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NBCS: A NOVEL OSTEOCONDUCTIVE SCAFFOLD FOR NEW BONE FORMATION IN ANIMAL SPINAL FUSION MODELS Yu Qian This thesis is presented for the degree of DOCTOR OF PHILOSOPHY IN MEDICAL SCIENCE SCHOOL OF SURGERY THE UNIVERSITY OF WESTERN AUSTRALIA 2009 The work presented in this thesis was performed in the University of Western Australia, School of Surgery, Centre for Orthopaedic Research, Queen Elizabeth II Medical Centre, Nedlands, Western Australia.
Transcript of NBCS: A NOVEL OSTEOCONDUCTIVE SCAFFOLD FOR NEW BONE ...€¦ · NBCS: A NOVEL OSTEOCONDUCTIVE...
NBCS: A NOVEL OSTEOCONDUCTIVE SCAFFOLD
FOR NEW BONE FORMATION
IN ANIMAL SPINAL FUSION MODELS
Yu Qian
This thesis is presented for the degree of
DOCTOR OF PHILOSOPHY
IN MEDICAL SCIENCE
SCHOOL OF SURGERY
THE UNIVERSITY OF WESTERN AUSTRALIA
2009
The work presented in this thesis was performed in the University of Western Australia, School of Surgery,
Centre for Orthopaedic Research, Queen Elizabeth II Medical Centre, Nedlands, Western Australia.
DECLARATION
This is to certify that all work contained herein was performed by myself, except where
indicated otherwise.
______________________
Yu Qian
______________________
Prof. Ming-Hao Zheng
(Supervisor)
______________________
Prof. Jiake Xu
(Co-supervisor)
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ACKNOWLEDGEMENTS
I would like to thank the Centre for Orthopaedic Research and Shaoxing People’s
Hospital for providing such a precious opportunity to study, and allowing me to
complete my PhD.
Particular thanks go to my supervisors Prof. Ming-Hao Zheng and Prof. Jiake Xu. To
Zheng, thank you so much for accepting me into the Centre for Orthopaedic Research.
Your steering and support was instrumental towards the completion of this thesis, and
your guidance, enthusiasm and instruction during my PhD will positively influence me
all my career and life. Your support has been greatly appreciated. To Jiake, you have
always stayed optimistic and given me the encouragement and support to keep driving
me during my candidature. I thank you immensely for your words of wisdom, advice,
and technical help during my PhD.
A big thank you goes to Dr. Ying Fan for your scientific advice and efficient organising.
Particularly in those difficult situations during this project, your confidence and
calmness really encouraged me to face the difficulties.
To Lesley Gasmier, I have greatly appreciated all your assistance and support during the
last three years. I appreciate your smartness and humour.
A very warm thank you goes to past and present members of the Centre for Orthopaedic
Research. In particular, thanks to Tam for your patient support on micro-CT analysis
and manuscript writing. To Jamie, thanks for your kind understanding and for always
offering me your ear. Thanks to Dr. Zhen Lin for your technical support on tissue
culture. I have appreciated your critical thinking and invaluable suggestions. To Jim,
thank you for your excellent surgical assistance in theatre. To Sky, thank you for your
warm help on my initial settling into Perth. Further thanks go to the rest of the
orthopaedics crew including Nathan, Tak, Creig, Esta, Katherine, Jacky, Felix,
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Ee-cheng, Jasreen, Ben, Lois, Samuel and Katherine. Perth would have never been the
same without you, and I will miss all these sunny days we spent in Perth together.
Many thanks go to Dr. Martin Cake, Susan Lewis, Andy Wilon and Astrid Armitage for
your continuous support on the animal model. Without you, the animal experiments
could not have been completed. Thanks to Robert Day for your technical assistance
with biomechanical assessment; and Peter Self at Adelaide Microscopy for your
technical support with the micro-CT system.
Last but not least, a big thank you to my loving family. I am grateful for all the love and
support my family have given me all these years. To my parents-in-law, thank you for
your generous help to look after Tony. Without your reliable support, I definitely could
not have concentrated on my study and would not have travelled so much. To my wife
Molly and son Tony, thank you for your love and constant encouragement. Words can
not express how much I have appreciated your love and understanding during these
years. This journey has been made easier having you by my side and in my heart. My
success is attributed to you. To my dearest mum and dad, who have always believed in
me and been there to encourage me, I thank you from the very bottom of my heart for
all that you have done for me, not only during these past three years but throughout my
life. I hope that I have made you both proud of me and will continue to do so in the next
phase of my life.
“Science is a wonderful thing if one does not have to earn one’s living at it”
Albert Einstein 1879 - 1955
To my family
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TABLE OF CONTENTS
Page
Declaration i
Acknowledgements ii
Special Dedication iv
Table of Contents v
Publications and Awards xi
Abbreviations xiv
List of Figures xviii
List of Tables xx
Thesis Abstract xxii
Chapter 1: Spinal fusion: bone grafts and substitute 1
1.1 Spinal fusion: the solution for spine instability 2
1.2 Indications of spinal fusion 3
1.2.1 Trauma 3
1.2.2 Degenerative instability 6
1.2.3 Other indications of spinal fusion 8
1.3 Surgical approaches 8
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1.3.1 Cervical spinal fusion 9
1.3.2 Thoracic spinal fusion 10
1.3.3 Lumbar spinal fusion 11
1.3.4 Minimally invasive spinal fusion 13
1.3.4.1 Minimally invasive cervical fusion 13
1.3.4.2 Minimally invasive thorscopic fusion 14
1.3.4.3 Minimally invasive lumbar interbody fusion 15
1.4 Bone grafts for spinal fusion 16
1.4.1 Biology of spinal Fusion 16
1.4.2 Bone grafts for spinal fusion 17
1.5 Autologous bone graft in spinal fusion 20
1.5.1 Advantages of autograft 20
1.5.2 Drawdacks of autograft in spinal fusion 21
1.6 The use of growth factors in spinal fusion 22
1.6.1 Osteoinductive growth factors 22
1.6.2 The use of growth factors in animal models of spinal fusion 24
1.6.3 The use of growth factors in clinical trials of spinal fusion 25
1.6.4 Potential limitations of the use of growth factors in spinal fusion 27
1.7 Cell therapy and gene therapy in spinal fusion 28
1.7.1 Cell therapy for spinal fusion 28
1.7.1.1 Autologous bone marrow cells 30
1.7.1.2 Mesenchymal stem cell 31
1.7.1.3 Other cell sources 32
1.7.1.4 Potential issues related to using cell therapy for spinal
fusion 33
1.7.2 Gene therapy for spinal fusion 34
1.7.2.1 In vivo and ex vivo techniques 35
1.7.2.2 Combination of cell therapy and gene therapy 36
1.7.2.3 Potential risks of gene therapy 36
1.8 Osteoconductive scaffolds 37
1.8.1 Allograft 38
1.8.2 Demineralized bone matrix 39
1.8.3 Collagen 40
1.8.4 Calcium phosphate biomaterials 41
1.8.5 Biodegradable polymers 43
1.9 Summary and future directions 47
Chapter 2: Research Proposal 48
2.1 Rationale 49
2.2 Hypotheses 54
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3.2 Aims 54
Chapter 3: Evaluation of insoluble bone gelatin as a carrier for
enhancement of osteogenic protein-1 induced intertransverse
process lumbar fusion in a rabbit model (Spine. 33, 2008;
Published)
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Chapter 4: Natural bone collagen scaffold combined with
OP-1 for bone formation induction in vivo (Journal of
Biomedical Materials Research Part B: Applied Biomaterials,
2009 Mar 12. [Epub ahead of print] 71
Chapter 5: Natural bone collagen scaffold combined with
autologous enriched bone marrow cells for induction of
osteogenesis in an ovine spinal fusion model (Tissue
Engineering, 2009; Peer review) 89
Chapter 6: General Discussion 108
6.1 Discussion 109
6.2 Future Directions 114
Appendix A: Materials and Methods 119
1.1 Materials 120
1.1.1 Animals 120
1.1.2 Surgical instruments 121
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1.1.3 Animal drugs 123
1.1.4 Chemical reagents 123
1.1.5 Commercially purchased kids 125
1.1.6 PCR Primers 126
1.1.7 Anitbodies 126
1.1.8 Buffers and solutions 127
1.1.9 Laboratory Instruments 135
1.1.10 Software 136
1.2 General Methods 136
1.2.1 Animal anesthesia 136
1.2.1.1 For rabbit 136
1.2.1.2 For sheep 137
1.2.2 Surgical procedures 138
1.2.2.1 For rabbit 138
1.2.2.2 For sheep 143
1.2.3 Postoperative animal care 148
1.2.3.1 For rabbit 148
1.2.3.2 For sheep 148
1.2.4 Manual palpation 149
1.2.5 Radiography 149
1.2.6 Micro-CT 149
1.2.7 Biomechanics 150
1.2.7.1 For rabbit lumbar 150
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1.2.7.2 For sheep lumbar 151
1.2.8 Histology 152
1.2.8.1 H &E staining 153
1.2.8.2 Saffanin O staining 153
1.2.8.3 Von Kossa staining 154
1.2.9 Immunohistochemistry 154
1.2.10 Bone marrow aspiration and isolation 155
1.2.11 Cells co-culture with scaffold 156
1.2.12 Scanning electron micrograph 157
1.2.13 RNA extraction 157
1.2.14 Reverse transcription for cDNA 159
1.2.15 Standard PCR 160
1.2.16 Agarose gel electrophoresis 161
1.2.17 Preparation of NBCS 162
1.2.18 Statistical analysis 162
Appendix B: References 163
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PUBLICATIONS AND AWARDS
Journal Publications:
1. Yao G*, Qian Y*, Chen J, Fan Y, Stoffel K, Yao F, et al. Evaluation of insoluble
bone gelatin as a carrier for enhancement of osteogenic protein-1-induced
intertransverse process lumbar fusion in a rabbit model. Spine 2008;33:1935-1942.
(* Co-first authors)
2. Qian Y*, Yao G*, Lin Z, Chen J, Fan Y, Davey T, et al. Natural bone collagen
scaffold combined with OP-1 for bone formation induction in vivo. J Biomed Mater
Res B Appl Biomater 2009 Mar 12. [Epub ahead of print] (* Co-first authors)
3. Qian Y, Lin Z, Chen J, Fan Y, Davey T, et al. Natural bone collagen scaffold
combined with autologous bone marrow cells for induction of osteogenesis in an
ovine spinal fusion model. Tissue Engineering. 2009
4. Yao F, Seed C, Kiely P, Parker S, Qian Y, Farrugia A, Morgan D, Wood D, Zheng
MH. Trends in prevalence of viral infections in Australian musculoskeletal tissue
donors and projections of incidence and residual risk, 1993-2004. Transplantation.
2008 Sep 15;86(5):746-8.
Awards arising from this work include
1. Ad Hoc Scholarship offered by The University of Western Australia (2007-2009)
2. Western Australia Biomedical Research Institute (WABRI) Award (2007)
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Abstract entitled: “Evaluation of ISBG as a Carrier for Enhancement of OP-1
Induced Inter-transverse Process Lumbar Fusion in a Rabbit Model” Qian, Y,
Yao, GF, Chen, Jim, Xu, J, Zheng, MH., Australian Society for Medical Research
Week Symposium, Perth, Australia (2007).
3. Best student Poster Award (2007)
Abstract entitled: “ISBG as a Carrie of OP-1 Induced Inter-transverse Process
Lumbar Fusion in a Rabbit Model” Qian, Y, Yao, GF, Chen, Jim, Fan, Y, Xu, J,
Zheng, MH., The Seventeenth Annual Combined Biological Sciences Meeting,
Perth, Australia (2007)
4. Surgery and Pathology Poster Award (1st place) (2007)
Abstract entitled: “ISBG as a Carrie of OP-1 Induced Inter-transverse Process
Lumbar Fusion in a Rabbit Model” Qian, Y, Yao, GF, Chen, Jim, Fan, Y, Xu, J,
Zheng, MH., School of Surgery and Pathology Research Symposium, Rottnest
Island, Perth, Australia (2007).
5. Travel Award offered by Australia & New Zealand Orthopaedic Research Society
(2007)
Abstract entitled: “ISBG as a Carrie of OP-1 Induced Inter-transverse Process
Lumbar Fusion in a Rabbit Model” Qian, Y, Yao, GF, Chen, Jim, Fan, Y, Xu, J,
Zheng, MH., 13th Annual Scientific Meeting of Australia & New Zealand
Orthopaedic Research Society, Auckland, New Zealand (2007)
6. Travel Grant offered by Australia & New Zealand Bone & Mineral Society (2009)
Abstract entitled: “Autologous bone marrow cells implantation induces bone
formation in a sheep spinal fusion model” Qian, Y, Lin, Z, Fan, Y, Chen, Jim, Xu,
J, Zheng, MH., Australia & New Zealand Bone & Mineral Society Scientific
Meeting, Sydney, Australia (2009)
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7. Travel Grant offered by The University of Western Australia (2009)
Abstract entitled: “Autologous bone marrow cells implantation induces bone
formation in a sheep spinal fusion model” Qian, Y, Lin, Z, Fan, Y, Chen, Jim, Xu,
J, Zheng, MH., Australia & New Zealand Bone & Mineral Society Scientific
Meeting, Sydney, Australia (2009)
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ABBREVIATIONS
The following abbreviations are used throughout this thesis:
ACS Absorbable collagen sponges
AEC Animal Ethics Committee
AFSC Amniotic fluid-derived stem cell
ALIF Anterior lumbar interbody fusion
a-MEM Alpha modified eagles medium
Amp Ampicillin
ANOVA Analysis of variance
AP Anterior-posterio
β-TCP β-tricalcium phosphate
BMP Bone Morphogenetic Protein
BMM Bone marrow monocyte
BMC Bone mineral content
BMCs Bone marrow cells
BMD Bone mineral density
BSA Bovine serum albumin
BV Bone volume
C Cervical
cDNA Complementary deoxyribonucleic acid
CO2 Carbon Dioxide
CSF Cerebrospinal fluid
CT Computer tomography
dATP Deoxyadenosine triphosphate
DBM Demineralized bone matrix
dCTP Deoxycytidine triphosphate
ddH2O Double distilled water
dGTP Deoxyguanosine triphosphate
DNA Deoxyribonucleic acid
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dNTPs Di-nucleotide triphosphates
dTTP Thymidine triphosphate
EDTA Ethylene diamine tetra acetic acid
EGF Epidermal growth Factor
FBS Foetal bovine serum
FGFs Fibroblast growth factors
Fig. Figure
H & E Haematoxylin and eosin
HA Hydroxyapatite
HATDMSCs Human adipose tissue-derived mesenchymal stem cells
HBC Hepatitis B core
HBMDMSCs Human bone marrow-derived mesenchymal stem cells
HBV Hepatitis b virus
HCl Hydrochloric Acid
hESCs Human ESCs
HIV Human Immunodeficiency Viruses
Hrs Hours
HTLV Human t-cell leukemia virus
HUCPVC Human umbilical cord perivascular cell
IGF Insulin-like growth factor
IgG Immunoglobulin G
Ihh Indian Hedgehog
IFN-g Interferon-gamma
IL Interleukin
IMAC Immobilised metal affinity chromatography
IPTG Isopropyl-β-thiogalactopyranoside
ISBG Insoluble bone gelatin
L Lumbar
LT-Cage Lumbar Tapered-Cage
Min Minutes
MMP Matrix metalloproteinases
ml Millilitres
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mRNA Messenger ribonucleic acid
MSCs Mesenchymal stem cells
NBCS Natural bone collagen scaffold
NHMRC National Health and Medical Research Council
Nm Newton meter
NZ Neutral zone
OD Optical Density
OP-1 Osteogenic protein-1
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PDGF Platelet-derived growth factor
PEG Polyethylene glycol
PFA Paraformaldeyhde
PGA Polyglycolic acid
PLA Polylactic acid
PLA-PEG Poly-d,l-lactic acid–polyethylene glycol
PLGA Polylactic/glycolic acid
PLIF Posterior lumbar interbody fusion
rhBMP-2 Recombinant human bone morphogenetic protein-2
rhGDF-5 Recombinant human growth-derived factor-5
RNA Ribonucleic acid
RNAse Ribonuclease
RNAsin Ribonuclease inhibitor
ROM Range of motion
RT Reverase transcriptase
SDS-PAGE SDS-polyacrylamide gel electrophoresis
Sec Seconds
SEM Scanning electron micrograph
T Thoracic
TAE Tris-acetate EDTA
Taq Thermus aquaticus
TBS Tris-buffered saline
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TCP Tricalcium phosphate
TE Tris-EDTA
TGF-β Transforming growth factor β
TLIF Transforaminal lumbar interbody fusion
TNF-a Tumour Necrosis Factor-alpha
Micro-CT Micro CT X-ray tomography
US The United States
UV Ultra-violet
UWA The University of Western Australia
VATS Video-assisted thoracic surgery
VEGF Vascular endothelial growth factor
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LIST OF FIGURES
CHAPTER 3
Figure 3.1 Biomechanical data of fused lumbar spine.
