretreated
irradiated grafts at the graft-host bone junction. 58
Figure 34. Creeping substitution seen within the ribose-pretreated irradiated
grafts (new bone (green) infiltrating into the graft (blue)). 59
Figure 35. Backscatter electron microscopy images of the three graft groups. 60
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Figure 36. Histomorphometric analysis of the bone volume (normalized to
tissue volume) (BV/TV) within the grafts. 61
Figure 37. Histomorphometric analysis of the mineralizing surface (MS)
within the grafts. 62
Figure 38. Histomorphometric analysis of the mineral apposition rate
(MAR) within the grafts. 62
Figure 39. Spiral fracture pattern observed with torsional testing of the
reconstructed radius. 63
Figure 40. Failure at the level of the osteotomy site. 64
Figure 41. Necropsy images showing petechial on the inner stomach lining
and duodenal hemorrhage. 66
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LIST OF APPENDICES
APPENDIX Page Number
Appendix A. Radiographic scores of the critically-sized radial defects
reconstructed using bone allograft. 91
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1. INTRODUCTION
Massive bone defects, such as those greater than 10 cm, are difficult and challenging
problems for orthopedic surgeons to manage. They can commonly arise following
musculoskeletal tumour resection [4, 19, 34], peri-prosthetic bone loss in the setting of revision
hip and knee arthroplasty [11, 35], and high-energy trauma [24, 37]. Unfortunately, current
treatment options are associated with a number of drawbacks. Amputations often result in the
limb function loss and/or disability [25]. The use of free vascularized bone grafts can be
technically challenging and yields significant donor site morbidity (including motor weakness,
sensory deficits, increasing ankle pain over time, and graft fracture one year after bony union
[59, 88]). Bone transport distraction osteogenesis (such as the Ilizarov technique), which
involves performing an osteotomy with subsequent distraction to stimulate bone growth, can be
extremely inconvenient for patients and requires an extended period of time for healing [54].
Orthopedic oncologists have utilized massive endoprostheses to reconstruct the defects, but these
implants have been shown to fail due to aseptic loosening or fractures around the prosthesis [43].
The drawbacks of many of these aforementioned treatment options are likely the reason for
continued research into developing and engineering new biologics and biomaterials to address
critically-sized bone defects [21, 29, 70].
Large structural bone allografts have been described as a viable option in reconstructing
significant bone defects following musculoskeletal skeletal tumour resection [4, 6, 27, 65], peri-
prosthetic bone loss [11, 35], and post-traumatic injury [20, 24]. The use of bone allografts helps
to avoid donor site morbidity and maintain bone stock [26]. Intercalary bone allografts have been
shown to be acceptable successful options in tumour reconstruction. Aponte-Tinao et al. reported
76% survivorship at 10 years, with good functional outcome scores [6]. Similarly, Gupta et al.
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reported even higher 10 year survivorship in their cohort at 84.8%, with good reported functional
outcome scores [39].
1.1 Allograft Bone Healing
The incorporation of bone allografts with host bone has been well studied, and found to
occur through three phases: (1) Reactive Bone Formation, (2) Revascularization, and (3)
Perivascular New Bone Formation [9]. The first phase (“Reactive Bone Formation”) starts with
bridging of new bone (or callus) between the graft and host bone. This new callus functions to
eliminate or reduce the shearing forces at the graft-host bone interface. The stability imparted by
the callus then allows for penetration of vascular vessels into the graft during the second phase
(“Revascularization”). This increased vascularity into the graft helps to bring an influx of cells
involved in the reparative process towards the center of the graft. In the final third phase
(“Perivascular New Bone Formation”), the pre-existing bone matrix in the graft is sequentially
removed and replaced with new bone. This invasion and replacement of old graft matrix with
new bone is referred to as creeping substitution.
1.2 Irradiation of Bone Allografts
To avoid the risk of disease transmission, harvested bone allografts often undergo
sterilization with γ-irradiation before implantation. Among tissue banks, a minimum low-dose of
25 kGy is recommended for terminal sterilization of bone allografts to meet the sterility
assurance levels [66]. However, the use of irradiation has been shown to negatively affect the
biomechanical properties of the graft [73]. Russell et al. found that New Zealand white rabbit
humeri irradiated at a dose of 25 kGy had a 64% decrease in maximum torque (with torsional
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testing) and a 44% reduction in energy to failure (with three-point bending) when compared to
untreated controls [78]. Similarly, the study by Burton et al. compared the biomechanical
properties of irradiated (33 kGy on dry ice) to non-irradiated cortical bovine tibia through three-
point bending, and found that irradiation resulted in 62% loss of work-to-fracture and 45% loss
in failure strain [16]. Using human cadaveric femora, Hamer et al. showed that irradiation with
28 kGy resulted in a 64% reduction in strength when compared to non-irradiated controls [41].
These studies all demonstrated the negative effects of irradiation on bone through static testing.
It has been suggested that allograft material typically fails due to fatigue [22]. Again,
cyclic testing has also shown the deleterious effects of irradiation on the biomechanical
properties on bone. Russell et al. cyclically tested rabbit bone through three-point bending and
found that sterilization with γ-irradiation significantly decreased the cycles to failure when
compared to untreated controls (46,000 versus 1,500 cycles to failure, p=0.000) [77]. The study
by Akkus and Belaney investigated the fatigue stress of human femurs (cortical femoral
diaphyseal specimens) irradiated with 36.4 kGy of γ-irradiation through cyclic testing [2]. They
found a 99.5% reduction in cycles to failure with γ-irradiation. The work by Mitchell et al.
showed that the weakened fatigue properties of γ-irradiated cortical bone were due to their
significantly reduced ability to resist fatigue crack propagation (when compared to non-irradiated
bone) [60]. Based on these findings, they suggest cortical bone sterilized with γ-irradiation may
be more predisposed to fracture, even under normal loads experienced during every day
activities.
Studies have further shown that the negative effects of γ-irradiation on bone occurs in a
dose-dependent manner. Barth et al. reported that exposure to irradiation resulted in a dose-
dependent degradation of the mechanical properties of bone [8]. Specifically, for doses greater
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than 35 kGy, there was a dose-dependent reduction in bending strength, ductility, and fracture
toughness. Furthermore, they found that once doses reached greater than 80 kGy, the irradiated
bone no longer exhibited any plastic deformation characteristics but rather failed at its elastic
limit. Similarly, the study by Hamer et al. showed a dose-dependent reduction in strength and
capacity to absorb work with increasing doses of γ-irradiation [41]. Russell et al. also reported
dose-dependent decreases in maximum load when higher doses of irradiation were used [77, 78].
Clinically, the deleterious effects of irradiation on the mechanical properties of bone
allografts are significant. Lietman et al. reported that patients with irradiated bone allografts
fractured at rates twice that of those with fresh non-irradiated allografts [57]. The work by Sorger
et al. showed that the occurrence of allograft fracture was predictive of a worse functional
outcome [85]. Survivorship analysis showed that patients with allograft fractures had
significantly lower proportion of excellent (defined as having no pain, and normal function with
no limitations) and good (defined as some degree of functional impairment) outcomes compared
to those with no fracture. Thus, the implications on the quality of life and function of patients
following allograft fracture is significant.
1.3 Mechanism of Degradation
To understand how γ-irradiation deleteriously affects the biomechanical properties of
bone, its structure must first be examined. Bone is complex hierarchical structure, composed of
organic components (such as collagen, proteoglycans, matrix proteins), inorganic mineral
components (such as calcium hydroxyapatite), and water [8]. At the submicron level, it is
composed of type I collagen (or tropocollagen), which then forms mineralized collagen fibrils.
Between the tropocollagen molecules (as well as between the mineralized collagen fibrils), there
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are both enzymatic and non-enzymatic crosslinks. It is these crosslinks that impart the intrinsic
plastic properties of bone.
Previous biomechanical studies have shown that γ-irradiation does not alter or negatively
affect the elastic properties of cortical bone, but rather its plastic properties [2]. This would thus
suggest that γ-irradiation imparts its deleterious effects on the collagen phase of bone (and not
the mineral inorganic phase). Indeed, Barth et al. showed that exposure to 70 kGy of irradiation
altered the enzymatic crosslinks present in type I bone collagen [8]. Specifically, a decreased
ratio of mature to immature collagen crosslinks was seen with Fourier transform infrared
spectroscopy (FTIR) of the 70 kGy irradiated bone (when compared to unirradiated bone). These
findings suggested that irradiation may impart more damaging effects on mature crosslinks,
which the authors warned could lead to lower fracture loads and early failure. Our laboratory
also found that sterilization of bone with γ-irradiation reduced the overall connectivity of
collagen with evidence of collagen fragmentation [16]. The study by Nguyen et al. found that
with increasing doses of irradiation, there was loss of bone toughness, which the authors
attributed to the denaturation of collagen molecules (as measured by the hydroxyproline content
using high-performance liquid chromatography (HPLC)) [66].
Further research has been conducted to better elucidate the mechanism by which
irradiation affects bone collagen. One mechanism of action that has been suggested is that γ-
irradiation creates free radicals, which then cleaves the collagen backbone [67]. In the study by
Hamer et al., bone that was γ-irradiated (30.2 kGy) at -78 degree Celsius tolerated greater
maximum load and exhibited less collagen denaturation than bone irradiated at room temperature
[40]. The authors hypothesized that irradiating at lower temperatures helped to keep the water
molecules in a frozen state, making it more difficult for radiolysis to occur to create free radicals.
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Akkus et al. provided further evidence of the damaging effects of free radicals caused by
irradiation. In their study, equal amounts of (solubilized) collagen from irradiated and non-
irradiated bone samples were loaded into SDS-PAGE electrophoresis gels [3]. The irradiated
samples showed weaker bands of α1, α2, and β chains of collagen (compared to the non-
irradiated samples), with diffuse staining observed throughout the gel lane (across all molecular
weights). The authors concluded that irradiation likely cleaved the bone collagen such that less
intact molecules remained, while more degraded molecules of varying molecular weights were
formed. Interestingly, the addition of a free-radical scavenger (thiourea) to the irradiated bone
resulted in the reappearance of the α1, α2, and β chain bands.
1.4 Ribose Pre-Treatment
To combat the deleterious effects of γ-irradiation, our laboratory developed a novel
sterilization method in which bone allografts were pre-treated with ribose prior to irradiation.
The idea behind our novel technique was to increase the connectivity of the bone collagen via
ribose crosslinking prior to irradiation. In this way, the collagen connectivity, which would
otherwise be altered, cleaved, and/or reduced following irradiation [8, 16], could be reinforced
and strengthened by the newly created crosslinks. In doing so, we hypothesized that this would
help protect the biomechanical properties of the allograft when undergoing sterilization with γ-
irradiation.
Ribose was chosen because it is a small enough molecule (less than 300 Da) to be able to
diffuse through the cortex of bone [50, 87]. As well, it has been shown to be a crosslinker of
bone collagen via non-enzymatic glycation [91, 96]. Additionally, ribose-crosslinked collagen
matrices have demonstrated good cytocompatibility in a number of previous studies [36, 75].
