biocompatibility of bone allograft toughened with a novel ... · massive bone defects, such as...

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


3.3.2 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


4.2.2 Data Collection……………………………………………………... 32

4.2.3 μCT Analysis………………………………………………………... 33


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TABLE OF CONTENTS

Page Number

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


4.3.2 μCT Analysis………………………………………………………... 52


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

Page Number

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

x

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

xi

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

xiii

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.

2

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

3

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

4

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

5

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.

6

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].

7

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

8

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;

9

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.

10

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).

11

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

12

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.


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)


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.


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)


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)


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)


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


**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


**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.


Callus distance onto

graft (mm) 2.288 ± 0.931 1.171 ± 0.553 1.520 ± 0.673 0.048



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


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.


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



***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



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



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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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

biocompatibility of bone allograft toughened with a novel ... · massive bone defects, such as...

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