Figure 3.2 Plain radiographs of fused rabbit lumbar spines at 6 weeks after surgery
Figure 3.3 Micro-CT reconstruction views of fused rabbit lumbar spines at 6 weeks
after surgery.
Figure 3.4 Bone volume, trabecular thickness, trabecular number, and trabecular
separation of new bone formation in fusion masses.
Figure 3.5 Photomicrographs of the histology of fused rabbit lumbar spines at 6
weeks after surgery.
CHAPTER 4
Figure 4.1 Macroscopic and microscopic evaluation of the structure of NBCS
Figure 4.2 3D-Micro-CT reconstruction of fused tissue regions at the lumbar spine
of rabbits 6-weeks postoperatively
Figure 4.3 Comparison of bone parameters assessed by micro-CT of new bone tissue
of each group
Figure 4.4 Saffranin O/fast green staining of spinal fusion masses
Figure 4.5 Comparison of individual tissue areas of each group using quantitative
histological analysis
Figure 4.6 Immunohistochemical detection of collagen type I expression
Figure 4.7 Immunohistochemical detection of osteonectin expression.
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Figure 4.8 RT-PCR results demonstrating gene expression levels of collagen type I,
II, III, X in fusion masses from each group.
CHAPTER 5
Figure 5.1 Population expansion of ovine BMCs in NBCS
Figure 5.2 New bone matrix generation and mineralization
Figure 5.3 Biomechanical properties of lumbar segments at 6 weeks and 10 weeks
postoperatively
Figure 5.4 Micro-CT imaging of spinal fusion segments
Figure 5.5 Histology of sheep spinal fusion
APPENDIX A
Figure A.1 Surgical procedure for rabbit spinal fusion
Figure A.2 Landmarks of skin incision of sheep anterior lumbar fusion
Figure A.3 Surgical procedure for sheep lumbar interbody fusion
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LIST OF TABLES
CHAPTER 1
Table 1.1 Properties of bone graft materials
Table 1.2 Overview of members of BMPs and their bioactivities
Table 1.3 Cell sources for preclinical and clinical spinal fusion
Table 1.4 Summary of scaffolds used for preclinical and clinical spinal fusion
CHAPTER 3
Table 3.1 Manual palpation and radiographic scores of rabbit lumbar fusion masses 6
weeks postoperatively
Table 3.2 Physiological range of motion (ROM) data of rabbit lumbar fusion spines
loaded to a maximum of 0.27 Nm
CHAPTER 4
Table 4.1 The sequences for the primers used for RT-PCR
Table 4.2 Comparison of NBCS and DBM
CHAPTER 5
Table 5.1 The sequences for the primers used for RT-PCR
Table 5.2 Manual palpation and radiographic scores (mean±SD) of ovine spinal
fusion
Table 5.3 ROM and NZ data of ovine spinal fusion loaded to a maximum of 0.475
Nm
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APPENDIX A
Table A.1 Antibodies for immunohistochemistry
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ABSTRACT
Spinal fusion is a commonly used surgical procedure for the treatment of various spinal
disorders, such as trauma, degeneration and tumor. The use of autologous bone graft in
spinal fusion has been the “gold standard” for decades. However, the use of the
autograft is associated with limitations and complications. Consequently, significant
research now focuses on developing substitutes for the autograft for bone tissue
applications. An osteoconductive scaffold is an essential element of bone tissue
engineering to provide an optimal environment for new bone formation. Natural bone
collagen scaffold (NBCS), also known as insoluble bone gelatin (ISBG), is a newly
developed osteoconductive scaffold for bone tissue engineering. This thesis focuses on
the osteoconductive property of NBCS, and the efficacy of NBCS combined with
osteogenic protein-1 (OP-1) or bone marrow cells (BMCs), to induce new bone
formation in rabbit and sheep spinal fusion models.
To investigate the efficacy of NBCS as a scaffold/carrier for OP-1 to induce new bone
formation, a rabbit postero-lateral lumbar fusion model was used. Spinal fusion masses
were evaluated by manual palpation, biomechanical testing, radiographic assessment,
micro-computer tomography (CT) scanning and histological examination 6 weeks
postoperatively. Animals treated with a combination of NBCS and OP-1 achieved 100%
solid fusion, which was higher than the control groups (autograft, NBCS only and OP-1
only). Enhanced fusion outcomes were also evidenced in the NBCS + OP-1 group by
radiographic examination, micro-CT analysis (bone volume), and biomechanical testing.
Histological assessment also demonstrated that treatment of NBCS + OP-1 induced new
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continuous bone formation in the interval between the transverse processes which was
not observed in the other groups. These findings raise the possibility that NBCS can be
used as a scaffold/carrier for bone tissue engineering, and that combined with OP-1,
may serve as a substitute for the autograft in spinal fusion.
To provide insight into the mechanisms of NBCS enhancing new bone formation
induced by OP-1, NBCS was characterized by scanning electronic microscopy (SEM),
and the rabbit fusion masses were analyzed by quantitative histology,
immunohistochemistry and semi-quantitative RT-PCR. By SEM, we showed that NBCS
maintained a porous, interconnecting microarchitecture. Quantitative histological
analysis demonstrated that the NBCS combined with OP-1 significantly enhanced bone
matrix area and bone marrow cavity size with lower cartilage component as compared
to the controls. Immunohistochemical assessment and RT-PCR also demonstrated that
NBCS combined with OP-1 enhanced type I collagen and osteonectin expression. These
finding revealed that the porous, interconnecting microarchitecture of NBCS provides
the osteoconductive property. NBCS enhances new bone formation induced by OP-1
probably by the acceleration of the process of ossification.
To evaluate the efficacy of NBCS as a scaffold for cell therapy, the osteogenesis of
ovine enriched BMCs within NBCS was investigated in vitro, and the new bone
formation induced by a combination of NBCS and enriched BMCs was evaluated in an
ovine interbody fusion model. In vitro results showed that NBCS supported the
population expansion, and osteogenic differentiation of ovine BMCs in vitro, and
promoted the new bone matrix generation and mineralization. In vivo outcomes
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demonstrated that NBCS combined with autologous enriched BMCs enhanced spinal
fusion with higher fusion rate, biomechanical stiffness and bone volume than in all
control groups. Histological assessment also revealed that the combination of NBCS
and enriched BMCs induced new bone formation that integrated well with host bone
tissue. These findings indicate that NBCS is an effective scaffold of BMCs, and
potentially the combination of NBCS and enriched BMCs may be used as a substitute
for the autograft used in achieving spinal fusion.
Viewed together, the studies presented in this thesis suggest that NBCS is an effective
osteoconductive scaffold for bone tissue engineering, and that combined with growth
factors such as OP-1, or enriched BMCs, is able to induce effective new bone formation.
Thus, the combination of NBCS and OP-1 or BMCs might be a potential substitute for
autologous bone graft in cases of spinal fusion.
CHAPTER 1
SPINAL FUSION: BONE GRAFTS
AND SUBSTITUTES
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1.1 Spinal fusion: the solution for spine instability
Spinal fusion is a common surgical procedure for the treatment of spinal diseases such
as trauma, degeneration, tumor and inflammation or infection, with the aim of
eliminating the instability caused by these pathologies, and achieving a bony union
between the involved vertebrae. Spinal fusion is ranked as the second most common
spine procedure, with approximately 46,500 lumbar spinal arthrodeses performed each
year in the United States. The use of spinal-fusion surgery in the United States is rapidly
increasing. National survey data indicate that the annual number of spinal fusion
operations rose by 77 percent between 1996 and 2001 (Stromqvist 2002). In contrast,
hip replacement and knee arthroplasty increased by 13 to 14 percent during the same
interval. Spinal fusion surgery is expensive, with the average hospital bill coming to
more than $34,000, excluding professional fee (Stromqvist 2002). Similarly in Australia,
spinal fusion procedures place a significant socioeconomic burden on both the
individual and the community.
There are two main types of spinal fusion, intertransverse process fusion and interbody
fusion. In intertransverse process fusion, the bone graft is implanted between the
transverse processes from the posterior approach; whereas in interbody fusion, the bone
graft is implanted between the vertebra, which is usually occupied by the intervertebral
disc tissue. In either intertransverse process fusion or interbody fusion, a bone graft
must be implanted into the fusion site, to induce new bone formation connecting the
involved vertebra, and to prevent soft tissue ingrowth. The autologous bone graft is the
most commonly used bone graft for spinal fusion, and has been considered a “gold
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standard” for decades. However, an accumulating body of evidence regarding the
limitations and complications associated with harvesting the autograft has been reported.
As a result, recent research now focuses on developing substitutes for the autograft for
tissue engineering applications to eliminate these limitations and complications.
This chapter will introduce indications and approaches for spinal fusion, and review the
use of the autograft and its substitutes in spinal fusion.
1.2 Indications of spinal fusion
The goal of spinal fusion is to re-establish the spinal stability in the segments with
unstable motion. Spinal instability, defined by White and Panjabi (White, Johnson et al.
1975), is the loss of ability of the spinal motion segments under physiological loads to
maintain normal anatomic relationships between the vertebrae. As a result, over-range
motion of unstable spinal segments may lead to clinical symptoms such as low back
pain and/or leg pain. Instability may be acute or chronic. Acute instability is normally
caused by bone or ligament disruption with trauma, and chronic instability is the result
of progressive degenerative deformity. The indications for spinal fusion are listed below.
1.2.1 Trauma
Spinal instability caused by trauma is the main indication for spinal fusion at the lumbar,
cervical or thoracic level. Due to their tight anatomical relationship, spinal column
injuries normally combine with spinal cord injuries resulting in paraplegia or
quadriplegia. A survey conducted in 650 trauma centers in the United States showed that
the incidence of cervical spine injury without spinal cord injury was only 3%, and of
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spinal cord injury without spinal fracture was as low as 0.7% (Grossman, Reilly et al.
1999).
According to a report from the National Institute of Disability and Rehabilitation
Research, 14,000 Americans suffer spinal cord injury each year, with about 8000 to
10,000 left paralyzed (Holdsworth 1970; Hopkins and White 1993). It is estimated that
50 people in 1 million suffer a spinal cord injury in America each year (Kraus, Franti et
al. 1975). This figure has been stable during the last three decades, although the number
of individuals who survive with spinal cord injuries has increased with the development
of regional trauma centers, and increased training to paramedics and emergency medical
technicians (Fisher, Noonan et al. 2006). In 1997-2000, 15.8% of individuals died en
route to hospital, a 50% decrease on the number observed in the early seventies (Dryden,
Saunders et al. 2003). However, despite improvements, traumatic spinal injuries place a
heavy burden on the public heathcare system.
Accidental falls and traffic injuries are the two most common causes of spinal fractures
(Hu, Mustard et al. 1996). Accidental falls become more prevalent with old age and are
more prevalent in women compared to men. Motor vehicle and transport injuries are
much higher in the younger male population, with an incidence rate of 35.3/100,000 in
the 20- to 29-year-old age group (Hu, Mustard et al. 1996).
Assessing the stability of the injured spine is crucial in immediate treatment and also
subsequent treatment of traumatic spinal injuries, including spinal fusion procedure. The
modern system to assess spinal injuries, which is based on Denis’ three-column concept
(Denis 1983), can clearly identify the stability of the injured spine. With a good
understanding of the biomechanical mechanisms underlying spinal injuries, Dennis
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separated the intact spinal column into three independent columns; 1) the posterior
column, formed by the posterior bony complex with the posterior ligamentous complex,
supraspinous ligament, interspinous ligament, capsule and ligamentum flavum; 2) the
middle column, formed by the posterior longitudinal ligament, posterior annulus
fibrosus and the posterior wall of the vertebral body, and 3) the anterior column, formed
by the anterior longitudinal ligament, the anterior annulus fibrosus and the anteriorpart
of the vertebral body.
A new classification system for assessing spinal injuries was subsequently established
based on this three-column concept (Denis 1984). This classification system is now
widely accepted, and can easily identify spinal instability. Major spinal injuries are
classified into four different types. These four types are compression fracture, burst
fracture, seat-belt-type spinal fracture and fracture-dislocation, and all are definable in
terms of the degree of involvement of each of the three columns. Furthermore, spinal
instability is further subdivided into three classes based on complications associated
with the spinal injury (Denis 1984). Instability degree one is defined as a mechanical
instability with risk of chronic kyphosis; degree two is defined as is a neurologic
instability; and instability of the third degree consists of both a mechanical and
neurologic instability. All spinal injuries with a mechanical and/or neurologic instability
are indications of spinal fusion.
The purpose of surgical treatment for unstable spinal injuries is to re-establish the
stability, and eliminate neurological compression. Spinal fusion is an effective approach
to achieve bony stability within the involved segments, either by intertransverse process
fusion or interbody fusion.
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1.2.2 Degenerative instability
Spinal degeneration, which leads to the symptom of low back pain or neck pain, is the
second main indication for spinal fusion. Spinal degeneration occurs most commonly in
the lumbar spine, which leads to back pain with or without leg pain. It occurs commonly
in the general population, with up to 80% of the population being affected by these
symptoms at some time in their lives (Canale 1998). Statistical data collected by the
National Center for Health Statistics (Kostuik and Bentivoglio 1981) indicated that low
back pain was the most frequent cause limiting the activity of people younger than 45
years. Furthermore, the lifetime incidence of low back pain is 61% and its prevalence is
31%, according to a survey conducted by Svensson and Andersson (Svensson and
Andersson 1983), with 40% of those reporting back pain also reporting sciatica. The
annual cost for the treatment of low back pain has been estimated at approximately
$11.1 billion in the United States (Webster and Snook 1990), with the cost for each case
increasing from $6807 in 1986 to $8321 in 1989 (Webster and Snook 1994). On top of
treatment costs, additional costs are required in disability payments.