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The formation of these crosslinks occur as the carbonyl aldehyde group on ribose (a
reducing sugar) readily reacts with the free amino group from a protein (such as on collagen)
through the Maillard reaction (Figure 1) [1, 83].
Figure 1. The Maillard reaction.
The subsequent glycated proteins undergo further reactions to form a number of advanced
glycation end-products (AGEs). Not all AGEs have been identified as there are a vast and
significant number. However, of the ones identified, they can be divided into three categories:
(1) fluorescent cross-linking AGEs (such as pentosidine); (2) non-fluorescent cross-linking
AGEs; and (3) non-crosslinking AGEs [1].
Biomechanical testing of cortical bone pretreated with our novel sterilization technique
showed protection of the mechanical properties otherwise lost to conventional irradiation [95].
Both human and bovine bone samples treated with ribose prior to irradiation showed 100%
protection of ultimate strength. As well, the ribose pre-treatment yielded 52% and 60%
protection of strain-to-failure of bovine and human samples, respectively. Furthermore, there was
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57% protection of work-to-fracture in the bovine treated bone, and 76% protection in the human
samples.
1.5 In-Vivo Testing
With the in vitro studies demonstrating that the ribose pretreatment was able to provide
protection of the biomechanical properties lost to irradiation, the next step was to test the
biocompatibility of these grafts in vivo. In order to advance engineered biomaterials and implants
such as our ribose-pretreated grafts for potential clinical use, preclinical testing in animal models
must first be performed. In vivo animal models serve to reproduce the physiological conditions
and measure the pre-clinical outcomes of new therapies that cannot be simulated using in vitro
models. This is especially important when testing the biocompatibility, osteoconduction, and
degradation properties of novel implant and graft materials [64].
The New Zealand White rabbit (NZWr) is a commonly used animal to study bone healing
because its bone exhibits Haversian remodeling and bone densities similar to human bone [64,
74, 93]. Furthermore, the issues of permanently open growth plates associated with rodent
models are avoided [74]. Their ease in handling, housing, availability, and economical cost
compared to larger animals also make the NZWr an ideal animal to use [72]. Indeed, the
International Organization for Standardization (ISO) practice guide for Biological Evaluation of
Medical Devices states that the rabbit model is the animal of choice for implant testing [45].
The NZWr radius segmental model serves as an appropriate long bone defect model for a
number of reasons. These include, but are not limited to: (1) the tubular cross-sectional shape of
the radius makes it easier to create implants of similar shape; (2) the model has been well
studied; (3) the reproducibility of the model allows for comparison of different graft materials;
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and (4) reportedly no fixation is required due to the support of the ulna [5]. However, we would
argue that stable fixation is required when testing the biocompatibility of graft materials in the
NZWr radius model.
1.6 Thesis Hypotheses and Objectives
This thesis was composed of two separate studies, each with their own objectives:
STUDY 1
To test the biocompatibility of ribose pre-treated irradiated bone allografts in vivo, a
proper animal model was required. We hypothesized that fixation (within the NZWr radius
model) was needed to help reduce excessive motion at the osteotomy sites and prevent the
occurrence of non-union as a result of instability. This would then allow the true effects of the
biological interactions of the graft material on union and osteoconduction to be assessed.
The objective of the first study was to evaluate and establish the use of intramedullary
wire as a fixation method within the NZWr radius segmental defect model, using bone allograft
as the model biomaterial.
STUDY 2
Our hypothesis was that the ribose-pretreated irradiated allografts could achieve bony
union with host bone, similar to that of conventionally-irradiated and fresh frozen controls.
Furthermore, we hypothesized that the quality of bony union of ribose-pretreated irradiated bone
allografts would be better than conventionally-irradiated allografts, but inferior to fresh-frozen
untreated grafts.
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Using the in vivo rabbit model established from Study 1, the objective of the second study
was to determine if the ribose-pretreated irradiated bone allografts could heal through bony
union with host bone (when compared with conventionally-irradiated and fresh frozen bone
allografts). Bony union would be assessed using radiographic measures, histomorphometry (to
characterize the cellular interactions at the union site), and biomechanical testing (to determine
the strength of the union).
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2. MATERIALS AND METHODS (Common to Both Studies)
2.1 New Zealand White Rabbit
Female retired NZWr breeders (Charles River, St. Constant, PQ, Canada) aged 7-9
months old and average weight of 4.02 kg (range: 3.1-5.4 kg) were used. Only female rabbits
were used as they were more tractable than their male counterparts at our institution. Younger
rabbits with open growth plates were excluded to avoid potential complications associated with
epiphyseal slipping [51]. The rabbits were maintained on a 12:12 hour light cycle with rabbit
chow (Harlan Laboratories Inc., Indianapolis, IN) and water ad libitum at a Canadian Council on
Animal Care accredited vivarium at the University of Toronto. Rabbit diets were supplemented
with hay, Critical Care (Oxbow, Murdoch, NE, USA), and fruit and vegetables as needed.
The surgical procedures performed on the NZWr strictly followed institutional,
provincial, and federal guidelines for the care and use of laboratory animals. All procedures were
subject to review and approved by the local Animal Care Committee/Institutional Ethics Review
Board.
2.2 Donor Bone Allograft Preparation
Bone allografts were first harvested from both radii of donor age-matched female retired
breeder NZWr (Charles River, St. Constant, PQ, Canada). Following euthanasia of donor NZWr
under heavy sedation (Atravet, Boehringer Ingleheim, Baie d’Urfe, PQ, Canada) with T-61 (T-
61, Intervet, Kirkland, PQ, Canada) both thoracic limbs were prepped by clipping the hair from
the axilla to the distal metacarpals, and the skin was subsequently disinfected using two
successive applications of ethanol followed by betadine. The feet were then wrapped with sterile
Tegaderm (3M Healthcare, Neuss, Germany), betadine was reapplied, and both limbs draped in a
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sterile fashion. An anterior skin incision on the forelimb was used, with soft tissue dissection
down to the radius and ulna. Small baby Hohmann retractors were used to expose the forelimb
bones, and a high-speed micro-oscillating saw under irrigation with physiological saline was
used to cut the proximal and distal aspects of the radius and ulna to ensure as much bone length
as possible was obtained. This resulted in an approximately 6-7 cm long segment of bone. The
radius was then separated from ulna. Any soft tissue attachments were removed from the radius,
and the IM canal was cleaned and irrigated using sterile normal saline in 10 cc syringes with 22
gauge needles to remove as much of the bone marrow contents as possible. Once cleaned, the
harvested radius samples were wrapped in saline soaked gauze, placed in sterile containers, and
stored in a -30° Celsius freezer for later use.
The harvested bone allografts then underwent one of three treatments: (1) No treatment
(fresh frozen controls) (C); (2) Conventional γ-irradiation (I); and (3) Ribose-pretreatment
followed by γ-irradiation (R) [95]. For the C allografts, these grafts remained wrapped sterile in
saline-soaked gauze in a -30° Celsius freezer. For the R group, ribose media was prepared prior
to incubation at concentration of 1.2 M moles/L in PBS. The harvested bone was then placed in
appropriate tubes and ribose incubation solution added. The tubes were placed in a shaking water
bath for 24 hours at 60° Celsius. The ribose-pretreated grafts were evident as the colour of the
bone changed from white to brown, indicating the presence of non-enzymatic glycation [53, 91].
Finally, the irradiation sterilization of the I and R allografts occurred with the grafts packed on
dry ice and irradiated at 33 kGy using a 60Co source (Isomedix Steris, Whitby, Ontario, Canada)
[95].
13
2.3 Surgical Procedure
The surgical reconstructions were performed by an Orthopedic Surgery resident with
assistance from a veterinarian and veterinary technical staff. The surgical procedure was based
on a previously described protocol by An and Friedman [5], but with modifications to the
surgical approach and the use of fixation for the graft.
The recipient NZWr underwent anesthetic induction using ketamine 35 mg/kg
intramuscular (IM) (Ketaset, Wyeth, Guelph, ON, Canada) and xylazine 5 mg/kg IM (Rompun,
Bayer, Toronto, ON, Canada) to allow for placement of a laryngeal mask airway (LMA North
America Inc., San Diego, CA, USA). The thoracic limb was then prepped for surgery by clipping
the hair and disinfecting with ethanol and betadine (as described in the previous section for the
donor NZWr). The marginal ear vein was catheterized for delivery of intravenous fluids (lactated
Ringer’s solution) at a rate of 3 ml/kg/hr. The recipient NZWr received a single pre-operative
prophylactic dose of cefazolin 20 mg/kg IM (Sandoz, Boucherville, PQ, Canada). The rabbit was
subsequently placed under a balanced anesthetic with isoflurane in 1 L/min O2 (Aerrane, Baxter,
Mississauga, ON, Canada), buprenorphine 0.05 mg/kg subcutaneous (SC) (Temgesic, RB
Pharmaceuticals, Berkshire, UK), ketoprofen 3 mg/kg SC (Anafen, Merial Canada, Baie d’Urfe,
PQ, Canada) and 0.02mg/kg glycopyrrolate SC (Sandoz, Boucherville, PQ, Canada). Multiple
sterile Tegaderms were used to cover the foot, and the forelimb was re-prepped with betadine,
and draped in a sterile fashion. The incision site was infiltrated with bupivacaine 1-2 mg/kg
(Marcaine, Hospira, Montreal, PQ, Canada).
A longitudinal skin incision was made over the distal anterior aspect of the forelimb over
the radius (Figure 2).
14
Figure 2. Longitudinal skin incision along the distal anterior forelimb.
The subcutaneous tissue was undermined to create medial and lateral skin flaps. The tendon of
the extensor digitorum communis (EDC) was initially identified to help locate the more medial
structures of the extensor carpi radialis (ECR) and abductor pollicis longus (APL). The distal
aspect of the bony radius could then be identified medially to ECR and laterally to flexor carpi
radialis (FCR) tendon. A surgical plane was created between ECR and FCR from distal to
proximal to better expose the full length of the radius. This plane went approximately 2 cm
proximal to the insertion of the pronator teres. Two small baby Hohmann retractors were used to
provide exposure of the radius (Figure 3).
Figure 3. Diaphysis of the radius exposed following surgical dissection.
15
Once the radius was exposed, a minimum 15-mm critically-sized defect (CSD) was measured in
the mid-diaphysis of the radius. While the accepted CSD of rabbit long bones in the literature is
two times its diameter of 5-6 mm [5], equivalent to about 10-12 mm, a minimum 15-mm defect
was created to ensure a true CSD was created. A micro-oscillating saw under irrigation with
saline was used to cut out the mid-diaphysis defect in the radius (Figure 4). The defect site was
then cleaned and thoroughly irrigated with saline.
Figure 4. Critically-sized bone defect created in the mid-diaphysis of the radius.