Currently, the most widely accepted classification system used to characterize spinal
degeneration is that originally established by Kirkaldy-Willis et al. (Kirkaldy-Willis
and Farfan 1982). According to clinical symptoms and radiographic measurement,
Kirkaldy-Willis et al. divided spinal degeneration into three stages. Stage 1 includes
dysfunction, and is associated with low back pain with nonspecific symptoms. In this
stage, the facet capsule may be lax and disc degeneration is of grade 1 to 2 on a scale of
1 to 4. Stage 2 is named as instability, which is marked by increased facet joint laxity
and moderate disc degeneration (grades 2 to 3). Clinical symptoms can be identified,
and the instability can be measured by functional X-ray. Stage 3 is classed as
7
restabilization, and is characterized by fibrosis in posterior joints and osteophytic
formations leading to decreased overall motion. Disc degeneration has reached the final
stage (grades 3 to 4).
The use of spinal fusion in the treatment of spinal degeneration is usually considered in
patients with mechanical pain, an associated Grade II or higher spondylolisthesis, and
radiographically documented instability with pain or progressive neurologic deficits
(Herkowitz and Kurz 1991; Sonntag and Marciano 1995; Detwiler, Marciano et al.
1997). Radiographic instability is an important indication for spinal fusion. Panjabi et al.
(Panjabi, Abumi et al. 1989) defined radiographic instability as translational motion at
one spinal motion segment that is more than 3mm in the lumbar spine or 5mm at L5 -
S1 or as angulations of one motion segment more than 10° on a lateral flexion-extension
radiographic. However, the indications for spinal fusion are still controversial since
multiple factors need to be considered, including curve flexibility, curve progression,
radiculopathy, loss of lumbar lordosis, and extent of intraoperative decompression
(Herkowitz and Sidhu 1995).
Clinical outcomes have demonstrated functional improvement using spinal fusion to
successfully treat spinal degeneration. For example, in a clinical trial for spinal
degeneration (Kornblum, Fischgrund et al. 2004) patients with a solid fusion were
found to have significantly improved long-term clinical outcomes in comparison with
patients with pseudarthrosis. Ghogawala et al. also reported that superior outcomes can
be obtained with fusion compared with laminectomy without fusion in patients with
Grade I spondylolisthesis and stenosis. Herkowitz and Kurz (Herkowitz and Kurz 1991)
reported the first prospective study that compared decompressive laminectomy with and
without intertransverse process fusion in 50 patients with spinal stenosis and
8
degenerative spondylolisthesis. The results demonstrated that a statistically significant
better outcome was achieved by spinal fusion than by decompression alone.
1.2.3 Other indications for spinal fusion
In addition to the treatment for trauma and degeneration, spinal fusion can also be used
in the treatment of instability caused by other clinical presentations, such as tumor and
infection. However, since the normal fusion process may be influenced by the progress
of tumor, the indications for spinal fusion should be stricter, with careful consideration
of the patient’s age, life expectancy, and prognosis (Heary and Bono 2001). Infective or
inflammatory diseases have also been treated with spinal fusion procedure; examples
include Pott’s disease (Ghadouane, Elmansari et al. 1996), (Khan, Vaccaro et al. 1999)
and blastomycrosis (Hadjipavlou, Mader et al. 1998). However, these infective diseases
are still not definitive indications for spinal fusion. In addition, spinal fusion can also be
used as treatment for various spinal deformities, such as scoliosis in children,
adolescents and adults. Bony fusion is able to maintain the surgically modified spinal
alignment in these cases.
1.3 Surgical approaches
There are two basic surgical approaches in spinal fusion, the anterior and the posterior
approach. Due to the anatomical differences, the approaches used vary at different
levels of the spine. The anterior approach is normally used for interbody fusion at all
levels, while a posterior approach is used for posterior fusion, posterolateral fusion
(intertransverse process fusion) and interbody fusion. In 1958, Cloward (Cloward 1958;
Cloward 1958) first described an anterior approach as a treatment for ruptured cervical
9
discs. This approach soon became a standard procedure in treatment at the cervical
spine. However, at the levels of the thoracic and upper-lumbar spine, an anterior
approach is more difficult due to the presence of the rib cage and diaphragm. However,
using either an anterior or posterior approach, interbody fusion can be achieved below
the level of the conus. Intertransverse process spinal fusion can be achieved using a
posterior approach at all levels. However, the approach and technique selected is
dependent on the type and location of the pathology and the surgeon’s experience and
knowledge (Gunzburg, Mayer et al. 2002).
1.3.1 Cervical spinal fusion
Historically, a posterior approach at the cervical spine was first used for laminectomy in
patients with cervical spondylosis. However, outcomes were poor. In 1958, Cloward
(Cloward 1958) first published his technique of disc excision and dowel graft fusion via
an anterior cervical approach for patients with cervical disc rupture. In the same year,
Smith and Robinson (Smith and Robinson 1958) also described an operative technique
using an anterior approach to achieve cervical fusion using a horseshoe-shape graft.
This procedure was later named the Smith-Robinson technique. The horseshoe-shape
graft implantation provided greater mechanical support to the cervical column compared
with other bone grafts. As a result, the anterior surgical procedure became readily
accepted to implant the horseshoe-shape bone graft into the intervertebral space,
restoring the disc height and preventing neural impingement by decreasing excessive
motion (White and Hirsch 1971).
Up to now, the use of the anterior approach in the cervical spine remains the most
commonly used approach for the treatment of various cervical disorders. However, the
10
role of cervical fusion using the anterior approach to treat anterior cervical column
instability is controversial (Caspar, Barbier et al. 1989; Goffin, van Loon et al. 1995).
Aebi et al. (Aebi, Zuber et al. 1991) suggested that the anterior cervical fusion technique
using a bone graft was straightforward, atraumatic, and reliable after the analysis of 86
patients with cervical spine injuries. Conversely, Stauffer and Kelly (Stauffer and Kelly
1977) believed that posterior stabilization and fusion was suitable for posterior complex
lesion in the cervical column, since anterior fusion may not re-establish of the stability
of posterior column. The benefits of cervical fusion in restoring disc height and
stabilization of the pathologic cervical segment using a bone graft are supported by
recent studies. Therefore, cervical fusion is presently the preferred technique (Gore and
Sepic 1984; Chesnut, Abitbol et al. 1992).
1.3.2 Thoracic spinal fusion
Both anterior and posterior approaches can be applied in the thoracic spine to treat
spinal decompression and spinal instability. However, the selection of approach is
controversial. Advocates of the anterior approach believe that the anterior approach
allows more thorough anterior decompression by exposing the spine anteriorly, and
provides better biomechanical support between vertebral bodies. Thus, better clinical
outcomes can be obtained from the anterior approach neurologically and
biomechanically (Kostuik 1984; Bohlman, Freehafer et al. 1985; McAfee, Bohlman et
al. 1985; Dunn 1986). Bradford (Bradford 1979) reported that significantly improved
neurologic function was achieved using the anterior approach which facilitated better
spinal decompression in patients with thoracic fractures, retrospectively compared with
posterior decompression. In a retrospective study conducted by Siegal and Siegal
(Siegal and Siegal 1985), results demonstrated that patients treated with anterior
11
vertebrectomy and fusion had significantly improved neurologic function as compared
with patients treated with posterior laminectomy and fusion. McLain and Weinstein
(McLain and Weinstein 1990) also reported that anterior decompression and
stabilization produced significantly better outcomes than historical reports using
posterior decompression.
On the other hand, those who support the posterior approach believed that the
complications associated with the anterior approach may limit its use in thoracic spinal
fusion. These complication include potential morbidity and the possibility of
development of late post-traumatic kyphosis with a loss of postoperative alignment
(Edwards and Levine 1986; Riska, Myllynen et al. 1987; Westfall, Akbarnia et al. 1987;
Gertzbein, Court-Brown et al. 1988). Moreover, Edwards and Levine (Edwards and
Levine 1986) reported that canal diameter improved by an average of 32%, and
correlated with significant neurologic improvement, in patients with thoracic injuries
treated using indirect posterior decompression of the spinal canal.
However, other studies, showed no differences in neurologic outcome between
decompression and fusion using both anterior and posterior approaches. Gertzbein et al.
(Gertzbein, Court-Brown et al. 1988) compared anterior and posterior thoracic
decompression and fusion, and noted no significant differences regarding neurologic
outcome, although the outcomes of surgical intervention were significantly better than
that of nonoperative treatment.
1.3.3 Lumbar spinal fusion
More options are available to apply spinal fusion in the lumbar region, including the
12
anterior approach, posterior approach, posterolateral approach and combined
approaches. Applying the anterior retroperitoneal approach to lumbar vertebral bodies is
most common in lumbar interbody fusion, particularly for extensive resection,
debridement, or grafting at multiple levels in the lumbar spine, based on the fact that the
anterior aspect of the lumbar spine can be exposed well using an anterior approach.
However, the anterior approach is not suitable for patients with pathologic conditions in
the posterior complex, or with anatomic variation bifurcation, such as high bifurcation
of the aorta. Furthermore, due to the tight anatomic relationship between the anterior
aspect of the lumbar column and major blood vessels, lumbar fusion via the anterior
approach involves technical challenges with respect to properly manipulating major
vessels, including the iliolumbar vein, vena cava, common iliac vein, and accompanying
arteries (Herkowitz and International Society for Study of the Lumbar Spine. 2004).
Compared with the anterior approach, the posterior approach provides direct access to
the posterior complex of lumbar spine, including spinous processes, laminae, and facet
joints. As the posterior approach cannot provide good exposure to the anterior aspect of
the lumbar spine, pathologies located at the anterior lumbar column are
contraindications for the posterior approach (Herkowitz and International Society for
Study of the Lumbar Spine. 2004). Notably, nerve root injury is a common and severe
complication associated with the posterior approach.
In addition, lumbar transverse processes and pedicles can also be reached via
posterolateral approach through the potential gap between the longitudinal sacrospinalis
muscle group. This approach is especially useful in intertransverse process fusion for far
lateral disc herniation, decompressing a “far-out” syndrome, and inserting pedicle
screws (Herkowitz and International Society for Study of the Lumbar Spine. 2004).
13
1.3.4 Minimally invasive spinal fusion
One of the newest approaches to spinal surgery is minimally invasive spinal fusion,
which is characterized by small skin incisions, normally with the assistance of a
video-assisted endoscope. The main goal in minimally invasive surgery is to minimize
iatrogenic trauma during the surgical procedure, and to retain optimal muscle function
after surgery. Furthermore, the minimally invasive technique improves patient comfort,
hastens the postoperative recovery, and decreases postoperative morbidity. The
development of minimally invasive spinal surgery relies much on endoscope techniques.
Endoscopic surgery was first introduced in visceral surgery in the late 1980s, and in the
early 1990s a video-assisted endoscope was used in thoracic surgery. Subsequently,
video-assisted endoscopic techniques were introduced in spinal surgery. More recently,
as a result of further development of techniques and instruments, this operating
procedure has become widely accepted.
1.3.4.1 Minimally invasive cervical fusion
Due to anatomic particularity, the applications of minimally invasive spinal fusion in the
cervical region have been limited. In 2003, Wang and coworkers (Wang, Prusmack et al.
2003) were the first to report the use of the minimally invasive fusion technique at the
cervical spine. Percutaneous lateral mass screw placement combined with anterior
decompression and fusion was utilized in two patients. In 2005, Fong and Duplessis
(Fong and Duplessis 2005) also reported the use of a similar technique, and discussed
the feasibility of the minimally invasive technique; however, there was no follow-up
data in their reports. Although minimally invasive cervical fusion is an appealing
technique as it preserves the posterior tension band, additional evidence is needed to
14
assess whether the technique is superior to conventional open surgery.
1.3.4.2 Minimally invasive thorscopic fusion
The minimally invasive spinal fusion technique has been widely used in the thoracic
region, based on the development of video-assisted thorscopic techniques. In the early
1990s, video-assisted thoracic surgery (VATS) was applied to the treatment of spinal
disorders once the superiority of video-assisted thoracic surgery (VATS) over traditional
thoracotomy was recognized in thoracic surgeries. Currently, the indications of VATS
have widened from thoracic spine trauma initially (Hertlein, Hartl et al. 1995), to
anterior release as part of scoliosis repair (Nymberg and Crawford 1996; Newton,
Wenger et al. 1997) and vertebral corpectomy for osteomyelitis or metastatic tumor
(Han, Kenny et al. 2002).
The major advantage of combining minimally invasive thoracic fusion with VATS is
minimization of approach-related morbidity, and low incidence of pneumonia or
pneumothorax (Han, Kenny et al. 2002). Anand and Regan (Anand and Regan 2002)
reported minor complications in 21 out of 100 patients undergoing VATS thoracic disc
surgery. No major complications were reported and a clinical success rate of 70% was
achieved. Khoo et al. (Khoo, Beisse et al. 2002) reported on a large study which treated
371 patients with fractures between T3 and L3 using VATS fusion. Major complications,
including aortic injury, splenic contusion, neurologic deterioration, cerebrospinal fluid
(CSF) leak, or wound infection were reported in only 1.3%. Additionally, animal studies
utilizing VATS have demonstrated that adequate mechanical stability is achieved using
this technique (Cunningham, Kotani et al. 1998).
15
1.3.4.3 Minimally invasive lumbar interbody fusion
Similar to open surgical procedures, a number of options for minimally invasive lumbar
fusion are available and include posterior lumbar interbody fusion (PLIF),
transforaminal lumbar interbody fusion (TLIF) and anterior lumbar interbody fusion
(ALIF). The minimally invasive PLIF technique was first described by Khoo et al.
(Khoo, Palmer et al. 2002) in 2002. With the assistance of endoscopic visualization,
whole PLIF procedures were performed, including hemilaminectomy, discectom, end
plates preparation, interbody distraction, bone graft implantation and pedicle screws
system placement. In 2005, the minimally invasive TLIF technique was reported by
Schwender et al. (Schwender, Holly et al. 2005) and Isaacs RE et al. (Isaacs, Podichetty
et al. 2005). Interbody fusion was achieved through the transforaminal channel with a
video-assisted endoscope, and an instrument of pedicle screws and rod system was also
placed percutaneously. From radiographic assessment, follow-up data demonstrated that
all patients achieved solid fusions.
The minimally invasive ALIF technique was applied relatively earlier than PLIF. In the
mid-1990s, several groups (Mathews, Evans et al. 1995; Olsen, McCord et al. 1996)
reported on studies using a laparoscope in lumbar interbody fusion. Meanwhile, Mahvi
and Zdeblick (Mahvi and Zdeblick 1996) presented encouraging results using minimal
ALIF. The technique resulted in a shorter hospital stay, a reduced pain relief rate of 60%
and a satisfying fusion rate. However, the minimally invasive ALIF technique is still not
favored given its association with severe complications, including iliac vein injury
(Mathews, Evans et al. 1995), bladder injury (Olsen, McCord et al. 1996) and
retrograde ejaculation (Olsen, McCord et al. 1996).