The previously harvested donor allograft (which had been thawed for 30 minutes, soaked
in betadine for 15 minutes, soaked in enrofloxacin 50 mg/ml solution (Baytril, Bayer, Toronto,
ON, Canada) for 15 minutes prior to implantation) was then cut and sized to fit the radial defect.
To ensure anatomic reconstruction, the curve of the allograft was matched to the curve of the
bone defect that had been removed with the micro-oscillating saw. It was important to match the
curve and length of the graft to the original host bone because short grafts resulted to poor
cortical contact at the osteotomy sites and slight recurvatum of the radius, while longer grafts
caused opening at the osteotomy sites and procurvatum of the radius. The anatomically-matched
allograft was then inserted into the defect.
16
In order to place the IM wire, the start point for the wire had to be established. This could
be achieved in two techniques. One method was retracting the ECR muscle laterally, exposing
the medial aspect of the distal radius. While somewhat of an easier technique, this method
allowed for more prominent hardware and less soft tissue coverage. The alternative was to incise
the fascia of the APL muscle and then retract the muscle belly over medially to expose the dorsal
aspect of the distal radius. Following insertion of the wire, the coverage with the APL muscle
and closure of its fascia allowed for good soft tissue coverage. Using either method, a 0.8 mm
smooth trocar tip Kirschner wire was placed as an IM device for fixation of the graft (Figure 5).
Figure 5. Intramedullary fixation of the bone allograft using a 0.8-mm Kirschner wire.
Careful check was done to ensure there was cortical contact between the host and allograft bone
at both proximal and distal osteotomy sites, no soft tissue was caught in the osteotomy sites, and
the Kirschner wire was within the IM canal. A thorough wash of the reconstructed site and the
Kirschner wire insertion site was done. Multiple layer closure of the fascial layers was performed
using 4-0 Polysorb (Covidien, Mansfield, MA, USA). Because of the irritation of staples or
exposed sutures, a subcuticular closure using 6-0 Vicryl (Johnson & Johnson, Markham, ON,
Canada) was done. A dressing was then placed on the operated forelimb.
17
2.4 Post-Operative Care
After completion of the surgical procedure, the NZWr was maintained under anesthesia
in order to obtain an immediate post-operative radiograph. Placement of the IM Kirschner wire
and adequacy of the reconstruction was confirmed on this radiograph. The rabbit was then
recovered and monitored during the immediate post-anesthesia period.
The NZWr were weight bearing as tolerated, and no restrictions in terms of their activity
status following surgery. The rabbits, which had been slowly habituated to flexible Elizabethan
collars in the 1-2 weeks prior to surgery, were placed into the collars continuously for 7 days in
the post-operative period. These flexible collars allowed for undisturbed healing of the surgical
incision, while still permitting eating, drinking, and limited species-specific behavior such as
grooming and coprophagy. All NZWr were observed several times daily with changes in
appetite, gait, body weight, body temperature, appearance and behavior recorded. Inappetent
rabbits were supplemented with Critical Care (Oxbow, Murdoch, NE, USA), and additional hay,
fruits and vegetables. The initial surgical dressings were changed on post-operative day 3 and
removed entirely by post-operative day 7-10. During the first 2-3 days following surgery,
analgesia with buprenorphine 0.05 mg/kg SC (Temgesic, RB Pharmaceuticals, Berkshire, UK)
every 12 hours and ketoprofen 3 mg/kg SC (Anafen, Merial Canada, Baie d’Urfe, PQ) every 24
hours was administered. Additional doses of analgesics were given as indicated. If the rabbits
exhibited stridorous breathing secondary to placement of the laryngeal mask airway, an anti-
inflammatory dose of dexamethasone 1.0 mg/kg SC (Dexamethasone, Dominion Veterinary
Laboratories, Winnipeg, MB, Canada) was administered.
To enable dynamic bone histomorphometry for later analysis in Study 2, a fluorescent dye
marker was administered prior to euthanasia of the rabbit subjects. This would allow for
18
quantification of the rates of bone formation and mineralization. The dye of choice for this study
was calcein green. Approximately 10 ml IV calcein green (20 mg/kg body mass) was
administered in the marginal ear vein at 2 weeks and 1 week prior to euthanasia. The time points
selected for dye administration were based on pilot data from Study 1.
2.5 Data Collection
Twelve weeks after surgery, the rabbits were euthanized under heavy sedation (Atravet,
Boerhinger Ingleheim, Burlington, ON, Canada) with T-61 (Intervet, Kirkland, PQ, Canada).
The study period of 12 weeks was selected as this was the time point at which there was
radiographic evidence of union based on pilot data. This ensured an objective and consistent time
point to evaluate the reconstruction. The study by Bodde et al. [13] further supported the 12
week time point (further explained in the Discussion section). After euthanizing the rabbits, the
entire surgically operated distal forelimb (including the ulna and reconstructed radius) was
carefully dissected out. The smooth Kirschner wire in situ was removed with care to not disrupt
the osteotomy sites and mineralized callus. The harvested forelimb was then cut into two
segments: (1) the distal segment, containing the distal osteotomy site; and (2) the proximal
segment, containing the proximal osteotomy site.
2.6 Radiographic Analysis
Bony healing in the post-operative period was monitored using radiographs. The
radiographs (Min X-ray Inc., model HF8015, Northbrook, IL, USA; digital processor, model CR
30-X, AGFA HealthCare, Peissenberg, Germany) were taken with the NZWr placed under
19
isoflurane (Aerrane, Baxter, Mississauga, ON, Canada), and acepromazine (Atravet, Boehringer
Ingleheim, Baie d’Urfe, PQ, Canada) sedation 0.3 mg/kg SC.
The radiographs were analyzed by a fellowship-trained musculoskeletal radiologist.
Healing at the osteotomy sites was quantified based on four parameters outlined in the modified
radiographic scoring system described by An and Friedman [5]: periosteal reaction, proximal
osteotomy union, distal osteotomy union, and bone remodeling (Table 1).
Table 1. Radiographic scoring system, modified from An and Friedman [5].
Category Scores
Periosteal reaction
Full (across the defect)
Moderate
Mild
None
3
2
1
0
Proximal osteotomy union
Union
Moderate bridge (>50%)
Mild bridge (<50%)
Non-union
3
2
1
0
Distal osteotomy union
Union
Moderate bridge (>50%)
Mild bridge (<50%)
Non-union
3
2
1
0
Remodeling
Full remodeling cortex
Intramedullary canal
No remodeling
2
1
0
Maximum total score: 11
Consensus on how to quantitatively evaluate the radiographs was reached by reviewing and
applying the modified scoring system to radiographs of surgical reconstructions from our pilot
study. In the case of periosteal reaction and bone remodeling, the description outlined by An and
20
Friedman were easily applicable. For scoring of bony union, we used the definitions described by
Johnson et al. [47]. Union was defined as a complete bridge across bone ends. Incomplete union
was characterized as having less than 50% (mild bridge, according to the An and Friedman
scoring system) or greater than 50% (moderate bridge) circumferential bone bridge across bone
ends. These definitions were applied to our radiographs by reviewing multiple orthogonal views.
Furthermore, given that healing was to occur by secondary bone healing (as fixation with IM
wire would achieve relative stability), the definition of union was not necessarily based on the
absence of the fracture (osteotomy) lines.
2.7 μCT Analysis
The harvested bone specimens were first scanned with μCT prior to static bone
histomorphometric analysis. The graft-host bone junction and mineralized callus at the
osteotomy site were scanned using the Skyscan 1174 compact X-ray μCT scanner (Skyscan,
Belgium) with the following parameters: beam of 50 kV and 800 μA and isometric voxel size of
14.4 μm. In addition, phantoms of 750 and 1,300 mgHA/cm3 were also scanned each day the
bone samples underwent μCT. Theses phantoms were used for bone mineral density calibration
purposes. Skyscan NRecon software was used to reconstruct the images and subsequently
analyzed using Skyscan CTan software.
2.8 Histomorphometric Analysis
Following μCT scanning, each reconstructed radius was cut distal and proximal to the
osteotomy site to obtain a smaller sample with the region of interest. The undecalcified samples
of reconstructed bone were then fixed in 70% ethanol for at least 1 week. These specimens were
21
subsequently dehydrated in increasing concentrations of 70%, 90%, 100%, and 100% acetone,
followed by infiltration with 50%, 80%, 100%, and 100% unpolymerized Spurr (Marivac,
Halifax, NS, Canada), with each step lasting 4 days under vacuum. Following the dehydration
and infiltration processes, the samples were embedded in blocks of Spurr and polymerized at
600C for 2 days. The polymerized Spurr blocks were then cut using the Leica RM 2265 rotatory
microtome (Leica Microsystems Canada, Inc., Richmond Hill, Ontario, Canada) to create 7-μm
thick sections of each sample and placed on gelatinized slides. The sectioned slides were dried
for 3 days at 600C, and subsequently stained with Goldner’s Trichrome (Hematoxylin Sigma
H3136; Acid Fushin, Fisher #733299; Phosphomolydbic Sigma P7390; Light green, Sigma
L1886) for static bone histomorphometric analysis, or cover slipped with no counterstain for
dynamic bone histomorphometric analysis. For all sections, histomorphometric analyses were
performed using BioquantOsteo Nova Prime software (Bioquant Image Analysis Corporation,
Nashville, TN).
22
3. STUDY 1
3.1 Study Design
A pilot study using four recipient NZWr (and two donor NZWr sacrificed for allografts)
was initially performed to determine the pre-operative protocol, surgical plan, and post-operative
protocol. Since our laboratory had not used this model previously, it was important to optimize
all parameters to ensure success with the NZWr radius defect model. Pre-operative parameters
included determining the number of days required for NZWr to be acclimatized, pre-operative
diet, and protocol for obtaining pre-operative radiographs. Surgical parameters included the
surgical approach, method by which the osteotomy of the radius would be performed, and multi-
layer closure of the wound. Post-operative parameters included determined post-operative diet
and analgesics, monitoring for complications, when to perform dressing changes, time interval
for administering fluorescent dye markers for later dynamic histomorphometry, time to
radiographic bony healing, and protocol for euthanasia.
Once the appropriate protocols were established, the use of intramedullary wire as a
fixation method within the NZWr radius defect model was evaluated using 10 NZWr, with five
additional donor age-matched female NZWr used to obtain bone allografts from (harvesting both
radii from each donor rabbit).
3.2 Methods (Study 1)
3.2.1 Radiographic Analysis
To evaluate the efficacy of intramedullary wire fixation within the NZWr radius defect
model, bi-weekly radiographs of the reconstructions were performed. This data helped to provide
information about a number of factors related to in vivo bony healing within the model, such as
23
when the first appearance of bony callus occurred, healing patterns of the graft and at the
osteotomy sites, and time to bony fusion that is evident radiographically. As well, the stability of
the intramedullary-fixated construct could be assessed.