16
1.4 Bone Grafts for Spinal Fusion
1.4.1 Biology of Spinal Fusion
In 1995, Boden et al. (Boden, Schimandle et al. 1995) established an excellent spinal
fusion model in the rabbit. Using this model, a good understanding of spinal fusion
biology was achieved. In this model, intertransverse process fusion was induced at the
L5-L6 level using autologous bone graft harvested from the iliac crest. A fusion rate of
60-70% was achieved, which is comparable with that observed in humans. Therefore,
this model is now widely used for various studies on spinal fusion.
To study the biological processes involved in spinal fusion, Boden et al. (Boden,
Schimandle et al. 1995) assessed newly formed tissue within fusion masses by
histological analysis. Results demonstrated that the process of new bone formation was
most advanced at the areas near the transverse processes, while ossification in the
central zone was delayed in time. This result indicated that new bone formation
occurred from transverse processes, but not from the soft tissue surrounding. Boden et
al. also noted that spinal fusion could not be achieved in the absence of decortication. In
addition, a vascular injection study also confirmed that the primary blood supply to the
fusion mass originated from the decorticated transverse processes, not the surrounding
soft tissues (Toribatake, Hutton et al. 1998). Collectively, this data suggests that
osteoprogenitor cells and osteoinductive factors necessary in new bone formation are
provided by the bone marrow in decorticated transverse processes, but not from the
surrounding soft tissue.
Osteoblast-related gene expression in different regions of the fusion mass during the
fusion process has also been studied using RT-PCR analysis (Morone, Boden et al.
17
1998). Consistent with histological findings, a delayed gene expression pattern is
detected in the central zone compared with the peripheral zone. For example, BMP-2
expression peaks at weeks three and four in the peripheral zones, but not until week five
in the ral zone.
1.4.2 Bone grafts for spinal fusion
There are three critical elements involved in the process of new bone formation during
spinal fusion: osteogenic cells, osteoinductive growth factors and osteoconductive
scaffold. Osteogenic cells have the capacity to synthesize new bone, osteoinductive
factors promote the osteoblastic differentiation of pluripotential stem cells, and an
osteoconductive scaffold supports the ingrowth of bone and facilitates
neovascularization. The process of new bone formation depends on the co-operative
work of these three elements. As mentioned in section 1.4.1, osteogenic cells and
osteoinductive growth factors are originally from host bone marrow from decorticated
transverse processes (intertransverse process fusion) or vertebral bodies (interbody
fusion). However, low concentrations of autologous cells and growth factors may not be
enough to induce sufficient new bone formation. Thus supplemental cells or factors
originating from implanted grafts can enhance new bone formation during spinal fusion.
Additionally, an osteoconductive graft is crucially important for spinal fusion to provide
a suitable environment for new bone formation in the application space and to prevent
soft tissue ingrowth into the fusion site. In other words, an osteoconductive scaffold
plays an important role in regulating the competition between new bone formation and
the surrounding soft tissues which encroach on the fusion site.
Autologous bone graft is the most commonly used material because it contains all three
18
critical elements for new bone formation mentioned above, and autograft has been
accepted as the “gold standard” for spinal fusion for several decades. Autograft includes
cortical bone graft and cancellous bone graft. Cortical bone provides better mechanical
support, while cancellous bone offers greater osteogenic potential due to its porous
structure and availability of bone marrow tissue. However limitations in quantity and
quality, and postoperative morbidities associated with autologous harvest have been
reported (Reid 1968; Cowley and Anderson 1983; Kuhn and Moreland 1986; Kurz,
Garfin et al. 1989; Summers and Eisenstein 1989; Younger and Chapman 1989;
Fernyhough, Schimandle et al. 1992; Banwart, Asher et al. 1995; Goulet, Senunas et al.
1997). In an attempt to address the limitations and complications of the autograft,
current research has focused on the development of substitutes for the autograft. These
substitutes include biomaterials with osteogenic potential, or a combination of two or
more materials, including the application of growth factors and osteoprogenitor cells
with the scaffold. Currently, commonly used substitutes include allograft, recombinant
osteoinductive growth factors, mesenchymal stem cells, gene therapy and
osteoconductive scaffolds. The properties of autografts and commonly used substitutes
are listed in Table 1.1 (Whang and Wang 2003). The advantages and potential issues
associated with each will be discussed extensively in section 1.5 – 1.8.
Table 1.1 Properties of bone graft materials
-: negative; +: weak positive; ++: medium positive; +++: strong positive
Bone graft substitute Osteogenic
cells
Osteoinductive
factors
Osteoconductive
structure
Initial bio-
mechanical strength
Donor-site
morbidities
Autogenous bone, cancellous +++ ++ +++ - ++
Autogenous bone, cortical + + + +++ ++
Osteoinductive growth factors - +++ - - -
Bone marrow cells ++ + - - +
Mesenchymal stem cells +++ - - - +
Gene therapy, in vivo - +++ - - -
Gene therapy, ex vivo ++ +++ - - +
Allograft - + + ++ -
Demineralized bone matrix - ++ + - -
Collagen sponge - - +++ - -
Calcium phosphate - - +++ + -
19
20
1.5 Autologous bone graft in spinal fusion
Autologous bone grafts are defined as bone tissue that is harvested from and implanted
back into the same individual. The autograft is the most commonly used graft material
in spinal fusion procedures, and has been accepted as the “gold standard” for decades
(Arrington, Smith et al. 1996; Sandhu, Grewal et al. 1999). The main source of
autograft is the iliac crest, since it can provide both cortical and cancellous bone.
Harvesting of bone tissue from the iliac crest has only minor effects on the stability of
the skeletal system (Arrington, Smith et al. 1996). Other sources for the autograft
include the proximal tibia, the fibula and the rib.
1.5.1 Advantages of autograft
Autografts are widely used for spinal fusion as they exhibit a number of positive
features. Firstly, the safety of the use of autograft is superior to the allograft or other
biomaterials. Harvesting bone from the same individual guarantees there is no risk of
disease transmission during implantation. Histocompatibility of the autograft also
precludes the risk of inflammation and immune rejection by host individual. Moreover,
autografts satisfy the three key elements required for new bone formation, including a
source of viable osteogenic cells, osteoconductive factors and osteoinductive structure
(Arrington, Smith et al. 1996; Sandhu, Grewal et al. 1999). Autografts, especially
cancellous bone chips are a source of bone marrow which “houses” types of cells
required in new bone formation during spinal fusion. Noncollagenous growth factors
provided by autograft, such as BMPs, are able to induce osteoblastic differentiation and
are primarily responsible for the osteoinductivity of the autograft (Boden 1998). Finally,
autograft has osteoconductive properties, based on its porous structure and organic
21
components, which provide a suitable environment for new bone formation and guide
new bone matrix generation (Sandhu, Grewal et al. 1999).
1.5.2 Drawbacks of autograft in spinal fusion
The drawbacks associated with the use of autograft, which have limited its wider
application, include donor-site complications, limited quantity and quality, and
unsatisfying fusion rate.
Postoperative donor-site complications are the main concern in harvesting autograft,
including chronic donor-site pain, infection, hematoma, nerve or vascular injury,
fracture, abdominal herniation and pelvic instability. (Kuhn and Moreland 1986;
Banwart, Asher et al. 1995; Arrington, Smith et al. 1996; Goulet, Senunas et al. 1997;
Seiler and Johnson 2000; Skaggs, Samuelson et al. 2000). It has been reported that up to
30% of the patients from whom iliac crest bone is harvested experience postoperative
donor-site complications (Seiler and Johnson 2000). and 15% of patients suffer
complications that affect daily living activities (Skaggs, Samuelson et al. 2000).
Donor-site complication is correlated with surgical approach. For example, deep
infection and heniation are normally associated with autograft harvest from the anterior
iliac crest; while dislocation is associated with the posterior approach (Kurz, Garfin et al.
1989).
The quantity of autograft may be limited in children, as well as in adults who require
multiple levels of fusion. The quality of autograft supplied by aged patients or patients
with metabolic disorders is also a concern. Although autograft can attract osteogenic
cells to the fusion site, the viability of cells is challenged once they are separated from
22
the blood supply (Sandhu, Grewal et al. 1999). Since the surviving cells receive oxygen
and nutrients by diffusion only at the implantation site, apoptotic cell death led by
ischemia may result in failure to achieve fusion (Bauer and Muschler 2000). Therefore,
the fusion process may be delayed or even blocked. This effect may account for the
variability in spinal fusion success rates (60% to 70%) yielded by autograft (Morone,
Boden et al. 1998).
1.6 The use of growth factors in spinal fusion
1.6.1 Osteoinductive growth factors
The process of bone formation is regulated by numerous factors, including
platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), insulin-like
growth factor 1 and 2 (IGF-1 and IGF-2), transforming growth factor-beta (TGF-β), and
vascular endothelial growth factor (VEGF). However none of the above are as
osteoinductively potent as bone morphogenetic proteins (BMPs), which are members of
the TGF-β superfamily. In 1965, Urist (Urist 1965) first reported on the osteoinductivity
of BMPs. In the late 1980s, several members of the BMP family were purified and
sequenced (Wozney, Rosen et al. 1988). The members of the BMP family are listed in
Table 1.2 (Groeneveld and Burger 2000). Currently, several BMPs are commercially
available and have been applied in preclinical and clinical spinal fusion. These include
recombinant human bone morphogenetic protein-2 (rhBMP-2), recombinant human
morphogenetic protein-7 (rhBMP-2), (also called recombinant human osteogenic
protein-1 (OP-1)), and a highly purified extract from a mixture of bovine bone
morphogenetic proteins (bovine BMP extract).
23
Table 1.2 Overview of members of BMPs and their bioactivities
BMP Bone induction (Ref.)
BMP-2 (BMP-2a)
Bone induction in subcutaneous pockets in rats
(Wang, Rosen et al. 1988; Wozney, Rosen et al.
1988)
BMP-3 (osteogenin)
Bone induction in subcutaneous pockets in rats
(Wang, Rosen et al. 1988; Wozney, Rosen et al.
1988)
BMP-4 (BMP-2b)
Bone induction in subcutaneous pockets in rats
(Wang, Rosen et al. 1988; Wozney, Rosen et al.
1988)
BMP-5 No report on osteoinductivity
BMP-6 (VGR-1) Bone induction in subcutaneous pockets in nude
mice (Gitelman, Kobrin et al. 1994)
BMP-7 (OP-1) Bone induction in subcutaneous pockets in rats
(Sampath, Maliakal et al. 1992)
BMP-8 (OP-2) No report on osteoinductivity
BMP-9 (GDF-2) Osteogenic differentiation in C2C12 cell line in
vitro (Bessa, Cerqueira et al. 2009)
BMP-10 Osteogenic differentiation in C2C12 cell line in
vitro (Bessa, Cerqueira et al. 2009)
BMP-11 Osteogenic differentiation in C2C12 cell line in
vitro (Bessa, Cerqueira et al. 2009)
BMP-12 (GDF-11,
CDMP-3) No report on osteoinductivity
BMP-13 (GDF-6, CDMP-2) No report on osteoinductivity
BMP-14 (GDF-5) Osteogenic differentiation in C2C12 cell line in
vitro (Bessa, Cerqueira et al. 2009)
BMP-15 (CDMP-1) No report on osteoinductivity
24
1.6.2 The use of growth factors in animal models of spinal fusion
To evaluate the efficacy of growth factors for spinal fusion, various animal models have
been selected and tested, including mouse, rabbit, dog, sheep and monkey models
(Khan and Lane 2004). The use of these animal models has established a systematic
path to evaluate the efficacy of bone tissue engineering using BMPs for induction of
spinal fusion, and has produced extensive high-quality data. The data from animal
studies are critical in defining BMPs’ clinical potential.
The most commonly used spinal fusion model is a rabbit intertransverse process lumbar
fusion model, as discussed in section 1.4.1 (Boden, Schimandle et al. 1995). Based on
its comparable fusion rate with patients, this model has been widely used for evaluation
of bone graft substitutes in spinal fusion. Using this model, the efficacy of rhBMP-2 for
spinal fusion was evaluated by manual palpation and radiography assessment at five
weeks postoperatively (Schimandle, Boden et al. 1995). The results showed that all
rabbits implanted with rhBMP-2 achieved solid spinal fusion in comparison to the
autograft which only achieved a fusion rate of 42%. Grauer et al. (Grauer, Patel et al.
2001) evaluated the efficacy of rhOP-1 in this rabbit model compared with the use of
the autograft or collagen-CMC carrier alone. Results demonstrated that at five weeks,
all eight rabbits (100%) in the OP-1 group achieved a solid fusion compared to five out
of eight (63%) in the autograft group and zero out of eight (0%) in the carrier alone
group. Using the same rabbit model, my colleagues and I tested the efficacy of OP-1
with and without a biomaterial scaffold, NBCS. The results showed that OP-1 alone
only yielded a successful fusion in three out of seven cases, compared to seven of seven
cases of solid fusion induced by OP-1 combined with bone gelatin scaffold (Yao, Qian
et al. 2008) (Chapter 3).
25
The ovine interbody fusion model is another popular model used to assess the anatomic
and biomechanical similarities between sheep and human lumbar vertebrae (McLain
and Weinstein 1990; Becker, Maissen et al. 2006; Easley, Wang et al. 2008). Human
surgical instruments and techniques can easily be applied to ovine surgical procedures,
and the results gained from the ovine model can be compared to those in humans
(Blattert, Delling et al. 2002; Toth, Wang et al. 2002). Using this ovine model, Sandhu
et al. (Sandhu, Toth et al. 2002) evaluated the efficacy of rhBMP-2 compared with the
autograft. The results showed that 100% of animals treated with rhBMP-2 reached
histological union, compared to a 37% fusion rate in the animals treated with autograft.
This ovine interbody fusion model was also used by Magin et al. (Magin and Delling
2001) to evaluate the efficacy of OP-1. The results demonstrated that by four months,
new bone volume was statistically higher in the OP-1 group than in either the autograft
group or in the hydroxyapatite group at four months (P<0.05). The fusion masses
induced by OP-1 exhibited increased mechanical stiffness and histological maturation
compared to control groups.
The primate model has a high degree of similarity with humans biologically, but is
limited in source. Hecht et al. (Hecht, Fischgrund et al. 1999) obtained 100% interbody
fusion in six rhesus monkeys treated with rhBMP-2, compared to only one solid fusion
out of three animals treated with autologous bone graft. Similarly, successful spinal
fusion was induced in five rhesus monkeys treated with rhBMP-2 by Boden et al.
(Boden, Martin et al. 1998), while the treatment of two animals with scaffold alone
failed to yield fusion.
1.6.3 The use of growth factors in clinical trials of spinal fusion
26
The first clinical application of BMPs in spinal fusion was in 1996 (Boden, Zdeblick et
al. 2000). A pilot study using rhBMP-2 for lumbar spinal fusion was conducted with
fourteen patients with single-level lumbar degenerative disc disease. Patients underwent
the lumbar interbody fusion with cages filled with either rhBMP-2/collagen sponge or
autogenous iliac crest bone. The results showed that all 11 patients treated with
rhBMP-2 achieved successful fusion, as determined by CT scans, whereas one out of
three control patients treated with autograft failed to exhibit solid fusion.
Following the successful pilot study, a prospective, multicentre, randomized trial for
interbody lumbar spinal fusion was conducted (Burkus, Gornet et al. 2002; Burkus,
Transfeldt et al. 2002). One hundred and forty-three patients were enrolled in the
experimental group and treated with rhBMP-2, while 136 patients were treated with
autograft as a control group. The fusion rate at six months, as determined by X-ray, was
99.2% in the rhBMP-2 group, and 96.7% in the autograft group. The clinical success
rate evaluated with Oswestry Index was 84.4% in the experimental group compared
with 82.4% in the control group.
Furthermore, rhBMP-2 was also used for cervical spinal fusion (Baskin, Ryan et al.
2003) and posterolateral lumbar fusion trials (Boden, Kang et al. 2002). The results
from these clinical trials showed that superior fusion outcomes were achieved in the
rhBMP-2 group compared to the outcomes achieved in the autograft group.
OP-1 has also undergone clinical evaluation. In a clinical trial conducted by Vaccaro et
al.(Vaccaro, Whang et al. 2008), 36 patients with degenerative lumbar spondylolisthesis
27
and spinal stenosis underwent a laminectomy and posterolateral fusion with OP-1 putty
or autograft. Follow-up of 28 patients was recorded at 24 months. Clinical success, as
determined by the Oswestry Index, was achieved in 14 out of 19 patients (73.7%) in the
OP-1 putty group, and 4 out of 7 patients (57.1%) in the autograft group. Radiographic
fusion was achieved in 11 out of 16 (68.8%) patients treated with OP-1, and 3 out of 6
(50%) patients in the autograft group.
1.6.4 Potential limitations of the use of growth factors in spinal fusion
The use of BMPs for spinal fusion has produced encouraging outcomes in both
pre-clinical studies and clinical trials; however, relevant limitations have also been
reported. The major concern regarding the use of BMPs for spinal fusion is overgrowth
and uncontrolled bone formation given the linear relationship between the amount of
new bone formation and the dose of BMPs (Wang, Rosen et al. 1990). Miyamoto et al.
(Miyamoto, Takaoka et al. 1992) implanted rhBMP-2 in the lumbar extradural space in
a rodent model for spinal cord compression. After eight weeks, degeneration and
demyelination of the spinal cord was observed due to ossification and hypertroph of the
ligament. Mimatsu et al. (Mimatsu, Kishi et al. 1997) noticed mild spinal cord
compression by new bone growth in the ligamentum flavum induced by rhBMP-2 in
rabbits with L5 laminectomy. Paramore et al. (Paramore, Lauryssen et al. 1999) reported
overgrowth of bone tissue induced by the leakage of OP-1, which resulted in spinal
stenosis.
Apart form overgrowth, additional complications associated with BMPs used in spinal
fusion have also been reported. Sandu et al. (Sandhu, Kanim et al. 1995) reported a
local inflammatory over-response with the use of rhBMP-2 in combination with an
28
open-cell polylactic acid (OPLA) polymer carrier. In addition, clinical trials of spinal
fusion with OP-1 putty have demonstrated variable results. Vaccaro et al. (Vaccaro,
Stubbs et al. 2007) reported that the fusion rate induced by OP-1 was 68.8% (11 of 16),
which was only a little higher than that by autograft (50%, 3 of 6). Furthermore, a
clinical trial conducted by Jeppsson (Jeppsson, Saveland et al. 1999) was ceased
because the first four patients failed to reach fusion.
So far, there are only two types of BMP commercially available for spinal fusion,
BMP-2 and OP-1 (BMP-7), and the high costs of the use of BMPs also limit the wider
application of growth factors in spinal surgeries.
1.7 Cell therapy and gene therapy in spinal fusion
1.7.1 Cell therapy for spinal fusion
Cell therapy is defined as the process of introducing new cells into a tissue for the
purpose of treatment of a certain disease. Initially, cell therapies focued on the treatment
of hereditary diseases, and they have currently been employed in bone tissue
engineering applications, including spinal fusion. The cell therapies for spinal fusion in
preclinical and clinical studies are new listed in table 1.3.
Table 1.3 Cell sources for preclinical and clinical spinal fusion
Cell source Species Fusion type Outcome (fusion rate) Reference Unfractionated bone marrow and vein blood
Sheep Interbody fusion Only the bone marrow group showed bone formation inside the scaffold
(Becker, Maissen et al. 2006)
Unfractionated bone marrow and enriched bone marrow cells
Sheep Posterolateral fusion Enriched bone marrow cells (3/12), unfractionated bone marrow (0/12)
(Gupta, Theerajunyaporn et al. 2007)
Bone marrow MSCs and scaffold alone Rat Posterolateral fusion New bone formation (5/5) by MSCs, no bone formation (0/2) by scaffold
(van Gaalen, Dhert et al. 2004)
Bone marrow MSCs and autograft Macaque Interbody fusion Bone marrow MSCs (4/6), autograft (5/6) (Wang, Dang et al. 2005)
Bone marrow MSCs and autograft Macaque Posterolateral fusion Bone marrow MSCs (5/6), autograft (4/6) (Orii, Sotome et al. 2005)
Bone marrow stromal-derived osteoblasts and autograft
Rabbit Posterolateral fusion Osteoblasts (6/6), autograft (4/6) (Kai, Shao-qing et al. 2003)
Cloned osteoprogenitor cells and mixed marrow stromal cells
Rat Posterolateral fusion Cloned osteoprogenitor cells (8/8), mixed marrow stromal cells (4/8)
(Cui, Ming Xiao et al. 2001)
Bone marrow MSCs (1 or 100 million) Rabbit Posterolateral fusion 100 million(5/7), 1 million (0/7) (Minamide, Yoshida et al. 2005)
Bone marrow MSCs and MSCs + rhBMP-2
Rabbit Posterolateral fusion Bone marrow MSCs (8/12), MSCs + rhBMP-2 (11/12)
(Fu, Chen et al. 2009)
Bone marrow MSCs with and without osteogenic differentaiation
Rabbit Posterolateral fusion Osteogentic MSC(4/5), bone marrow MSC (2/6)
(Nakajima, Iizuka et al. 2007)
Enriched bone marrow cells + corticocancellous bone allograft
Human Posterolateral fusion Fusion from C3 to C7 (1 pateint) (Vadala, Di Martino et al. 2008)
Enriched bone marrow cells Human Posterolateral fusion Clinical lumbar fusion rate (39/41) (Gan, Dai et al. 2008)
29
30
1.7.1.1. Autologous bone marrow cells
Bone marrow cells (BMCs), which contain mesenchymal stem cells (Reddi 1995), have
pluripotent potential to differentiate into multiple mesenchymal lineages, including
osteoblasts, chondrocytes and adipose cells (Jorgensen, Djouad et al. 2008), have been
used for bone tissue engineering applications for spinal fusion, bone defect repair and
nonunion healing. The osteogenic potential of autologous bone marrow has been well
documented in various in vitro and in vivo studies (Nade, Armstrong et al. 1983; Paley,
Young et al. 1986; Connolly, Guse et al. 1989; Ohgushi, Goldberg et al. 1989; Goshima,
Goldberg et al. 1991; Grundel, Chapman et al. 1991; Wolff, Goldberg et al. 1994;
Werntz, Lane et al. 1996). Autologous bone marrow cells were initially used in the form
of unfractionated bone marrow (Healey, Zimmerman et al. 1990; Connolly, Guse et al.
1991; Garg, Gaur et al. 1993). However, in an ovine intertransverse process fusion study,
unfractionated bone marrow failed to induce solid fusion in 12 segments, while enriched
bone marrow cells yielded 25% (3/12) fusion (Gupta, Theerajunyaporn et al. 2007). The
major concern of unfractionated bone marrow is its low concentration of MSCs. The
bone marrow of healthy adults contains only one MSC for every 50,000 nucleated cells
(Kahn, Gibbons et al. 1995; Inoue, Ohgushi et al. 1997; Muschler, Boehm et al. 1997;
Muschler, Nitto et al. 2001) and this population is further diminished if bone marrow is
diluted by peripheral blood during aspiration (Muschler, Boehm et al. 1997).
To increase the concentration of MCSs in BMCs, the centrifugation enrichment method
has been developed to eliminate red blood cells and plasma. This approach has been
used in preclinical studies and clinical trials (Connolly, Guse et al. 1989; Hernigou and
Beaujean 1997; Gangji, Hauzeur et al. 2004). The enrichment of bone marrow cells
obtained from centrifugation avoids the need for manipulation by cell culture and
31
avoids potential risks such as contamination, carcinogenicity and rejection from cell
culture (D'Amour and Gage 2000). When human embryonic stem cells are cultured in a
medium containing animal serum or serum replacement, the binding of nonhuman sialic
acid Neu5Gc with human sera antibodies can result in cell death in vivo (Martin, Muotri
et al. 2005). Additionally, the enriched BMCs isolated by centrifugation contain a mixed
population which includes MSCs as well as other mononuclear cells, which have been
shown to be a potential source of angiogenic and osteogenic cytokines which positively
influence osteogenesis (Tateishi-Yuyama, Matsubara et al. 2002).
1.7.1.2 Mesenchymal stem cells
Due to their capability to differentiate into various different cell types, MSCs can be
used in tissue engineering applications to produce various mesenchymal tissues,
including bone, cartilage, muscle and connective tissues. The differentiation of the
MSCs depends on the specific inductive signal provided by local growth factors and
cytokines in vitro or in vivo. For example mature osteoblasts can be induced from MSCs
with BMP or dexamethasone treatment (Dennis, Haynesworth et al. 1992; Jaiswal,
Haynesworth et al. 1997; Pittenger, Mackay et al. 1999) (Grigoriadis, Heersche et al.
1988; Cassiede, Dennis et al. 1996; Kadiyala, Young et al. 1997). Using special
purification and subcultivation techniques, MSCs can be separated from bone marrow
and expanded in vitro, retaining their multilineage potential (Haynesworth, Goshima et
al. 1992; Bruder, Jaiswal et al. 1997; Jaiswal, Haynesworth et al. 1997). In addition,
ectopic bone and bone marrow were induced by subcutaneous implantation of MSCs
into immune deficient mice, which indicated the osteogenesis potential of MSCs
(Abdallah and Kassem 2008).
32
In bone tissue engineering, the use of MSCs enhances bone production by augmenting
the number of osteogenic cells available. As expected superior bone formation is
induced by MSCs amplified by cell culture compared to that obtained using
unfractionated bone marrow (Kahn, Gibbons et al. 1995; Inoue, Ohgushi et al. 1997;
Muschler, Boehm et al. 1997; Muschler, Nitto et al. 2001). For example, the healing of
critical-sized long-bone defects in animal models is enhanced with the treatment of
culture-expanded MSCs rather than unfractionated bone marrow (Bruder, Kraus et al.
1998).
MSCs are generally combined with a carrier/scaffold which delivers and maintains
these cells at the fusion site and provides osteoconductivity to support new bone
formation. MSCs are autologous and therefore do not subject the patient to the risk of
disease transmission and immunological rejection. However, unlike autograft and
unfractionated bone marrow, this approach carries a risk of in vitro contamination from
the procedure of in vitro cell culture.
1.7.1.3 Other Cell sources
In addition to adult-derived MSCs, the potential of non-autologous cell sources for
therapeutic applications is also being explored, for example amniotic fluid-derived stem
cell (AFSC), human umbilical cord perivascular cell (HUCPVC) and human ESCs
(hESCs). It has been reported that AFSCs can differentiate along the osteogenic,
adipogenic, myogenic, endothelial, neurogenic, and hepatic pathways in vitro. For
example, human AFS cells, cultured in an osteogenic inductive medium, support the
formation of highly mineralized tissue upon implantation in immunodeficient mice (De
Coppi, Bartsch et al. 2007).
33
Human umbilical cord perivascular cell (HUCPVC) is another recently identified MSC
source (Sarugaser, Lickorish et al. 2005). The frequency of MSCs in HUCPVC is 0.3%,
far exceeding the frequency of 1:30 million in umbilical cord blood-derived
mononuclear cells (Erices, Conget et al. 2000) and an approximate ratio of 1:100,000 in
bone marrow (Pittenger, Mackay et al. 1999). The capability of HUCPVC forming
osteoblasts, adipocytes, chondrocytes, myoblasts, and fibroblasts has been confirmed by
Sargaser et al. (Sarugaser, Lickorish et al. 2005).
An alternative source of MSCs is pluripotent human ESCs (hESCs). ESC lines are
characterized by high telomerase expression, normal and stable karyotype (Hyslop,
Armstrong et al. 2005). The potential of osteogenic differentiation of hESCs has been
confirmed in multiple conditions, such as in the EB system (Cao, Heng et al. 2005),
monolayer system (Karp, Ferreira et al. 2006), as well as in co-culture with primary
bone-derived cells (Ahn, Kim et al. 2006).
1.7.1.4 Potential issues related to using cell therapy for spinal fusion
Although the data from in vitro and in vivo studies strongly highlight the potential of
cell therapy for bone regeneration, there are still quite a few issues that need to be
resolved before these cells can be widely used for clinical applications. One of the
major obstacles is the risk of contamination during the procedure of cell culture. The
medium used in cell culture commonly contains non-human serum or other animal
derived substances, which may serve as a source of contamination for foreign proteins
(e.g., xeno-carbohydrate N-glycolylneuraminic acid) (Martin, Muotri et al. 2005), and
may also increase the risk of transmitting infectious diseases (xenozoonosis). To address
34
this limitation, serum-free media, or conditioned media, is being developed (Ludwig,
Levenstein et al. 2006).
Another issue limiting the use of cell therapy for spinal fusion is the potential
carcinogenicity from cultured stem cells (Gertow, Wolbank et al. 2004). Therefore, the
use of optimized, defined culture conditions for the maintenance of selected cells is
critical for the generation of pure populations of functional cells in a directed and
controlled manner.
1.7.2 Gene therapy for spinal fusion
Gene therapy is the insertion of a specific DNA sequence into an individual's target cells
and tissues that subsequently express the therapeutic proteins to treat diseases, initially
for genetic diseases. However, gene transfer systems typically generate only temporary
protein production, and as a result, such chronic diseases as genetic diseases cannot
benefit from an instant expression of specific factors or cytokines. Temporary gene
expression is likely to be more appropriate for the purpose of enhancing spinal fusion,
because the specific growth factor is only demanded during the period of new bone
formation. The growth factor expressed by genetically modified cells stimulates new
bone formation at the fusion site, and once sufficient fusion is achieved, prolonged
exposure to these biologically active proteins is usually not necessary. Compared with
delivering growth factors to the application site directly, gene therapy may provide a
more potent and stable osteoinductive signal.
Three key elements are required in gene therapy: DNA encoding the protein, target cells
and a vector that transfers the gene into the cells. Viral and nonviral vectors are two
35
basic ways to deliver genetic material into target cells. Viral vectors, such as
retroviruses, adenoviruses and adeno-associated viruses, are often favoured over
nonviral ones based on their superior transduction efficiencies, while the safety of the
infectious agents is the major concern for viral vectors. Nevertheless, nonviral vectors,
such as liposomes, are more stable and easier to produce than viruses.
1.7.2.1 In vivo and ex vivo techniques
The two basic techniques for the insertion of therapeutic genes into cells are in vivo and
ex vivo techniques. In vivo genetic transfer means the DNA-containing vector is directly
introduced into the patient or animal. This process is relatively simple and convenient. A
study of spinal fusion using in vivo gene therapy was conducted by Alden et al. (Alden,
Pittman et al. 1999). They injected an adenoviral vector carrying the DNA sequence for
the BMP-2 protein into the paraspinal musculature of rats, and observed new bone
formation at the injection site indicating that in vivo gene transfer may facilitate spinal
fusion. However, there are still several issues involved with the use of in vivo gene
therapy, including inefficient gene delivery, nonspecific target cells and the possibility
of host inflammatory response.