3.2.2 Data Collection
Analysis of the harvested bone segments comprised of μCT and static bone
histomorphometry testing. μCT of the osteotomy sites was utilized to confirm the radiographic
findings of bony union, as well as to visualize the degree of bony callus formation and
remodelling of the graft. Static histomorphometry further confirmed if union was achieved at the
tissue level.
3.2.3 μCT Analysis
The graft and host bone junction at the osteotomy sites were first rendered into three-
dimensional (3D) images. Presence or absence of a bony callus at the osteotomy site was
qualitatively examined. A region of interest (ROI) was then drawn to produce a sagittal view of
the volumetric 3D images. This allowed for assessment of bony union at the osteotomy site,
characterization of the callus, and degree of remodeling as evident by graft porosity.
3.2.4 Histomorphometric Analysis
Static bone histomorphometry was used to further confirm if union was achieved at the
tissue level. Analysis was done qualitatively and assessed for the presence or absence of bony
union.
24
3.2.5 Statistical Analysis
The statistical analyses were conducted using SPSS version 21 (IBM, New York, NY).
Statistical significance was achieved when the p-value was less than 0.05.
Kruskal-Wallis statistical testing was used to determine if there were differences in the
categorical radiographic scores (based on the parameters of periosteal reaction, proximal union,
distal union, and cortical bone remodeling) between the bi-weekly radiographs. If statistical
significance was observed, post hoc statistical comparisons between the bi-weekly time intervals
was conducted using Mann-Whitney test.
3.3 RESULTS (STUDY 1)
Gross inspection of the radial defect reconstruction following harvesting of the rabbit
forelimb showed good integration of the allograft to host bone at the proximal and distal
osteotomy sites in the presence of visible bony callus (Figure 6).
Figure 6. A harvested reconstructed forelimb with Kirschner wire in situ.
3.3.1 Radiographic Analysis
Stable intramedullary (IM) wire fixation of bone allograft within a critically-sized radial
defect was initially achieved in nine of the ten NZWr. Most NZWr exhibited partial weight
bearing immediately following surgery. All NZWr were fully weight bearing on the operated
25
limb within 7 days after surgery. The reconstructions were anatomic in that the curvature of the
radius was matched, cortical contact at the proximal and distal osteotomy sites was obtained
(Figure 7, yellow arrows), and the interosseous space between the radius and ulna was
maintained (Figure 7, red arrow).
Figure 7. Post-operative radiographs at 2 weeks of a critically-sized radial defect reconstructed
using bone allograft and fixed with Kirschner wire
(yellow arrows point to the cortical contact achieved at the distal and proximal osteotomy sites;
red arrow indicates the interosseous space maintained between the reconstructed radius and
ulna).
By the 12 week endpoint, the osteotomy sites in all ten rabbits went on to bony union.
Analysis of the radiographic scores of the reconstructions (Appendix A) showed that at 2
weeks post-surgery, no periosteal reaction or union was evident (Figure 7). By 4 weeks, there
was mild to moderate periosteal bridging callus localized to the osteotomy sites with less than
50% bridging union (Figure 8).
26
Figure 8. Radiographs at 4 weeks post-operative. Callus formation can be seen localized to the
osteotomy sites.
From 6 to 8 weeks, significant callus formation over the osteotomy sites and greater than 50%
bridging union (more so at the proximal than distal osteotomy sites) was observed (Figure 9).
Figure 9. Radiographs at 8 weeks post-operative. Greater callus formation can be observed.
By 10 weeks, cortical remodeling was noted (Figure 10).
Figure 10. Radiographs at 10 weeks post-operative demonstrate cortical remodeling occurring.
27
In the final 12 week radiographs, there was complete proximal osteotomy site union, near full
circumferential union at the distal osteotomy site, periosteal callus formation extending over the
entire reconstruction site, and significant cortical remodeling was observed (Figure 11).
Figure 11. Radiographs at 12 weeks show evidence of bony union and cortical remodeling.
3.3.2 μCT analysis
μCT analysis revealed bony union at the proximal and distal osteotomy sites in all ten
rabbits. Furthermore, significant porosity was found in the allografts on axial cuts at both
proximal and distal osteotomy sites, indicating cortical remodeling had occurred. The μCT scans
also confirmed that the interosseous space between the radius and ulna was maintained, except at
the osteotomy sites where the bony callus had localized (Figure 12).
28
Figure 12. Three-dimensional reconstruction of μCT scan of the graft-host bone junction at 12
weeks post-operative (sagittal cut shown). Density gradient scale provided in image.
3.3.3 Static Bone Histomorphometry
Static histomorphometry of the osteotomy site revealed significant callus formation by 12
weeks. There was also evidence of osteoid formation within the healing callus, indicating
ongoing bone formation (Figure 13).
29
Figure 13. Static bone histomorphometry (stained with Goldner’s Trichrome) of the graft-host
bone junction at 12 weeks post-operative.
30
3.3.4 Complications
There were no infections or mortality in any of the ten NZWr that underwent surgical
reconstruction. The graft in one rabbit was found to have displaced after slight distal migration of
the Kirschner wire at 2 weeks post-surgery (Figure 14).
Figure 14. Radiographs at 2 weeks showing graft displacement after proximal Kirschner wire
migration in one NZWr.
This rabbit was nevertheless fully weight bearing on the operated limb and had an uneventful
recovery. No further displacement of the Kirschner wire occurred in this rabbit and later went on
to bony union by 12 weeks.
31
4. STUDY 2
4.1 Study Design
Twenty-four female NZWr were randomized to receive one of three graft types: (1)
Untreated fresh frozen control allografts (C); (2) Allografts irradiated with conventional
irradiation (33 kGy γ-irradiation on dry ice) (I); and (3) Ribose-pretreated irradiated bone
allografts (ribose + 33 kGy γ-irradiation on dry ice [95]) (R) (Figure 15).
Figure 15. Study design for testing the union of ribose-pretreated irradiated bone allograft.
Determining the sample size for Study 2 was difficult. To the best of our knowledge,
there are no previous in vivo studies that have tested a similar biomaterial such as our ribose
treated graft. This made it difficult to perform an a priori power analysis to determine the
appropriate sample size for our study. Thus, we relied on the International Organization for
Standardization (ISO) practice guidelines for biologic evaluation of medical devices. Under Part
32
6 of the ISO guide (titled “Tests for Local Affects After Implantation”), a minimum of three
animals are required for each test interval [45]. Additionally, a review of the literature revealed
that previous studies examining the efficacy of different methods to repair segmental bone
defects had sample sizes between four and ten rabbits per treatment group [63, 79, 81, 92, 97]. A
sample size of eight rabbits per treatment group was selected for our study. This appropriately
met the ISO guidelines for biologic evaluation of medical devices and was consistent with other
segmental defect studies in the literature.
4.2 Methods (Study 2)
4.2.1 Radiographic Analysis
Upon better understanding the healing pattern of the reconstruction in Study 1, the time
points for which radiographs would be taken in Study 2 were determined. Only pertinent time
points were considered. For instance, if there were no apparent differences in radiographs two
weeks apart, then only one set of radiographs would be required. Based on this, less time points
were selected. Radiographs were only take at 2-, 6-, and 12-weeks.
4.2.2 Data Collection
To allow for biomechanical testing and histomorphometric analysis, the harvested
forelimb was cut so that the distal segment contained as much of the graft as possible while
approximately 2 mm of graft remained for the proximal segment (Figure 16).
33
Figure 16. Summary of all analyses performed on reconstructed samples in Study 2.
Obtaining a long length of graft within the distal segment was important because adequate bone
stock was needed to be able to be potted for biomechanics testing. The distal segment was
ultimately selected for torsion testing because there was less curvature to its bony anatomy
compared to the proximal end, allowing for better testing and easier analysis. As for the proximal
segment, this bone end first underwent μCT scanning, followed by histomorphometry, and then
back scatter electron (BSE) microscopy.
4.2.3 μCT Analysis
The Skyscan CTan software was used to first generate the volumetric 3D image of the
bone sample. A ROI was then drawn to allow for sagittal cuts of the bone sample. This allowed
34
identification of the graft-host bone junction (osteotomy site). The associated z-position line of
the osteotomy site within the computer software was then recorded. This was done for both the
distal and proximal bone sample segments.
Analysis of the proximal segment of bone sample involved creating a ROI of 2 mm
centered at the previously referenced osteotomy site. Thus, 1 mm of bone sample proximal and 1
mm of bone sample distal to the osteotomy site was included in the ROI. In order to draw the
ROI around the bone sample, the shrink-wrap function of the Skyscan CTan software was
utilized. This function allowed the ROI to wrap around and encase the entire bone area of
interest. The shrink-wrapping of the bone was based on the histogram threshold for what was
considered bone. To set the threshold for bone, each sample was analyzed. It was important to
properly set the bone threshold to avoid background noise being inadvertently picked up as bone.
The average threshold level of bone was determined to be 52.9. Thus, the histogram threshold
parameters were set between 52.9 and 255. Once the shrink-wrapped ROI was drawn, three
morphometric parameters were measured: volumetric bone mineral density (vBMD), bone
volume (BV), and tissue volume (TV).
The proximal bone segment underwent additional assessment by examining the extent
and degree of new bony callus formation on to the graft (Figure 17).
35
Figure 17. Three-dimensional rendered image illustrating new bony growth onto the graft.
Analysis of bone growth on to the graft was done by determining the distance from the
osteotomy site to the furthest leading edge of the callus. This measurement was taken from the
radial most side of the callus (as the ulnar side underwent significant bony remodeling). The
calculation involved identifying the z-position line of the osteotomy site and the z-position line
of the furthest leading edge of the callus, and determining the difference in distance between the
lines (Figure 18).
36
Figure 18. Calculating distance from osteotomy site to furthest leading edge of callus.
The difference in z-position lines was then converted into millimeters.
4.2.4 Static Bone Histomorphometry
To examine the cellular interactions at the graft-host junction, a 4 mm ROI was centered
at the osteotomy. The top and bottom limits of the ROI differed with each sample, but these
boundaries were drawn such that it included the callus bone and up to the where the callus
formed against the ulnar cortex (Figure 19).
37
Figure 19. A 4 mm ROI (green outline) drawn centered at the osteotomy site for static bone
histomorphmetric analysis.
The area and perimeter of bone were then determined using thresholding techniques. Areas of
white space (considered artifacts secondary to fragile bone sections) within and around the bone
samples were subtracted from the actual bone area to avoid having inflated values for the bone
volume. Following this, the perimeter of any ostetoid matrix was manually traced out. The
BioquantOsteo software then allowed the following parameters to be calculated: bone volume
standardized to tissue volume (bone volume (BV) / total tissue volume (TV)), ratio of osteoid
matrix to bone (osteoid volume (OV) / bone volume (BV)), and ratio of osteoid surface to bone
surface (osteoid surface (OS) / bone surface (BS)).