The procedure of ex vivo gene therapy is more complex, involving the harvesting of
target cells, transduction of genetic material in vitro, and implantation of modified cells
into the patient. Compared to in vivo techniques, ex vivo approaches are considered
more efficient and safer, because the process of genetic transduction is performed in
vitro and the genetically altered cells may be thoroughly screened before they are
reimplanted into the patient.
36
1.7.2.2 Combination of cell therapy and gene therapy
Combination of cell therapy and gene therapy involves using pluripotential cells as
vehicles for gene therapy. Used in bone tissue engineering, it provides both
osteoprogenitor cells as well as osteoinductive growth factors, because these transduced
stem cells not only provide their own osteogenic proteins, but also express
osteoinductive factors to induce the osteoblastic differentiation of surrounding cells.
This novel approach has been used for spinal fusion in several animal models. Boden et
al. (Boden, Titus et al. 1998) first employed this combined technique in a rat model. In
their study, bone marrow cells were genetically encoded in vitro with LIM
mineralization protein (LMP-1), and then implanted into rats to induce single-level
posterior fusions. Consistent fusions were obtained in all of the animals treated with
genetically modified bone marrow cells, whereas no bone formation was observed in
controlled animals with implantation with cells carrying an inactive copy of the gene. In
2003, Wang et al. (Wang, Kanim et al. 2003) transduced the BMP-2 gene into
autologous bone marrow cells, in combination with DBM, for induction of
posterolateral spinal fusions in rats. The results demonstrated that animals treated with
BMP-2–producing marrow cells generated solid fusion masses comparable to those
treated with recombinant BMP-2 protein locally. Similar spinal fusion was induced with
buffy-coat cells transduced with the LIM-1 gene in a rabbit model, and all the treated
animals achieved solid fusion (Viggeswarapu, Boden et al. 2001).
1.7.2.3 Potential risk of gene therapy
Although the efficacy of gene therapy has been confirmed in preclinical studies, there
are still several potential issues needing to be resolved before it can be readily applied
37
clinically. The safety of gene therapy is the major concern, especially using a viral
vector. The capability of replication of these viruses has been eliminated by deleting
portions of their genome essential to this process, however, the risk of uncontrollable
infection still exists if viruses can regain the ability to propagate. In addition, the
cost-effectiveness of gene therapy for spinal fusion has not been evaluated and
documented. These safety and economic issues should be thoroughly addressed before
gene therapy can be acceptable as a substitute for autograft in spinal fusion.
1.8 Osteoconductive scaffolds
Osteoconductive materials alone are not considered an effective alternative to autograft,
since they cannot induce efficient bone formation in the absence of osteoinductive
factors or osteogenic cells (Sandhu, Kanim et al. 1995; Cornell and Lane 1998).
However, a suitable osteoconductive scaffold is essential for bone tissue engineering,
especially in critical surgical situations such as spinal fusion.
Numerous materials have been selected or designed as an osteoconductive scaffold for
bone tissue engineering, including allograft, demineralized bone matrix, collagen,
ceramics and biodegradable polymers. However, it is not clear which scaffold may be
optimal for spinal fusion, given the requirements for an ideal scaffold vary and are
based on different fusion environments, including combination with osteoinductive
factors or osteogenic cells (Boden 2001; Erbe, Marx et al. 2001; Louis-Ugbo, Murakami
et al. 2004), the location and placement of bone graft, in intertransverse process or
interbody fusion (Boden, Martin et al. 1999), and the species of animal model (Boden,
Kang et al. 2002; Seeherman, Wozney et al. 2002). In particular, studies revealed that
scaffold materials can profoundly influence the efficacy of BMP in inducing new bone
38
formation induction (Fischgrund, James et al. 1997; Boden 2001; Saito, Okada et al.
2001; Suh, Boden et al. 2002; Akamaru, Suh et al. 2003; Geiger, Li et al. 2003).
The osteoconductive scaffold should provide an optimal environment for the adhesion
and differentiation of osteogenic cells, and guide new bone matrix generation within its
framework. In this section, we will discuss commonly used scaffolds in spinal fusion.
1.8.1 Allograft
Allograft was first applied as an autograft extender in spinal fusion. Based on its porous
structure, it was then used as an osteoconductive scaffold for bone tissue engineering.
Cancellous allograft is favoured because of its osteoconductive, interconnecting porous
structure which promotes new bone formation. The cortical allograft is normally used
for mechanical support and is favoured as an osteoconductive scaffold.
Allograft has shown variable efficacy in spinal fusion compared with autograft. For
example, in a prospective clinical trial, lower fusion outcomes were noted by allograft
implantation than by autograft, 1 year postoperatively (Jorgenson, Lowe et al. 1994).
Similar results were also observed in a another prospective clinical trial conducted by
An et al. (An, Simpson et al. 1995). However, Dodd et al. (Dodd, Fergusson et al. 1988)
reported a 100% fusion rate in patients with idiopathic scoliosis treated with allograft.
Although the role of cancellous allograft as an osteoconductive scaffold is conceptually
reasonable there has been no data reported on it in combination with osteoinductive
factors or osteogenic cells for the induction of spinal fusion.
The concern associated with the use of the allograft in spinal fusion is not negligible.
Given that it is harvested from different individuals, the issues of immunogenicity and
39
disease transmission are always going to be of concern (Asselmeier, Caspari et al. 1993;
Ehrler and Vaccaro 2000), although these risks can potentially be lessened during tissue
processing, preparation and disease screening of donors.
1.8.2 Demineralized bone matrix
Demineralized bone matrix (DBM) was first reported in 1965 as an acid extraction, with
osteoinductivity properties (Urist 1965), as it contains BMPs. Consequently, BDM was
studied as an osteoinductive material to induce spinal fusion in preclinical experiments.
However, conflicting results arose from these studies (Vaccaro, Stubbs et al. 2007;
Alanay, Wang et al. 2008; Urrutia, Thumm et al. 2008). This may be due to differences
in preparation procedures which vary the biological properties of the DBM.
More recently, it was reported that DBM also has osteoconductive properties, related to
the collagenous and noncollagenous proteins left after the demineralization process. The
porous structure that remains also contributes to its osteoconductivity. Martin et al.
(Martin, Boden et al. 1999) studied the osteogenic potential of different formulations of
DBM in a rabbit lumbar intertransverse process fusion model, and observed that the
improved structure of DBM may enhance the osteoconductive and osteoinductive
aspects of bone formation. In addition, DBM, in combination with rhBMP-2, can induce
ectopic bone formation in a mouse model (Niederwanger and Urist 1996). In our study
(Chapter 3), we also reported that natural bone collagen scaffold (NBCS), a form of
DBM, was used as a scaffold for OP-1 to induce spinal fusion in a rabbit model, and
enhanced the new bone formation induced by OP-1 (Yao, Qian et al. 2008). When
combined with bone marrow cells, it also induced superior new bone formation to that
induced by autograft (Chapter 5).
40
1.8.3 Collagen
Collagen, the major nonmineral component of organic bone, is optional as an
osteoconductive scaffold for spinal fusion. Most collagen materials have affinity to
osteoprogenitor cells through specific cell-surface receptors and diffusible signaling
molecules growth factors (Hollinger, Uludag et al. 1998). Furthermore, collagen type I
has also been shown to promote osteoblastic differentiation via the BMP signaling
pathway (Jikko, Harris et al. 1999; Streuli 1999; Wang, Dang et al. 2005). To improve
its osteoconductivity, the three-dimensional porous structure of collagen can be
maintained or reestablished using a variety of chemical or physical techniques during
the manipulation process (Geiger, Li et al. 2003).
Collagen products can be manufactured in various forms. Collagen sponge is the most
commonly used form in bone tissue engineering. Many groups (Boden, Martin et al.
1999; Hecht, Fischgrund et al. 1999; Martin, Boden et al. 1999; Alam, Asahina et al.
2001; Suh, Boden et al. 2002) have studied the efficacy of absorbable collagen spongy
(ACS) as an osteoconductive scaffold (growth factor carrier), in combination with
rhBMP-2, for the induction of spinal fusion in the rabbit intertransverse process fusion
model. The results from these studies suggest that the rhBMP-2/ACS construct yielded
spinal fusion with a high fusion rate and fewer adverse events than the autograft. A
recent integrated analysis of clinical trials by Burkus et al. (Burkus, Heim et al. 2003)
showed that compared with the autograft, rhBMP-2/ACS (IN-FUSE;
Medtronic-Sofamor Danek, Memphis, TN) achieved superior outcomes in terms of
surgery length, blood loss, hospital stay, reoperation rate, median time to return to work
and fusion rates. Combined with growth factors, ACS not only has the capability to
41
retain the growth factors but also to release them at specific rates, thus, maintaining
bioactivity of osteoinductive factors at the fusion site during the fusion (Damien, Grob
et al. 2002; Geiger, Li et al. 2003). This was also confirmed in a rabbit ulna osteotomy
model. ACS maintained rhBMP-2 more effectively than buffer (32% vs. 3%,
respectively) at the implantation site after seven days (Li and Wozney 2001).
Collagen has been selected as a scaffold in cell or gene therapies for spinal fusion. In a
rat spinal fusion model (Miyazaki, Zuk et al. 2008), a collagen sponge containing
human adipose tissue-derived mesenchymal stem cells (HATDMSCs) or human bone
marrow-derived mesenchymal stem cells (HBMDMSCs), transduced with an
adenovirus containing the cDNA for bone morphogenetic proteins (BMP)-2, was
implanted to induce of spinal fusion. The results demonstrated that at eight weeks, all
animals treated with collagen sponge containing cells achieved solid fusion, which
indicated that collagen sponge can be used as a scaffold to deliver cells into fusion site
and maintain the bioactivities of these cells with or without genetic modification.
Collagen sponge was also used as a scaffold for rat bone marrow cells transfected with
bone morphogenetic protein (BMP)-2-containing lentivirus to induce posterolateral
spinal fusion in a rat model (Miyazaki, Sugiyama et al. 2008; Miyazaki, Sugiyama et al.
2008).
1.8.4 Calcium phosphate biomaterials
Calcium phosphate is a type of inorganic osteoconductive material that is the main
mineral composition of organic bone. Calcium phosphate in natural or synthetic forms
closely resembles the microarchitecture of human cancellous bone and has a high
affinity for binding proteins. The commonly used calcium phosphate materials include
42
hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), biphasic calcium phosphate
(combination of TCP and HA), and calcium-deficient apatite forms (Tay, Patel et al.
1999), and these materials have been used for spinal fusion in preclinical studies and
clinical trials.
Studies have demonstrated that pore size is a key parameter in the osteoconductivity of
materials, and the optimal pore size for neovascularization and bone ingrowth is 300μm
to 500μm (Kuboki, Takita et al. 1998; Jahng, Fu et al. 2004), which is comparable to the
pore size in cancellous bone (Jahng, Fu et al. 2004). HA can be manufactured with
different pore size, for example, ProOsteon 200 and ProOsteon 500 (Interpore Cross,
Irvine, CA). ProOsteon 200 provides greater mechanical support, whereas ProOsteon
500 is superior in terms of its osteoconductive properties. Several investigations have
assessed the role of HA as a scaffold, in combination with BMPs, or stem cells, for the
induction of spinal fusion. Using a rabbit spinal fusion model, HA combined with
rhBMP-2 was implanted into the intertransverse process space to induce new bone
formation. At six to nine weeks, mineralization was observed using dual X-ray
densitometer in the porous structure of HA pellets (Ono, Gunji et al. 1995). Porous HA
was also used as an osteoconductive scaffold for cell therapy. In 1999, Chistolini et al.
(Chistolini, Ruspantini et al. 1999) implanted autologous bone marrow stromal cells
combined with porous HA to induce new bone formation for repair of long bone defects.
Results showed that a combination of HA and bone marrow stromal stem cells achieved
higher mechanical stiffness than scaffold alone.
Beta-TCP is another type of calcium phosphate used as an osteoconductive scaffold for
bone tissue engineering. In a canine lumbar spine model, Ohyama et al. (Ohyama, Kubo
et al. 2004) assessed the efficacy of the combination of β-TCP and rhBMP-2 for
43
induction of new bone formation, and showed it yielded superior results in terms of
trabecular bone area and volume, percentage of trabecular bone formation and
mechanical stiffness compared to the autograft. Β-TCP has also been selected as a
scaffold in cell therapy for spinal fusion. Orii er al. (Orii, Sotome et al. 2005) assessed
the efficacy of β-TCP/MSCs hybrid as an inducer of spinal fusion in a macaque lumber
posterolateral fusion model. Superior spinal fusion induced by the β-TCP/MSCs hybrid
to that by autograft was detected by manual palpation, radiography, micro computed
tomography, peripheral quantitative computed tomography, and histology. More recently,
β-TCP combined with bone marrow cells has also been used in clinical trials of spinal
fusion and achieved encouraging outcomes (Gan, Dai et al. 2008).
1.8.5 Biodegradable polymers
Biodegradable polymers were first used as suture materials, since they are to be
degraded after the sutured tissue heals. With the development of manufacturing
techniques, additional forms of biodegradable polymers have been designed and used as
scaffold materials in bone tissue engineering. When biodegradable polymers are
combined with growth factors, factors can be released slowly as the polymers degrade
(Alpaslan, Irie et al. 1996; Hollinger and Leong 1996; Uludag, Norrie et al. 2001;
Kuboki, Kikuchi et al. 2003; Vaccaro, Singh et al. 2003). The commonly used
biodegradable polymers included polylactic acid (PLA), polylactic/glycolic acid (PLGA)
and polyglycolic acid (PGA).
For several years biodegradable polymers have been widely used in bone tissue
engineering, including in the treatment of long bone defects (Murakami, Saito et al.
2003; Yoneda, Terai et al. 2005; Matsushita, Terai et al. 2006) and cranial defects
44
(Suzuki, Terai et al. 2006). The use of PLGA, in combination with rhBMP-2, in a canine
model of spinal fusion, induced greater bone formation than the autograft (Fischgrund,
James et al. 1997). Biodegradable polymers can be made into injectable forms based on
their temperature-sensitive properties, facilitating minimally invasive spinal fusion
procedures (Saito, Okada et al. 2003). However, there are no reports on the use of
biodegradable polymers in cell therapy or gene therapy.
A limited number of issues are associated with the use of degradable polymers. It has
been reported that PLA led to aggressive foreign body giant cell reaction (Hollinger,
Uludag et al. 1998). High molecular weight polymers may also result in inflammatory
response (Kirker-Head 2000). In addition, the breakdown products of these polymers
might influence the environment at the application site by changing the local pH value
(Lu, Peter et al. 2000).The summary of scaffolds used for spinal fusion is listed in Table
1.4.
Table 1.4 Summary of scaffolds used for preclinical and clinical spinal fusion Products Formulation Category Advantage Disadvantage Conjunction
with Species Outcomes References
Allograft Particle Natural polymers
Bioactivities and porous structure
Diseases transmission
DBM and autograft
Rabbit and human
Allograft + DBM (49/63), autograft (39/59)
(An, Simpson et al. 1995; Urrutia, Thumm et al. 2008)
DBM Flowable gel and putty
Natural polymers
Bioactivities and porous structure
Immunological reaction
Autograft and allograft
Rabbit amd human
Conflicting fusion rate 33.3 -100%
(Martin, Boden et al. 1999; Urrutia, Thumm et al. 2008; Weinzapfel, Son-Hing et al. 2008)
Collagen Sponge and sheet
Natural polymers
Potent bioactivities
Weak mechanical strength
rhBMP-2 Rabbit (pseudarthrisis) and macaque
collagen + BMP-2 (15/17), autograft (5/17) or lower dose BMP-2 required
(Barnes, Boden et al. 2005; Kraiwattanapong, Boden et al. 2005; Lawrence, Waked et al. 2007)
HA Mesh and compact body
Inorganic material
Immunological inertia, biodegradability
Weak mechanical strength
rhBMP-2 Rat, rabbit and human
Mesh (5/5), compact body (0/4)
(Boden, Kang et al. 2002; Konishi, Nakamura et al. 2002; Morisue, Matsumoto et al. 2006)
Beta-TCP Porous block
Inorganic material
Immunological inertia, biodegradability
Weak mechanical strength
MSCs and Enriched bone marrow cells
Macaque, Sheep and Human
Clinical lumbar fusion rate (39/41)
(Orii, Sotome et al. 2005; Becker, Maissen et al. 2006; Gan, Dai et al. 2008)
45
46
Table 1.4 (continuous) Summary of scaffolds used for preclinical and clinical spinal fusion
Products Formulation Category Advantages DisadvantagesConjuncti
on with Species Outcomes References
PLGA Slide and block
Synthetic polymer
Controlled microstructure
Inflammatory response
rhBMP-2 Dog PLGA was associated with more void formation compared with collagen
(Muschler, Hyodo et al. 1994; Fischgrund, James et al. 1997)
PLA Injectable fluid and block
Synthetic polymer
Avoiding the surgical invasion
Giant reaction rhBMP-2 Mouse, rabbit and dog
Ectopic and orthotopic boneformation by injection. Rabbit fusion rate (2/5), and dog (4/9)
(Saito, Okada et al. 2003; Takahashi, Saito et al. 2003; Namikawa, Terai et al. 2005)
Collagen + HA Porous sponge
Composite Porous structure and bioactivity
Complicity of preparation
MSCs + rhBMP-2
Rabbit And macaque
Fusion rates varied 92 - 100%
(Suh, Boden et al. 2002; Fu, Chen et al. 2009)
Collagen + TBC Porous particle
Composite Porous structure and bioactivity
Various quality of TBC
rhBMP-2 Rabbit Scaffold + BMP (5/5),autograft (3/7)
(Minamide, Tamaki et al. 1999; Minamide, Kawakami et al. 2001)
47
1.9 Summary and future directions
Spinal fusion, which has been performed for more than a century, is still playing an
important role in the treatments of various spinal disorders. Numerous innovations have
been developed with respect to instruments, approaches, devices and materials to
improve the safety and effectiveness. One of the most significant innovations is the
development of substitutes for the autologous bone graft, which is available only in
limited quantities and is associated with considerable morbidity. Recently, significant
achievements have been made in the design and development of various bone
substitutes via bone tissue engineering means. These have demonstrated potent
potential for clinical application.
Although most newly developed substitutes have been confirmed to be capable of
promoting spinal fusion in preclinical studies and clinical trials, the safety of the use of
these materials is still to be addressed before they can be readily applied in clinical
situations. Furthermore, randomized, controlled clinical trials should be performed to
confirm the efficacy, safety and cost-effectiveness of these existing strategies, before
they can be applied to spinal procedures.
CHAPTER 2
RESEARCH PROPOSAL
49
2.1 Rationale
Spinal fusion is a common surgical procedure involving the combination of two or more
vertebrae, to treat spinal disorders resulting from degenerative changes, trauma or
tumour. Spinal fusion is ranked as the second most common spinal procedure performed
worldwide, with approximately 46,500 lumbar spinal fusion procedures performed each
year in the United States (Frymoyer, Matteri et al. 1978). The use of spinal-fusion
surgery in the United States is rapidly increasing. National survey data indicates that the
annual number of spinal-fusion operations rose by 77 percent between 1996 and 2001
(Stromqvist 2002). In contrast, hip replacement and knee arthroplasty only increased by
13 to 14 percent during the same interval. Spinal fusion surgery is expensive, with the
average hospital bill mounting to more than $34,000 (Stromqvist 2002). Similarly in
Australia, spinal fusion procedures place a significant socioeconomic burden on both
the individual and the community. Information obtained from this study will provide
valuable information to improve the treatment regime for injury or degeneration to the
lumbar spine, and possibly provide economic and health benefits to the patient and the
health care system.
The goal of the spinal fusion procedure is to obtain a solid bony fusion between two or
more vertebrae with infilled graft materials (Edwards, Zura et al. 2003). There are
basically two spinal fusion approaches, intertransverse process fusion and interbody
fusion. The intertransverse process fusion involves implantation of the bone graft
between the transverse processes in the back of the spine, while interbody fusion
50
involves implantation of the bone graft between the vertebral bodies in the area usually
occupied by the intervertebral disc. The process of intertransverse process fusion is an
easier approach than interbody fusion, although interbody fusion does provide a proven
logical solution to diseases of the intervertebral discs by eliminating motion of the
segment (Vishteh, Crawford et al. 2005; Sohn, Kayanja et al. 2006).
In either intertransverse process fusion or interbody fusion, graft material is infilled into
the fusion site to induce new bone formation and to prevent ingrowth of soft tissue. The
most commonly used graft material for spinal fusion is the autologous bone graft, bone
tissue that is harvested from the same individual and reimplanted at a different site. The
main source of autograft is the iliac crest, since it can provide both cortical and
cancellous bone tissue. Iliac crest is commonly selected as the donor site based on its
superficial location with little influence on the stability of the skeletal system
(Arrington, Smith et al. 1996). Other autograft sources include the proximal tibia, the
fibula and the rib.
The process of bone regeneration or new bone formation requires three critical
elements: osteogenic cells that have the capacity to synthesize new bone, osteoinductive
factors (ie, growth factors and cytokines) that promote the osteoblastic differentiation of
pluripotent stem cells, and an osteoconductive scaffold that facilitates
neovascularization and supports the ingrowth of bone. Autogenous bone possesses all
three of these properties essential for bone formation and is therefore considered the
51
“gold standard” as a graft material for spinal fusion (Arrington, Smith et al. 1996;
Sandhu, Grewal et al. 1999).
Although autogenous bone represents the most effective method of generating a solid
fusion and is presently the most common graft substance used in spinal surgery, its
procurement results in more extensive surgical trauma and frequently necessitates a
separate operative incision. Besides increasing operative time and blood loss, this
process is also associated with considerable donor site morbidity; up to 30% of all
patients undergoing the harvesting of iliac crest bone graft will experience
complications postoperatively, including infection, hematoma, nerve or vascular injury,
fracture, persistent pain, abdominal herniation and pelvic instability (Reid 1968;
Cowley and Anderson 1983; Kuhn and Moreland 1986; Kurz, Garfin et al. 1989;
Summers and Eisenstein 1989; Younger and Chapman 1989; Fernyhough, Schimandle
et al. 1992; Banwart, Asher et al. 1995; Goulet, Senunas et al. 1997). The amount of
autograft available for transplantation may also be insufficient in children as well as
adults requiring revision surgery or fusion of multiple spinal segments.
To address these limitations, significant research has focused on substitutes for the
autograft in bone tissue engineering and has included using osteoinductive growth
factors or osteogenesis cells to induce spinal fusion.
Recombinant human BMP-7, also known as osteogenic protein-1 (OP-1), is currently in
commercial development (Stryker Biotech, Hopkinton, MA). Although OP-1 has been
extensively evaluated and has demonstrated efficacious results in a variety of
52
applications (Cook, Salkeld et al. 2005) (Friedlaender 2001; Cunningham, Shimamoto
et al. 2002; Maniscalco, Gambera et al. 2002), OP-1 has not been thoroughly studied in
the lumbar spine. Cook et al. (Cook, Salkeld et al. 2005) performed an inter-transverse
process fusion study with OP-1 in a dog model and demonstrated fusion occurred more
rapidly with OP-1 than without. Furthermore, Grauer et al (Grauer, Patel et al. 2001)
performed an inter-transverse process fusion study with OP-1 in a rabbit model which
showed OP-1 reliably induced solid inter-transverse process fusion at 5 weeks, but was
tarnished by a high mortality rate. This thesis will focus on the efficacy of OP-1, in
combination with a suitable osteoconductive scaffold, to induce new bone formation in
a spinal fusion animal model.
Bone marrow cells (BMCs), containing mesenchymal stem cells (Gan, Dai et al. 2008),
which have pluripotent potential to differentiate into multiple mesenchymal lineages,
including osteoblasts, chondrocytes or adipose cells (Jorgensen, Djouad et al. 2008), are
now also being used for bone tissue engineering applications, including cartilage repair,
tendon healing and spinal fusion (Friedenstein, Gorskaja et al. 1976; Muraglia,
Cancedda et al. 2000; Colter, Sekiya et al. 2001; Pochampally, Smith et al. 2004; Gan,
Dai et al. 2008; Koga, Tokuhashi et al. 2008; Miyazaki, Zuk et al. 2008).
In combination with growth factors or BMCs, an osteoconductive scaffold is also
essential to provide a suitable environment for cell differentiation and bone matrix
generation. An optimal osteoconductive scaffold provides a suitable environment for
cells’ migration, attachment and differentiation into osteoblasts (Meijer, de Bruijn et al.
53
2007). The framework of the scaffold also guides the synthesis of the extracellular
matrix in a 3-D manner, and mediates integration of new tissue with host bone tissue
(Karageorgiou and Kaplan 2005).
A variety of biomaterials have been utilized as osteoconductive scaffolds in orthopaedic
surgery, and range from inorganic to organic materials including hydroxyapatites
(Blattert, Delling et al. 2002) and collagen putties (Grauer, Patel et al. 2001). However,
all have disadvantages which limit their use. Hydroxyapatite is a brittle material with a
low fracture resistance and is difficult to shape to bone defects (Barboza, Duarte et al.
2000). Additionally, biodegradation of hydroxyapatites is exothermic which may be
detrimental to the osteogenic cells that are attached to it (Caiazza, Colangelo et al.
2000). Collagen putty can serve as a carrier for growth factors but lacks osteoconductive
potential owing to the absence of a porous structure. Currently, a novel osteoconductive
scaffold, natural bone collagen scaffold (NBCS), also named insoluble bone gelatin
(ISBG), has been developed for bone tissue engineering. This material is prepared by
acid extraction of the mineralized bone components, and retains the porous structure
and collagen component of organic bone. The osteoconductivity of NBCS has not been
characterized, and the efficacy of NBCS combined with autologous BMCs to induce
new bone formation in a spinal fusion animal model has not been evaluated. Thus, the
other focus of this thesis is to evaluate the use of a novel osteoconductive scaffold to
support and promote new bone formation that is induced by autologous BMCs in vitro
and in vivo.
54
Taken together, the scope of this thesis is to characterize the osteoconductive properties
of NBCS, and the efficacy of NBCS combined with OP-1 or autologous BMCs for
induction of new bone formation in a rabbit intertransverse process fusion model and
ovine interbody fusion model.
2.2 Hypotheses
1. NBCS is an effective osteoconductive scaffold with a porous structure and
component which promotes new bone formation.
2. The use of NBCS combined with OP-1 in a rabbit model of spinal fusion will
enhance fusion rate, and biomechanical stiffness more than with the use of the
conventional autograft.
3. NBCS will enhance new bone formation induced by OP-1 in vivo, with higher bone
volume and acceleration of ossification process.
4. NBCS will support population expansion and osteogenesis differentiation of ovine
BMCs and guide the new bone matrix generation and mineralization in vitro.
5. NBCS combined with autologous BMCs will induce greater interbody spinal fusion
in an ovine model than the autograft.
2.3 Aims
Therefore, the aims of this project are to:
55
1. Evaluate the efficacy of NBCS in combination with OP-1, as a graft that induces
bone formation of spinal fusion in a rabbit model by measuring fusion rate,
biomechanics, bone volume, and gene expression.
2. Assess the efficacy of NBCS to support population expansion, osteogenesis
differentiation of ovine BMCs, and the new bone matrix generation and induction
osteogenesis by ovine BMCs in NBCS in vitro.
3. Evaluate the efficacy of NBCS in combination with ovine BMCs, as a graft that
induces bone formation of spinal fusion in an ovine model by measuring fusion rate,
biomechanics, bone volume, and gene expression.
Chapters 3, 4 and 5 could not be included in this digital thesis for copyright reasons.
Please refer to the print copy of the thesis, held in the University Library.
CHAPTER 6
GENERAL DISCUSSION
109
6.1 Discussion
Spinal fusion is a surgical technique used to combine two or more vertebrae with newly
formed bone tissue. The autologous bone graft has been used for spinal fusion for
decades as it has essential elements to induce new bone formation, including
osteoinductivity, osteoconductivity and a source of osteogenic cells. Significant
limitations and complications are associated with harvesting the autograft, including
increased operative time and blood loss, donor site complications, limited quantity and
various qualities (Kuhn and Moreland 1986; Banwart, Asher et al. 1995; Arrington,
Smith et al. 1996; Goulet, Senunas et al. 1997; Seiler and Johnson 2000; Skaggs,
Samuelson et al. 2000). Therefore, significant research now focuses on the application
of bone tissue engineering in spinal fusion to improve success rate and address
limitations (Morone and Boden 1998; Boden 2002; Carlisle and Fischgrund 2005;
Gupta, Theerajunyaporn et al. 2007; Handoll and Watts 2008).
At the fusion site, host bone can provide osteoinductive growth factors and osteogenic
cells from the bone marrow of decorticated transverse processes or vertebral bodies.
However, the low concentration and limited quantity of local growth factor and
osteogenic cells often do not induce sufficient new bone formation for spinal fusion. As
a result, spinal fusion procedures also combine osteoinductive factors (Boden, Martin et
al. 1999; Hecht, Fischgrund et al. 1999; Martin, Boden et al. 1999; Muschik, Schlenzka
et al. 2000; Erulkar, Grauer et al. 2001; Minamide, Kawakami et al. 2001; Cunningham,
Shimamoto et al. 2002; Alini, Li et al. 2003; Carlisle and Fischgrund 2005) or enriched
110
osteogenic cells (Friedenstein, Gorskaja et al. 1976; Muraglia, Cancedda et al. 2000;
Colter, Sekiya et al. 2001; Pochampally, Smith et al. 2004; Gan, Dai et al. 2008; Koga,
Tokuhashi et al. 2008; Miyazaki, Zuk et al. 2008) to enhance the new bone formation at
the fusion site. Additionally, a suitable osteoconductive scaffold is an essential
element, providing a suitable environment for cellular migration, attachment and
osteogenic differentiation (Meijer, de Bruijn et al. 2007). The framework of the scaffold
can guide the synthesis of the extracellular matrix in a 3-D manner, and mediate
integration of new tissue with host bone tissue (Karageorgiou and Kaplan 2005). In this
thesis, NBCS was evaluated as an osteoconductive scaffold to promote new bone
formation in spinal fusion animal models. This study is the first to show that NBCS may
serve as a stable osteoconductive scaffold, and in combination with OP-1 or autologous
BMCs, may offer a potential alternative to the traditional autograft.