38
4.2.5 Dynamic Bone Histomorphometry
Gross examination of the static bone histomorphometry had revealed significant callus
formation (and osteoid matrix) at the proximal osteotomy sites in all samples (discussed in the
Results section for STUDY 1; refer to Figure 13). Our laboratory had previously seen that
within newly formed callus around a fracture site, the dynamic bone formation parameters were
significant and no differences were appreciated when comparing such parameters. As such,
dynamic bone histomorphometry data of the callus was not included in their published studies
[68, 69]. Essentially, areas of high bone formation such as a callus were similar in terms dynamic
bone characteristics within the same animal model. Given this, we focused our dynamic bone
histomorphometric analysis to within the graft. In particular, the ROI was drawn such that it
encased only the cortical bone of the graft, starting from the osteotomy site and ending 2 mm
into the graft (Figure 20).
Figure 20. A 2 mm ROI (green outline) drawn within the graft for dynamic bone
histomorphometric analysis. Example shown is of a ribose-treated irradiated graft.
39
Due to the limitations of the BioquantOsteo software, the ROI had to be drawn for each
individual cortex of each graft. This meant there were two cortical analyses for each graft
sample. Furthermore, since the cortical anatomy of the graft often changed when compared to its
original anatomy due to bony remodeling (secondary to bony erosions and/or new bone
formation), the ROI for each graft was drawn to outline the original shape of the graft. If the
original shape was difficult to outline due to significant bone resorption, a region of the graft
away from the remodeling area was used as reference to determine the ROI. As well, in some
samples, it was difficult to separate new bone callus from graft bone. In these situations, the
corresponding Goldner’s Trichrome stained slides were used, as allograft bone could be
differentiated from new bony callus by the absence of osteocytes.
Once the ROI was drawn, the following parameters were calculated: tissue volume (TV),
bone volume (BV), and bone surface (BS). The tissue volume encompassed the entire area
within the ROI, representing the area of the original graft. The bone volume was the bone within
the ROI and showed how much graft bone remained following the 12 weeks of healing.
Therefore, if there was less bone volume than tissue volume, it indicated there had been
resorption of the graft. Finally, the bone surface measured within the ROI represented the surface
of bone that had been eroded/resorbed (Figure 21).
40
Figure 21. Example of graft resorption (in a ribose-treated irradiated graft). Tissue volume is the
area within the green ROI. Bone volume is the area within the yellow outline. The presence of
yellow lines indicate bone resorption has occurred.
Following this, single-labelled and double-labelled lines within the ROI were manually drawn.
This allowed for calculation of the following parameters: mineralizing surface (MS), mineral
apposition rate (MAR), and bone formation rate (BFR).
4.2.6 Backscatter Electron Microscopy
Following sectioning for histomorphometric analysis, the undecalcified bone specimens
embedded in Spurr blocks were polished using the Beuhler polisher (Phoenix BETA
Grinder/Polisher, Beuhler, Canada). Polishing occurred with increasing size of sandpaper grit.
41
Multiple samples were then loaded on to plates and lightly coated with carbon. These coated
samples were placed on the stage of a scanning electron microscope (FEI XL30 SEM; FEI, Best,
Netherlands). The incident beam was set at a voltage of 20 kV and the beam current at 4 nA. The
BSE detector (Solid state BSE detector, FEI Company, Hillsboro, OR) was adjusted to capture a
field of view that included the osteotomy site at a magnification of 200X. More detailed images
of the cortical graft-host bone junction were obtained by increased the magnification to 50X.
Bone mineral density distributions were not evaluated using quantitative backscattered electron
imaging given that the areas of new mineralizing bone and mature cortical bone were easily
differentiated: new mineralizing bone (often the bony callus) appeared gray, while the mature
cortical bone appeared white (refer to Figure 22).
Figure 22. Backscatter electron microscopy image. Gray areas are new mineralizing bone, while
white areas are mature, mineralized cortical bone.
42
4.2.7 Torsion Testing Set-Up Protocol
The reconstructed radii underwent biomechanical testing under torsion. Torsion was
selected as the mechanical test to use because the goal was to assess the strength of the callus
(and not the graft or host bone). By undergoing a torsional moment, the same torque is applied to
every cross-section of the callus, such that failure occurs at the weakest cross-sectional area [62].
For this reason, torsion often considered the gold standard for testing the mechanical properties
of bony callus. In contrast, other tests such as compression and three-point bending are not able
to accurately assess the strength of the callus.
Torsional testing was performed on the distal bone segment (containing the distal
osteotomy site) because it had a straighter bone anatomy to allow for easier testing. When the
distal bone segment was initially harvested, the reconstructed radii was still attached to the native
ulna due to the bony callus at the osteotomy site and the native interosseous membrane between
the two forelimb bones. To test the strength of the callus/graft-host bone junction at the
osteotomy site with the intact ulna attached would result in inaccurate biomechanical data.
Therefore, the reconstructed radius was separated from the intact ulna using a diamond wire by
hand, which helped to maintain the reconstruction and callus at the osteotomy site (Figure 23).
Thermal bone necrosis was avoided during this process with the addition of normal saline drops.
43
Figure 23. The distal reconstructed radius bone segment separated from the ulna.
Once separated, any remaining soft tissue was removed. The side of bony attachment of
the radius onto the ulna was further polished using coarse sandpaper by hand. The μCT data was
then used to identify the distal osteotomy site on the bone sample. The distance from the cut end
of the graft to the distal osteotomy site was measured on the μCT scans. This measured distance
was then used to mark out the location of the osteotomy site on the gross specimen. Following
this, a line 4 mm proximal and a line 4 mm distal to the marked osteotomy site was then drawn.
This 8 mm segment of bone comprised the working length of the specimen. A distance of 4 mm
(proximal and distal to the osteotomy site) was chosen as this provided the most amount of graft
to be available for the working length while still allowing enough graft end to be potted. Strong
adhesive glue was then used to attach sandpaper at either ends of the bone samples (Figure 24).
44
Figure 24. Sandpaper attached to ends 4 mm distal and 4 mm proximal to the osteotomy site.
The coarse sandpaper was used to ensure the polymethylmethacrylate (PMMA) could
interdigitate to keep the bone specimen potted during torsional testing. The PMMA was first
prepared and inserted to fill the screw hole of a hex nut. One end of the sandpaper-attached
radius was then inserted in to the PMMA-filled hex nut and left to harden. This was repeated for
the other end of the bone sample (Figure 25).
Figure 25. Reconstructed radius potted in to hex nuts using polymethylmethacrylate (PMMA).
45
The PMMA-potted radii samples were then placed into clamps on an Instron ElectroPuls E1000
biracial mechanical testing machine (Instron, Norwood, MA, USA) for torsion testing (Figure
26). Specifically, the hex nut containing the graft bone was placed in the machine such that it
was the static end, while the hex nut containing the host radius was the end that rotated.
Figure 26. The potted reconstructed radius samples secured into the Instron ElectroPuls E1000
mechanical testing machine.
4.2.8 Torsional Testing Data Analysis
Using the torsion testing data, the structural and material properties of the reconstructed
radii were calculated. Structural properties characterize the mechanical properties of the entire
bone in its original intact form, while material properties describe the mechanical behaviour of
the bone when normalized to shape and size (thus, independent of geometry) [7].
46
The raw torsion data was first reviewed and the data was cut off to 10% drop from the
maximum torque (N·mm).
4.2.8.1 Structural Properties
The structural properties were calculated directly from the torsion data, and included
maximum torque, maximum angle of deformation, total energy to failure (J), and torsional
stiffness. Maximum torque (N·mm) was determined by identifying the highest torque value in
the data set. Maximum angle of deformation (degrees) was identified as the highest rotatory
displacement in the data set. Total energy to failure (J) was calculated as the area under the
rotatory displacement versus torque curve. Torsional stiffness (N·mm2/degrees) was calculated
based on the slope of the linear portion of the rotatory displacement-torque curve, using 100 data
points to determine the slope range.
4.2.8.2 Material Properties
The material properties calculated for the biomechanically-tested reconstructed radii
included shear strain, shear stress, and shear modulus.
Shear strain (degrees) was calculated using Equation (1) [71]:
γ = ro
ι× ɵ𝑜𝑠 (1)
where,
γ = Shear strain (degrees)
ro = Outer radius of bone specimen (mm)
ι = Distance from potted end to osteotomy site (mm)
ɵos = Angular deflection at osteotomy site (degrees)
in which the angular deflection at the osteotomy site was calculated using Equation (2) [71]:
47
ɵ𝑜𝑠 = ɵ
L× ι (2)
where,
ɵos = Angular deflection at osteotomy site (degrees)
ɵ = Maximum angle of deformation (degrees)
L = Gauge length (mm)
ι = Distance from potted end to osteotomy site (mm)
The gauge length (L) was 8 mm, which was the working length of the potted bone sample (as
previously described in the Torsion Testing Set-up Protocol). ι was determined by measuring the
distance from the potted static bone end to the spike at the osteotomy site (Figure 27).
Figure 27. Measured distance (mm) from the potted static end to the bone spike at the osteotomy
site (ι).
48
Shear stress (MPa) was calculated using Equation (3) [71]:
τ = M×ro
J (3)
where,
τ = Maximum shear stress (MPa)
M = Maximum torque (N·mm)
ro = Outer radius of bone specimen (mm)
J = Polar moment of inertia (mm4)
In order to calculate the polar moment of inertia, Equation (4) could have been used [71]:
𝐽 = 𝜋
2(𝑟0
4 − 𝑟𝑖4) (4)
where,
J = Polar moment of inertia (mm4)
ro = Outer radius of bone specimen (mm)
ri = Inner radius of bone specimen (mm)
However, applying the above equation to an irregularly shaped bone such as the reconstructed
radii would result in inaccurate values. To address this limitation, the polar moment of inertia
was determined using the Skyscan CTan software. At the graft-host bone junction of each
sample (previously identified and the associated z-position line recorded), the shrink-wrap
function was utilized. The software then computed the polar moment of inertia, which was
subsequently used in Equation (3).
The shear modulus of the reconstructed bone was calculated using Equation (5) [71]:
G = τ
γ (5)
where,
G = Shear modulus (GPa)
τ = Maximum shear stress (MPa)
γ = Shear strain (degrees)
49
4.2.9 Statistical Analysis
The statistical analyses were conducted using SPSS version 21 (IBM, New York, NY).
Statistical significance was achieved when the p-value was less than 0.05.
Kruskal-Wallis test was used to compare the radiographic (categorical) scores between
the untreated, conventionally-irradiated, and ribose-pretreated irradiated graft groups at the 2-, 6-
, and 12-week post-operative time points. If significance was observed for any parameter, further
post hoc comparison between time points was conducted using the Mann-Whitney test.
Although the data obtained from the μCT analysis, static and dynamic bone
histomorphometry, and biomechanical torsion testing, was comprised on continuous values, the
small sample sizes of the graft groups meant we did not meet the requirements to use parametric
statistical testing. As a result, the nonparametric Kruskal-Wallis test was used to analyze the
data. Post hoc analysis was performed using Mann-Whitney test.
50
4.3 RESULTS (STUDY 2)
4.3.1 Radiographic Analysis
Radiographic analysis revealed no differences in the degree of union at the proximal and
distal osteotomy sites between the three allograft types at all three time points (Figure 28).
Figure 28. Radiographs at 2-, 6-, and 12-weeks, post-surgical reconstruction of the critically-
sized radial defect (red arrows point to the osteotomy sites; yellow arrows point to the newly
formed callus at the osteotomy sites).
There was initially less periosteal reaction observed in the irradiated graft group at 2 weeks, but
no differences seen in the subsequent time points after (Table 2).
51
Table 2. Radiographic analysis of periosteal reaction.
Time Point Untreated Control Irradiated Ribose-Irradiated p-value
2 week 15.50 8.00 14.00 0.028
6 week 12.50 11.06 13.94 0.238
12 week 14.00 12.50 11.00 0.334
*Statistical test: Kruskal-Wallis
**Mean rank values are shown in the table
Furthermore, less cortical remodeling was observed in the ribose-pretreated-irradiated and
conventionally-irradiated graft groups compared to the untreated controls at 6 weeks (Table 3).
Table 3. Radiographic analysis of cortical remodeling.
Time Point Untreated Control Irradiated Ribose-Irradiated p-value
2 week 12.50 12.50 12.50 1.000
6 week 18.50 9.50 9.50 0.002
12 week 15.50 9.50 12.50 0.082
*Statistical test: Kruskal-Wallis
**Mean rank values are shown in the table
However, by 12 weeks, the statistical significant differences were no longer present.
Between the time points, there were notable changes that occurred, which were similar to
all groups (Figure 28). At 2 weeks, the osteotomy sites were still evident. By 6 weeks, there was
evidence of periosteal bridging callus localized to the osteotomy sites. At the 12 week endpoint,
union was seen in all groups.
52
4.3.2 μCT Analysis
μCT analysis further confirmed union at the osteotomy sites, with the presence of
mineralizing callus in all three allograft groups (Figure 29).
Figure 29. Three-dimensional rendering of the μCT scans at the osteotomy sites.
The 3D rendered images showed the bony callus growing onto the graft and encapsulating it. The
sagittal cut views further showed that the cortices of the untreated control graft and ribose-
pretreated irradiated grafts maintained their shape and anatomy, while there was evidence of
remodeling and bone resorption of the cortices in the irradiated grafts. Analysis of the 2 mm ROI
centered at the osteotomy site revealed no statistically significant differences in volumetric BMD
or tissue volume (Table 4).
53
Table 4. μCT analysis of the volumetric bone mineral density, bone volume (normalized to
tissue volume), and tissue volume, at the osteotomy site ROI.
Untreated Control Irradiated Ribose-Irradiated p-value
vBMD
(g/cm3) 1.134 ± 0.061 1.145 ± 0.087 1.150 ± 0.120 0.981
BV/TV 0.697 ± 0.049 0.648 ± 0.050 0.718 ± 0.067 0.104
TV (mm3) 92.720 ± 20.566 86.191 ± 9.698 82.028 ± 6.756 0.400
*Statistical test: Kruskal-Wallis
**Mean ± standard deviation values are shown in the table
***vBMD = volumetric bone mineral density; BV/TV = bone volume / tissue volume; TV = tissue volume
When examining the distance of growth of the bony callus onto the graft, there were statistically
detectable differences noted between the groups (Table 5).
Table 5. Measured growth of bony callus on to the graft.
Untreated Control Irradiated Ribose-Irradiated p-value
Callus distance onto
graft (mm) 2.288 ± 0.931 1.171 ± 0.553 1.520 ± 0.673 0.048
*Statistical test: Kruskal-Wallis
**Mean ± standard deviation values are shown in the table
Post-hoc Mann-Whitney testing revealed that a statistically detectable difference existed only
between the untreated control and conventionally irradiated groups (p=0.020) (Figure 30).
54
Figure 30. Bony growth of the callus onto the graft.
*p < 0.05
4.3.3 Static Bone Histomorphometry
Analysis of the Goldner’s Trichrome-stained histology slides also confirmed union at the
graft-host bone interface, with the presence of callus formation and osteoid (Figure 31).
55
Figure 31. Static bone histomorphometry (stained with Goldner’s Trichrome) of the graft-host
bone junction at 12 weeks post-operative (red arrow indicating the osteotomy site; yellow arrow
indicating the callus).
Qualitatively, the bridging external callus at the union site appeared to originate from the host
bone. This was evident as there were osteocytes present in the bridging callus and host radius,
but no osteocytes in the allograft bone. The gap between the host and graft bone was also found
to have filled in with new bone originating from the host radius (as evident by the presence of
osteocytes). This new bone was orientated perpendicular to the long axis of the host and graft
bones, resulting in a distinct line at the graft-new bone interface. Along the outer surface of the
bone grafts, there were often pockets of graft resorption in areas where the graft was in contact
with muscle tissue. Primary bone union was not observed in any groups as there was formation
of a fracture callus in all samples and no evidence of cutting cones on histology.
Quantitatively, there were no statistically detectable differences were observed between
the graft groups with respect to osteoid volume (normalized to bone volume) and osteoid surface
(normalized to bone surface) (Table 6).
56
Table 6. Quantitative static bone histomorphometry of the three graft types.
Untreated Control Irradiated Ribose-Irradiated p-value
BV/TV 0.523 ± 0.091 0.386 ± 0.094 0.521 ± 0.128 0.083
OV/BV 0.017 ± 0.009 0.018 ± 0.008 0.012 ± 0.006 0.311
OS/BS 0.306 ± 0.102 0.237 ± 0.093 0.261 ± 0.068 0.399
*Statistical test: Kruskal-Wallis
**Mean ± standard deviation values are shown in the table
***BV/TV = bone volume / tissue volume; OV/BV = osteoid volume / bone volume; OS/BS =
osteoid surface / bone surface
However, there was a statistical trend showing that irradiated grafts had less BV/TV when
compared to untreated controls and ribose-irradiated grafts (p=0.083).
4.3.4 Dynamic Bone Histomorphometry
Dynamic bone histomorphometry further showed healing at the osteotomy site through
bony callus formation. There was diffuse calcein labeling within the callus, with both single- and
double-labels, consistent with new bone formation occurring. With the control and ribose-
pretreated grafts, calcein labeling appeared to predominantly surround the grafts, with less
labeling seen within the grafts. In contrast, the irradiated grafts showed diffuse labeling
throughout the graft, with evidence of graft remodeling/resorption (Figure 32).
57
Figure 32. Dynamic bone histomorphometry of the graft-host bone junction at 12 weeks post-
operative (red arrow indicating the osteotomy site; yellow arrow indicating the callus).
Analysis of the dynamic bone histology also made it easier to appreciate the invasion of new
bone into allograft bone at the graft-host bone interface. This creeping substitution was
especially evident with the ribose-pretreated irradiated grafts (Figure 33).
58
Figure 33. Graft healing via creeping substitution in the ribose-pretreated irradiated grafts at the
graft-host bone junction. Histologic image on the left shows the Goldner’s Trichrome-stained
slides, while image on the right shows the corresponding dynamic bone histology. Osteocytes
can be seen in the invading new bone into the graft, which corresponded to the green-coloured
new bone invading into the blue-coloured ribose graft on the dynamic bone histology slide.
Creeping substitution was observed within all of the ribose-pretreated allografts (Figure 34).
59
Figure 34. Creeping substitution seen within the ribose-pretreated irradiated grafts (new bone
(green) infiltrating into the graft (blue)).
4.3.5 Backscatter Electron Microscopy
The backscatter electron microscopy images showed similar findings as the radiographic,
μCT, and histomorphometry data. There was evidence of union at the osteotomy sites, with the
presence of less mineralized (relative to the graft and host bone) callus (Figure 35).
60
Figure 35. Backscatter electron microscopy images of the three graft groups. The top images
show the graft-host bone junction at 50X, while the bottom images show a magnified area
(200X) of the interface. White areas represent more mineralized bone, and gray areas represent
less mineralized bone.
Similar to the dynamic histomorphometry data, the less mineralized callus appeared to surround
the graft bone in the untreated and ribose-pretreated groups. The cortical anatomy appeared to be
maintained in the untreated and ribose-pretreated groups. In contrast, there was loss of graft
shape and areas of bone resorption in the irradiated group. Magnified views of the osteotomy
sites revealed healing via creeping substitution. Specifically, the less mineralized new bone (gray
areas in Figure 35) invading in to the graft bone (white-coloured bone in Figure 35).
61
4.3.6 Histomorphometric Analysis Within the Grafts
Based on the findings of the μCT, histomorphometry, and backscatter electron
microscopy analyses at the graft-host bone interface, it was important to examine the changes
occurring within the different grafts during the healing process. Histomorphometric analysis
within the graft bone revealed the irradiated grafts had statistically less bone volume (normalized
to tissue volume) compared to the untreated and ribose-pretreated grafts (Figure 36), indicating
greater graft resorption.
Figure 36. Histomorphometric analysis of the bone volume (normalized to tissue volume)
(BV/TV) within the grafts.
*p < 0.05
Interestingly, the ribose-irradiated grafts had more bone volume than the untreated controls.
In contrast, more mineralizing surface and higher mineral apposition rates were seen in
the irradiated grafts compared to the ribose-pretreated grafts (Figures 37 and 38).
62
Figure 37. Histomorphometric analysis of the mineralizing surface (MS) within the grafts.
*p < 0.05
Figure 38. Histomorphometric analysis of the mineral apposition rate (MAR) within the grafts.
*p < 0.05
63
The ribose-pretreated grafts had less mineralizing surface and lower mineral apposition rates of
the three groups.
4.3.7 Biomechanical Testing
Torsion testing of the distal segment of the reconstructed radii resulted in a spiral fracture
that broke at the level of the osteotomy site (Figure 39). The angle of the spiral component of
the fracture occurred in an oblique fashion.
Figure 39. Spiral fracture pattern observed with torsional testing of the reconstructed radius
(yellow arrows point to the oblique configuration of the fracture ends following testing; black
line represents the level of the osteotomy site).
Gross inspection of the tested samples showed that the failure occurred at the junction between
graft and host bone (Figure 40).
64
Figure 40. Failure at the level of the osteotomy site (red arrow indicates the level of the graft-
host bone junction).
The pattern of failure was similar between all three graft types.
Quantitative data of the torsion testing revealed no statistically significant differences
between the three groups with respect to their structural mechanical properties (Table 7).
Table 7. Structural properties of the three graft groups following torsion testing.
Untreated Control
Conventionally-
Irradiated Ribose-Irradiated p-value
Maximum Torque
(N·mm) 495.51 ± 196.34 292.63 ± 148.15 433.97 ± 191.29 0.085
Total Energy to
Failure (J) 2389.88 ± 1347.53 1255.20 ± 1207.80 2742.85 ± 1821.22 0.055
Torsional
Stiffness
(N·mm2/deg)
771.28 ± 250.86 628.66 ± 263.99 613.33 ± 319.10 0.404
*Statistical test: Kruskal-Wallis
**Mean ± standard deviation values are shown in the table
65
However, there were statistical trends noted showing less maximum torque (p=0.085) and total
energy to failure (p=0.055) in the conventionally-irradiated group.
Comparison of the material properties between the three graft groups also showed no
statistically significant differences (Table 8).
Table 8. Material properties of the three graft groups following torsion testing.
Untreated
Control
Conventionally-
Irradiated
Ribose-
Irradiated p-value
Shear Stress
(MPa) 17.31 ± 4.98 13.19 ± 3.66 15.75 ± 4.58 0.259
Shear Strain
(degrees) 2.61 ± 0.78 2.08 ± 1.03 4.33 ± 3.00 0.183
Shear Modulus
(GPa) 0.40 ± 0.13 0.41 ± 0.14 0.32 ± 0.25 0.164
Maximum Angle
of Deformation
(degrees)
7.22 ± 1.82 5.91 ± 3.11 11.48 ± 7.36 0.132
*Statistical test: Kruskal-Wallis
**Mean ± standard deviation values are shown in the table
4.3.8 Complications
With regards to complications, no infections were observed. However, two NZWr died
due to complications unrelated to the grafts. Both rabbits died on post-operative day 3-4. The
only notable aspect of their care that likely contributed to their deaths was that they had both
undergone pre-operative radiographs the day before surgery. Necropsy performed of these
rabbits showed petechiae on the inner lining of the stomach and serosal hemorrhage on the
duodenum (Figure 41).
66
Figure 41. Necropsy images showing petechiae on the inner stomach lining (left image) and
duodenal hemorrhage (right image).
67
5. DISCUSSION
5.1 STUDY 1
The results from our study demonstrated that intramedullary wire fixation of bone
allograft to reconstruct a critically-sized radius segmental defect can be successful with minimal
complications. Furthermore, our model addressed problems with previously published methods
of testing bony reconstructions of critically-sized NZWr radial defects. Many authors have
previously claimed that fixation is not required for the radius due to the intact load-bearing ulna
[5, 33, 42, 94]. We do agree that the adjacent intact ulna provides additional stability such that
any observed non-union would be less likely to be due to failure of stability and more likely a
failure of biology; making the NZWr radius model ideal for studying bony union. However, we
found that the significant curvature of the radius made it difficult for grafts to maintain position
by press-fit alone (with the rabbits weight bearing) and required at least some method of fixation.
Zhao et al. [98] had similarly reported implant failure with their method of tightly impacting
segmental bone allografts to repair 15 mm critically-sized defects in the mid-diaphysis of
bilateral radii. The authors described the failure as “… implant loosening of which was
characterized by segment floating accompanying by apparent reactive bone formation around of
the host bone ends without segment connection [98].” This description seems consistent with a
non-union secondary to failure of stability.
To better understand why fixation of the graft is required in this model, one must
understand the true degree of stability imparted by the intact ulna on the radius. Bodde et al. [13]
attempted to determine this by inserting titanium bone markers into the host bone at the proximal
and distal osteotomy sites, and then comparing the radial defect size immediately after surgery
and at sacrifice at 12 weeks. No structural bone graft or composite materials were inserted to fill
68
the defect in their study. By the 12 week endpoint, it was found that the distance between the
titanium markers had become significantly shorter (regardless of whether the defect was 15 mm
or 20 mm in size). These findings would suggest that the ulna is not able to immobilize the
radius as previously thought. The authors went on to conclude that the NZWr radius segmental
defect model was not a suitable model without additional fixation. Indeed, we found that even
with intramedullary fixation, the slight migration of the Kirschner wire in one rabbit resulted in
displacement of the bone allograft. In this case, the Kirschner wire did not completely fail and
was able to provide enough stability to eventually achieve union by 12 weeks. It was apparent
that the weight bearing activity of the rabbits, combined with the natural anatomic curvature of
the radius, made it difficult for graft stability to be maintained without the use of fixation in this
model.
There have been a number of studies implementing fixation of grafts or composite
materials into critically-sized long bone rabbit defect models. However, many of the fixation
methods described come with serious drawbacks that may hamper the ability to properly study
long bone critically-sized defect repairs or reconstructions. Shafiei et al. [81] used cerclage wire
fixation of bone allograft to the ulna to reconstruct the segmental defect. The result was an
undesired periosteal reaction of the ulna on the graft, creating a large callus enveloping both the
radius and ulna. With callus formation arising from the ulna, and not the host bone at the
osteotomy sites, assessing the ability of the graft being tested to unite with host bone may not be
accurately assessed in their model. The use of intramedullary wire fixation in our model created
an anatomic reconstruction that not only maintained the interosseous space but ensured cortical
contact of the graft with host bone occurred only at the osteotomy sites. Serial radiographs in the
post-operative period demonstrated that callus formation initially occurred and was localized to
69
the osteotomy sites. The localization of callus to the proximal and distal sites was more
representative of how allografts would heal clinically. The radiographic, μCT, and histologic
findings from our study support that union can be achieved in a more clinically representative
manner using our model.
Yoneda et al. [97] described the use of external fixators to test a critically-sized rabbit
femoral defect model. Similar use of external fixators in our model in the forelimb would not be
appropriate as it would not only make grooming difficult for rabbits, but high infection rates
have been reported with their use (likely due to self-inoculation from grooming and picking) [44,
48]. Additionally, the creation of screw holes in the bone could compromise the ability to
perform biomechanical testing and interfere with histological analysis of the union sites. Some
have argued that the use of plate fixation could circumvent the issues with grooming and
infection. However, plate fixation of grafts or composite materials would not address the
problems with screw holes. Furthermore, the plate fixation hardware for the small NZWr radius
defect model would be quite costly and may explain why no studies have described its use for
studying critically-sized long bone defects. These methodological weaknesses can all be avoided
with the use of a smooth intramedullary wire that could be pulled out without disrupting the
union sites.
While it is important to identify the ideal fixation method when studying bony defect
healing and regeneration in the NZWr defect model, there are a number of other factors that must
also be considered for this model to be successful. The age of the NZWr is one important factor
that must be recognized. In our study, mature NZWr aged 7-9 months were used, as it has been
shown that their skeletal growth ends between around 19 and 32 weeks (as determined by growth
plate closure radiographically) [51]. The use of rabbits with closed growth plates helped to avoid
70
potential complications associated with epiphyseal slipping. Furthermore, rabbits that are young
and skeletally immature may affect how large of a defect must be created to be considered
critically sized. Bodde et al. [13] found that 15- and 20-mm mid-diaphyseal radial defects in 4
month old NZWr were not critically sized. The authors concluded that such young NZWr likely
possess high bone regenerative capabilities, and recommended using rabbits older than 6 months
in studies using NZWr to assess bone regeneration. The study period length is also another
important factor to consider. Bodde et al. [13] recommended that at least 12 weeks was needed
for evaluating bone formation in critically-sized defect models. Pilot data from our study (not
included in our Results data) showed that 12 weeks was the approximate time point at which
radiographic union was objectively observed in 7-9 month old NZWr, and thus was the endpoint
used.
This study demonstrated that intramedullary wire fixation of bone allograft in the NZWr
radius segmental model was a successful, reproducible, and anatomically correct method for
studying reconstructions of critically-sized skeletal defects. We propose that this model is suited
to study the biology of large segmental defect reconstructions using bone allograft (and
potentially other structural graft materials) because it better avoids biomechanical instability and
associated non-unions. This in turn helps to isolate the biological effects of the tested
biomaterial(s) as much as possible.
5.2 STUDY 2
To successfully test in vivo the biocompatibility of the ribose-pretreated irradiated
allografts (as well as any other biomaterials) first requires the use of a proper animal model.
Stevenson et al. highlighted that there are two principal determinants in achieving successful
71
union between host and graft bone: (1) construct stability, and (2) cortical contact between host
and graft bone [86]. The authors found that not meeting these two conditions resulted in non-
unions or delayed unions of both autograft and allograft reconstructions in a number of animal
studies. The need to maintain stability and cortical contact in graft incorporation makes logical
sense, as both conditions provide the optimal environment for the initial reactive bone callus
formation stage to occur and the subsequent creeping substitution phase that follows [9]. We
previously showed that the use of intramedullary wire fixation within the NZWr radius model
provided stability and helped to maintain cortical contact between host and graft bone, thus
ensuring the highest chances of union to occur.
Using the intramedullary fixation NZWr radius model, the primary objective of this study
was to assess if ribose-pretreated irradiated bone allografts could achieve union with host bone.
A number of clinical and in vivo animal studies have examined the effects of non-enzymatic
advanced glycation end products (AGEs) in bone with respect to fracture healing. In these
studies, the glycation crosslinking of bone was achieved with subjects being in diabetic states.
Multiple clinical studies have reported significant increased time to (or delayed) union of
fractures in diabetic patients [23, 58]. Yet, the exact pathogenesis of why bony healing is
impaired in diabetes has not been fully determined [18]. Various in vivo and in vitro studies have
shown that systemic AGEs may work to decrease the proliferation and differentiation of
osteoblasts and bone mineralization, while also altering the activity of osteoclasts [12]. It is
important to understand that these effects on fracture healing are within the diabetic
environment. To the best of our knowledge, no studies have assessed the healing potential of
glycated bone within the non-glycemic state. The results from our study showed that the ribose-
pretreated irradiated bone allografts were able to union with host bone.
72
The histologic data further supported that the ribose-irradiated grafts exhibited the typical
biological reparative processes needed to achieve bony union. Comparison of our histological
findings with that of retrieved human allografts showed that similar reparative mechanisms were
exhibited [10]. In the observational study by Enneking et al., histologic analyses were performed
on 73 retrieved human allografts in an effort to better understand the incorporation and healing
of large allografts [30]. The authors found that bony healing of allografts in cortical contact with
host bone (such as in segmental diaphyseal defects of long bones) occurred through a bridging
external callus that only originated from the host bone. There was no evidence of callus
extension from the allograft side to the host bone. In addition, mature cortical bone (originating
from the host side) was found to have filled in to the gap between the host and graft bone. This
new bone was oriented in a perpendicular direction to that of the long axis of the graft, resulting
in a distinct “cement line” separating the host bone from allograft bone at the site of union. These
findings were similarly seen in our histologic samples. Furthermore, areas of resorption seen on
the external surface of the human allografts in contact with host muscle were also seen in our
samples. These small erosive cavities on the surface of the grafts were evident and better
appreciated on our three-dimensional-rendered μCT images.
While the primary objective of this study was to examine the ability of the ribose-
pretreated irradiated grafts to unite with host bone, our results unexpectedly showed that
conventional irradiation of grafts may impart some deleterious effects during bony healing. The
previous study by Jinno et al. found that sterilization of cortical bone allografts with 1.5 Mrad of
γ-irradiation did not compromise their ability to incorporate [46]. Zhao et al. also showed that γ-
irradiated (dosage not specified) bone allografts exhibited slow creeping substitution with
significant lymphocyte infiltration during healing [98]. Indeed, we similarly found that the
73
conventionally irradiated grafts were able to go on to bony union and exhibited creeping
substitution healing. However, our histomorphometric analysis and backscatter electron
microscopy images suggested that irradiation at 33 kGy may result in increased graft resorption
(as evident by the significantly decreased bone volume within the graft). This was a concerning
finding because increased resorption of the graft could lead to an increased risk of fracture or
failure, which has been reported to occur clinically with irradiated bone allografts [57]. One
possible mechanism for this increased bone resorption has been described by Russell et al. [76].
The authors examined the osteoconductivity of allograft bone chips sterilized with 25 kGy of γ-
irradiation by inserting them within a critically-sized NZWr tibial defect. At 2 weeks, increased
bone resorption (based on bone volumetric analysis using CT) was observed. While the
investigators were unclear about the exact mechanism for this, their immunohistochemistry data
suggested it could be due to increased osteoclast activity. In particular, they found there was an
upregulation of Cathepsin K, a protease synthesized by osteoclasts to degrade organic matrix
[15, 28]. Given that bone resorption is an important initial step in remodeling during the
incorporation of bone allografts [52, 67], it would be expected that osteoclast activity would
increase during graft healing. Our findings would suggest that bone sterilized with 33 kGy of γ-
irradiation may further increase this resorptive process. Unfortunately, due to the limitations in
the number of samples, we were unable to perform tartrate-resistant acid phosphatase (TRAP)
staining to assess the activity and effects of osteoclasts.
Interestingly, our results further showed that pre-treatment of bone with ribose prior to
irradiation may be protective against graft resorption (seen with the conventionally-irradiated
grafts). This was evident by the higher bone volume values seen within the ribose-irradiated
grafts. Consistent with our findings was the study by Valcourt et al. [89], which investigated the
74
effects of collagen AGEs on bone resorption. In their study, AGE formation was first induced in
vitro by incubating cortical bone slices (from 3 month old calves) in ribose solution. Mature
osteoclasts (harvested from the long bones of New Zealand rabbits) were then seeded and
cultured on to the AGE-containing bone slices for 8 days. Compared to the untreated control
bone slices, the total area resorbed per slice and the area degraded per resorption lacuna were
significantly decreased in the AGE-containing bone slices. Conversely, Miyata et al. found the
number of resorption pits significantly increased when osteoclasts were cultured on AGE-
modified dentin slices (versus when they were seeded on control slices) [61]. The authors
concluded that AGEs may enhance osteoclast-induced bone resorption. However, the study by
Valcourt et al. [89] additionally assessed bone resorption by measuring the release of collagen
fragments in culture media. They found a significantly decreased concentration of type I collagen
fragments in ribose-modified bone slices compared to controls. The authors ultimately concluded
that AGEs have an overall inhibitory effect on the degradation of bone. While the exact
mechanism for the decreased degradation of AGE-containing bone is unknown, Valcourt et al.
[89] proposed three hypotheses: (1) AGEs may block the cleavage sites within the collagen that
proteases released by osteoclasts bind to; (2) AGEs could accelerate apoptosis of mature
osteoclasts; or (3) the AGE-crosslinks could “trap” the collagen within the bone matrix.
Although our histologic data showed that pre-treating bone with ribose prior to γ-
irradiation may be protective against the increased graft resorption seen with the conventionally-
irradiated grafts, the dynamic histomorphometric analysis showed that this did not necessarily
impair or inhibit the incorporation of the ribose-irradiated grafts. In fact, evidence of creeping
substitution into the ribose-irradiated grafts was grossly observed in all samples, with bony union
achieved by the 12-week endpoint. However, the rate at which new bone formed into the ribose-
75
irradiated grafts was slowed, as less mineralization and decreased mineral apposition rate was
seen in this group. This may be due to decreased or impaired formation and/or function of
osteoblasts, of osteoclasts, or both. Katayama et al. found that AGE-modified type I collagen
inhibited osteoblast function and maturation (as evident by the suppression of calcified nodule
formation, alkaline phosphatase activity, and osteocalcin secretion from cultured osteoblasts)
[49]. The decreased mineralization and bone formation within the ribose-irradiated grafts may
also be a product of decreased osteoclast activity as previously discussed. With less resorption of
the bone graft, less new bone formation may be needed. At the same time, there is coupling
interaction between osteoblasts and osteoclasts [17, 82]. Unfortunately, osteoclasts were not
assessed histologically in our study. As well, the biochemical interactions between bone cells
(osteoblasts, osteoclasts, and osteocytes) were not investigated. Further research is needed to
better elucidate the interactions and pathways between cells within the ribose glycated bone.
Biomechanical torsion testing of the three allograft groups all resulted in a spiral fracture
of the reconstructed radii. The spiral configuration and oblique helical angle of the fracture line
were consistent with an external rotation force applied during torsion [14, 80]. Gross inspection
of the tested samples revealed failure most likely occurred at the graft-host bone junction. The
fracture line appeared to have also extended through the external bridging callus that united the
graft and host bones. Enneking et al. found similar results when biomechanically testing
retrieved human allografts used for reconstructing bone defects following musculoskeletal
tumour resections [30]. They observed that when the retrieved allografts were subjected to
torque, failure occurred at the junction between allograft and host bone.
Quantitatively, statistical comparisons of the three graft groups yielded no significant
differences in the biomechanical testing measures. However, statistical trends were observed as
76
the irradiated grafts exhibited lower maximum torque properties and total energy to failure
compared to the untreated controls and ribose-irradiated grafts. We hypothesize that this likely
reflected the graft properties, as opposed to the strength of the bony callus or graft-host bone
junction. Recall that PMMA-potted graft side was the static end, while the host bone end rotated
during torsion testing. Gross inspection of the samples just after failure revealed that the
initiation of the fracture line may have started in the graft end, which then propagated to the
graft-host bone junction resulting in ultimate failure. Unfortunately, real-time video capture
during torsion testing of the samples was not performed and so the exact mechanism by which
the samples failed was not fully elucidated. Further testing and analysis is required.
Our study was not without limitations and these must be considered when interpreting the
results. First, the sample size within each treatment group was small. Although we met the
requirements as outlined by the ISO guidelines, a greater sample size could have helped to detect
statistically significant differences (as opposed to statistical trends) between graft groups. A
greater number of samples could have also allowed for histologic assessment of osteoclast
activity through TRAP staining. This analysis could have provided more information on the
mechanisms by which differences in bony healing were observed between the graft groups.
Unfortunately, the limited number of samples available did not allow for such testing. Finally,
the endpoint of our study was only at 12 weeks. This made it difficult to assess the long-term
healing potential and mechanism of these grafts.
77
6. CONCLUSION
The application of ribose prior to irradiation of bone allografts has the potential for
helping patients (undergoing surgical reconstructions using allografts) by decreasing the rates of
graft fracture. Our study showed that within the NZWr radius segmental defect model using
intramedullary fixation, the ribose-pretreated allografts were able to union with host bone in the
short-term. The increase in graft resorption of irradiated bone grafts could explain the higher
rates of graft fracture/failure seen clinically. Ribose pre-treatment may be protective against such
graft resorption, but this sterilization technique may also result in delayed graft incorporation.
78
7. FUTURE DIRECTIONS
Long-term studies investigating osteoclast and osteoblast function, and their interplay, are
needed to fully elucidate the mechanism of healing within the ribose-pretreated irradiated bone
allografts. Such studies will help to determine the extent of graft incorporation and provide more
understanding of how these grafts will fare over time. As well, in order to advance this
technology into clinical trials, testing in larger animal models (such as canine or ovine) are
needed.
The ribose-pretreatment sterilization technique lends the potential to being able to restore
100% of normal bone strength and toughness while still undergoing γ-irradiation. Current
accepted standards are to use low-dose irradiation (less than 25 kGy) to protect against bacterial
transmission [90]. However, up to 50 kGy of irradiation is required to inactivate viruses such as
HIV (human immunodeficiency virus) [32, 38, 84]. Such high levels of radiation would
significantly impair the biomechanical properties of allografts. The addition of ribose could
allow for the use of higher doses of irradiation to sterilize bone allografts to better eliminate
infectious sources without compromising their biomechanical properties.
Once the biomechanical properties of irradiated allografts are optimized (while still
allowing for grafts to heal), the next step would be to revitalize these grafts. New biological
methods to revascularize these grafts, such as with endothelial progenitor cells, could work to
increase angiogenic factors (such as vascular endothelial growth factor) [56]. There is also
advancing research into tissue engineering periosteum that could induce vascularization and
osteogenesis [31, 55]. The creation of biomechanically sound, revascularized bone allografts
could provide new surgical options in the treatment of massive skeletal defects.
79
Ultimately, the advancement of this technology into clinical trials and use could help to
reduce the rates of allograft fractures, the need for subsequent surgery, and provide new viable
options in patients with significant bone defects.
80
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91
9. APPENDIX
Appendix A. Radiographic scores of the critically-sized radial defects reconstructed using bone
allograft.
Rabbit
ID
Time
point
(weeks)
Periosteal
reaction
Proximal
union
Distal
union Remodeling
Total
score:
14
2 0 0 0 0 0
4 1 0 0 0 1
6 2 1 1 0 4
8 2 1 1 0 4
10 3 2 1 0 6
12 3 3 2 0 8
15
2 1 0 0 0 1
4 2 1 1 0 4
6 2 1 1 0 4
8 2 3 2 0 7
10 2 3 2 0 7
12 2 3 2 0 7
16
2 0 0 0 0 0
4 1 1 1 0 3
6 2 2 2 0 6
8 2 2 2 0 6
10 3 3 3 0 9
12 3 3 3 2 11
17
2 1 1 1 0 3
4 2 2 1 0 5
6 2 3 2 0 7
8 2 3 2 0 7
10 2 3 2 1 8
12 2 3 2 2 9
18
2 1 1 1 0 3
4 2 2 1 0 5
6 2 2 1 0 5
8 2 3 2 1 8
10 3 3 2 2 10
12 3 3 3 2 11
19
2 0 0 0 0 0
4 1 1 1 0 3
6 2 1 1 0 4
8 2 2 2 0 6
10 2 2 2 0 6
12 2 2 2 0 6
92
20
2 0 0 0 0 0
4 2 2 1 0 5
6 2 3 2 0 7
8 3 3 3 1 10
10 3 3 3 2 11
12 3 3 3 2 11
21
2 0 0 0 0 0
4 1 1 1 0 3
6 2 2 1 0 5
8 3 3 2 1 9
10 3 3 2 2 10
12 3 3 2 2 10
22
2 0 0 0 0 0
4 1 1 1 0 3
6 2 2 2 0 6
8 2 2 2 0 6
10 2 2 2 0 6
12 3 2 2 2 9
23
2 1 1 0 0 2
4 2 2 1 0 5
6 2 2 2 0 6
8 2 2 2 1 7
10 2 3 2 2 9
12 3 3 2 2 10