Using a rabbit intertransverse process fusion model, the efficacy of NBCS, combined
with OP-1, for induction of new bone formation was evaluated (Chapter 3), and the
results demonstrated that the combination of NBCS with OP-1 improved spinal fusion
by enhancing spinal fusion rate, biomechanical stiffness and bone volume at the fusion
site when compared to the autograft. The osteoinductivity of OP-1 has been studied in
vitro (Cheifetz, Li et al. 1996; Yeh and Lee 1999) and in vivo (Bright, Park et al. 2006;
Tsiridis, Ali et al. 2007), but has had limited potential in spinal fusion procedures due to
the lack of a suitable osteoconductive scaffold. Paramore et al. (Paramore, Lauryssen et
al. 1999) reported overgrowth of bone and spinal stenosis induced by leakage of OP-1,
as a result of the lack of an effective scaffold which would retain and localize the
111
growth factors in situ. In addition, clinical trials of spinal fusion using OP-1 putty have
demonstrated variable results. Vaccaro et al. (Vaccaro, Stubbs et al. 2007) reported that
the fusion rate induced by OP-1 was 68.8% (11 of 16), which was only a little higher
than that achieved with autograft (50%, 3 of 6). Furthermore, a clinical trial led by
Jeppsson (Jeppsson, Saveland et al. 1999) was ceased because the first four patients
failed to reach fusion. Results from our rabbit spinal fusion model indicated that NBCS
is a suitable scaffold for OP-1, and is able to enhance the efficiency of new bone
formation induced by OP-1.
To provide further mechanistic insight into the osteoconductive potential of NBCS in
bone tissue engineering, the microstructure of NBCS was characterized and it was
revealed that NBCS did exhibit ideal properties that are required of an osteoconductive
scaffold. A porous structure and bioactivity are two key elements in the design and use
of osteoconductive materials (Kruyt, Dhert et al. 2004; Okamoto, Dohi et al. 2006; Li,
Habibovic et al. 2007). NBCS retains the original porous structure of organic bone and
therefore exhibits suitable pore size, porosity and interconnection to provide an optimal
environment for the population expansion and differentiation of MSCs (Karageorgiou
and Kaplan 2005). Secondly, NBCS retains the main collagen type I component from
organic bone which has been shown to promote osteoblastic differentiation via the BMP
signalling pathway (Jikko, Harris et al. 1999; Streuli 1999; Yang, Bhatnagar et al.
2004).
112
Osteogenic cells are readily utilized in bone tissue engineering applications to induce
new bone formation. For example, bone marrow cells (BMCs), containing
mesenchymal stem cells, which have pluripotent potential to differentiate into multiple
mesenchymal lineages (Jorgensen, Djouad et al. 2008), have been commonly used for
bone tissue engineering applications, such as experimental spinal fusion and long bone
defect repair (Friedenstein, Gorskaja et al. 1976; Muraglia, Cancedda et al. 2000;
Colter, Sekiya et al. 2001; Pochampally, Smith et al. 2004; Gan, Dai et al. 2008; Koga,
Tokuhashi et al. 2008; Miyazaki, Zuk et al. 2008).
Although osteoconductive materials alone are not considered an effective alternative for
the autograft (Sandhu, Kanim et al. 1995; Cornell and Lane 1998), their use together
with osteogenic cells is crucial in bone tissue engineering to provide an optimal
environment for new bone formation. In other words, the scaffold should provide a
permissive environment which allows bone cells to migrate into, and deposit bone
matrix in, the scaffold template. This study selected autologous BMCs as a cell source
and NBCS as an osteoconductive scaffold to induce spinal fusion (Chapter 5). In vitro
results showed that ovine BMCs displayed the capacity of population expansion and
were able to express collagen type I and produce mineralized matrix within the NBCS.
In vivo results showed that NBCS combined with BMCs enhanced spinal fusion and
was associated with a higher fusion rate, greater biomechanical stiffness and larger bone
volume than in all control groups. Histological findings also revealed that the newly
formed bone was well integrated with host bone tissue. Taken together, the results from
this study showed that NBCS supported the population expansion and osteogenic
113
differentiation of ovine BMCs and guided the bone matrix generation in vitro and in
vivo.
This thesis focused on the evaluation of the efficacy of NBCS, a novel biomaterial as an
osteoconductive scaffold in spinal fusion models. The encouraging results from this
thesis demonstrate that the combination of NBCS and growth factor or bone marrow
cells could be a competitive alternative of tissue engineering approaches for spinal
fusion. Previously, several studies have tested the use of biomaterials (Boden, Martin et
al. 1999; Hecht, Fischgrund et al. 1999; Martin, Boden et al. 1999; Alam, Asahina et al.
2001; Suh, Boden et al. 2002) or inorganic materials (Chistolini, Ruspantini et al. 1999;
Ohyama, Kubo et al. 2004; Orii, Sotome et al. 2005) as scaffold in bone regenerations,
and some of them also used animal spinal fusion models (David, Gruber et al. 1999;
Boden, Kang et al. 2002; Cunningham, Shimamoto et al. 2002). However, the outcomes
from these studies are various, and it might lay in the lack of an optimal
osteoconductive scaffold to support osteogenic differentiation and to guide the new
bone matrix generation. The results from current studies strongly indicated the potent
potential of NBCS for clinical spinal fusion applications.
There are several limitations in this project. Firstly, human NBCS was used as an
implantation material in a rabbit and ovine model. Although the xenograft material is a
collagen matrix and does not contain living cells, relevant immunological response may
still exist and potentially influences the results. Secondly, two animal models were used
to test the efficacy of NBCS, therefore, the species difference should be cautiously
114
considered before NBCS can be used in clinical applications. For example, an
inflammatory response to OP-1 (Chapter 3) has not been reported in previous clinical
trials. Human collagen I within NBCS demonstrated weaker positive
immunohistochemistry than ovine collage I derived by ovine bone marrow cells
(Chapter 5). These might be related to species difference, which potentially leads to
unnecessarily consensus outcomes from human and animal models. In addition, this
thesis concentrated on spinal fusion outcomes using bone tissue engineering
approaches, so the study on the relevant mechanisms was relatively weak. Although
using available technology, studying molecular mechanisms using a scaffolding
material is difficult. Additional in vitro studies, such as the release kinetics of OP-1
from NBCS may enhance understanding of interaction between scaffolds and growth
factors.
In summary, the results from this thesis suggest that NBCS is an efficient
osteoconductive scaffold that, when combined with OP-1 or autologous BMCs, can be
used as a substitute for the autograft to induce new bone formation in spinal fusion
procedures.
6.2 Future Directions
Work from this dissertation has led to the suggestion that NBCS is a potent
osteoconductive scaffold with potential use in clinical bone tissue engineering
applications. However, a number of issues regarding the characterization and potential
115
clinical applications of this novel osteoconductive scaffold remain unclear, and could
not be addressed in this thesis because of time constraints. Additional research is
required to investigate the clinical potential of NBCS, including the suitability of NBCS
in treating other orthopaedic procedures, the efficacy of NBCS and other scaffolds, and
the extent of availability of NBCS for clinical usage.
Although the efficacy of NBCS as an osteoconductive scaffold in spinal fusion animal
models has been evaluated in this thesis, there are additional surgical situations, such as
long bone defect repair and nonunion healing, which require the development and
availability of additional osteoconductive scaffolds which induce new bone formation.
Animal models of long bone defect (Knothe Tate, Ritzman et al. 2007; Viateau,
Guillemin et al. 2007) and nonunion fractures (Schmidhammer, Zandieh et al. 2006;
Kaspar, Matziolis et al. 2008) have been well established, and have been used to test the
efficacy of bone grafts/scaffolds. NBCS combined with either growth factors or BMCs
might have the potential to be used in these models. In addition, given NBCS has only
been tested as a scaffold for BMCs in a sheep model, the efficacy of NBCS as a scaffold
for human bone marrow cells needs to be evaluated. Since cell behavior does vary
between species assessing the interaction of NBCS and human cells will be important to
determine the clinical suitability of NBCS.
This thesis has already evaluated the efficacy of NBCS, combined with OP-1 or BMCs,
to induce formation in animal models of spinal fusion. However the interaction between
the scaffold, growth factor and BMCs still needs to be further investigated. For example,
116
an optimal scaffold/carrier, when combined with growth factors, may retain and release
growth factors slowly, thus maintaining sufficiently high concentration of growth
factors at the application site during the process of new bone formation. There are
basically two major mechanisms by which a scaffold may retain and release growth
factors: physical hindrance and chemical interaction (Duggirala, Mehta et al. 1996;
Uludag, Friess et al. 1999). Investigation of pharmacokinetics of growth factor released
from NBCS may provide an insight into the mechanism of how NBCS improves the
osteoinductivity of osteoinductive growth factors. In addition, the dose of OP-1 used in
rabbit model was designed based on the relevant literatures (Grauer, Patel et al. 2001;
Jenis, Wheeler et al. 2002). Whether the osteogenesis effect is dose dependent has not
been included in this thesis. Future dose dependent study will be useful to determine the
optimal dose for spinal fusion in clinical applications.
Although NBCS has been shown to be an efficient osteoconductive scaffold, combined
with BMCs, for induction of new bone formation in an ovine spinal fusion model, the
molecular mechanism by which NBCS promotes osteogenic differentiation of BMCs
has not been revealed. Further investigation of the signaling pathways activated by
NBCS would be interesting to characterize the molecular properties of NBCS. In
addition, tracking cellular migration, adhesion and differentiation within NBCS could
also detail the interaction between the scaffold and cells. The findings from these
studies would provide crucial information that may improve the design and manufacture
of NBCS.
117
Currently, materials such as collagen type I sheet and tricalcium phosphate have been
studied as scaffolds for bone tissue engineering in pre-clinical experiments and clinical
trials (Schumann, Ekaputra et al. 2007; Gan, Dai et al. 2008). The results from the
studies above indicate that these are favorable selections for use as an osteoconductive
scaffols in bone tissue engineering. A comparison of the physical and biological
properties and efficacy in vitro and in vivo, between NBCS and these scaffolds, may
provide valuable information for different clinical situations, as an aid to designing and
selecting an appropriate scaffold.
As a “type” of allograft, NBCS is associated with the potential risk of disease
transmission. Although entire donor materials have been screened for transmitted
diseases including HIV, HBV, HBC, HTLV, etc, and the preparation process of NBCS
eliminates pathogens, it will be valuable to investigate the potential approach in using
NBCS to further reduce the risk of disease transmission. In addition, the supply of the
NBCS is limited by donor availability. On the other hand, if animal derived NBCS
could be developed, the quantity limitation of NBCS from human sources could be
addressed. However, the risk of anthropo zoonosis transmission should be considered if
using animal sources of NBCS.
To eliminate the risk of disease transmission completely from the use of scaffolds,
artificial recombination may be a potential approach. The development of
nanotechnology has opened up new avenues in scaffold design, although there is still a
long way to go before an osteoconductive bone collagen scaffold is available clinically.
118
Furthermore, as a potential product for clinical application, there are some relevant
issues to be solved before NBCS can be made clinically available, including extension
of its shelf life, cost-effectiveness, malleability to fit the shape needed and user-friendly
availability.
In summary, further work to better characterize NBCS and to fully investigate the
mechanism of interaction between NBCS, growth factors and BMCs, as well as to
assess and develop NBCS for clinical applications, would bring the work from this
thesis to a fruitful conclusion and more importantly, facilitate the translation of basic
science research into future clinical applications.
APPENDIX A
MATERIALS AND METHODS
120
1.1 Materials
1.1.1 Animals
All the animals involved were approved for breeding after application to the
University of Western Australia (UWA) Animal Ethics Committee (AEC)
(RA/3/100/485 for the rabbit project and RA/3/100/579 for the sheep project). All
operative procedures and cage activities were conducted under strict guidelines detailed
by the National Health and Medical Research Council (NHMRC).Rabbits were bred
from a colony maintained in the M-block animal house facility, QEII Medical Campus,
Nedlands, and ewes were housed in the Large Animal Facility, Main Campus, Crawley.
Thirty-two outbreed Albino New Zealand White (oryctolagus cuniculis) rabbits were
used in this project. All rabbits were 52 weeks old at surgery. Adult weight was 3.5-4.5
kg in females. Rabbits were fed ad libitum with rabbit pellets, hay, and provided water
ad libitum. Rabbits were held in cages measuring 1.5m width by 0.75m length by 0.75m
height, with grid floors to prevent pododermatitis.
Thirty-six sheep were used in this project. Female sheep, around 2-year old with
average weight of 58.4 kg (range 54.5 kg ~64.2 kg) were obtained for interbody lumbar
fusion surgery. All sheep were fed with hay, and provided water ad libitum. The cages
for housing measured 2.0m widths by 1.0 m length by 1.2 m height.
121
1.1.2 Surgical instruments
Instruments Manufactory
Bone rongeurs (2 joints) Renli Orthopaedic Inc. Tianjing, China
Bone rongeurs (3 joints) Renli Orthopaedic Inc. Tianjing, China
Bone cutting forceps (18’’) Renli Orthopaedic Inc. Tianjing, China
Bone files Medical Instrument Inc. Shanghai, China
Bone wax (W810) Ethicon, NJ, USA
Caliper Measurement Instrument, Hangzhou, China
Chisels (22) Renli Orthopaedic Inc. Tianjing, China
Dermabond (DHV12) Ethicon, NJ, USA
Disc curettes (25’’) Medical Instrument Inc. Shanghai, China
Drainage bottle Renli Orthopaedic Inc. Tianjing, China
Dressing forceps Medical Instrument Inc. Shanghai, China
Electric drill (set) Renli Orthopaedic Inc. Tianjing, China
Elevators Medical Instrument Inc. Shanghai, China
Gouges Medical Instrument Inc. Shanghai, China
Hemostatic forceps (S) Medical Instrument Inc. Shanghai, China
Hemostatic forceps (meeker) (25’’, S) Medical Instrument Inc. Shanghai, China
Hemostatic forceps(spencer) Medical Instrument Inc. Shanghai, China
Ligature Suture (POLYsorb, L-12) Syneture Inc. MA, USA
Keiss (22) Medical Instrument Inc. shanghai, China
122
K-wires 1.5mm Renli Orthopaedic Inc. Tianjing, China
K-wires 2.0mm Renli Orthopaedic Inc. Tianjing, China
Needle holder Medical Instrument Inc. Shanghai, China
Needle holder (micro-slim) Medical Instrument Inc. Shanghai, China
Operating scissors (slim) Medical Instrument Inc. Shanghai, China
Probe Renli Orthopaedic Inc. Tianjing, China
Raspatories (23) Renli Orthopaedic Inc. Tianjing, China
Retractors (S) Medical Instrument Inc. Shanghai, China
Retractors (M) Medical Instrument Inc. Shanghai, China
Retractors (L) Medical Instrument Inc. Shanghai, China
Scalpel handle (3'') Medical Instrument Inc. Shanghai, China
Scalpel handle (2'') Medical Instrument Inc. Shanghai, China
Self-retaining retractors Medical Instrument Inc. Shanghai, China
Sharp curettes (21) Renli Orthopaedic Inc. Tianjing, China
Spinal abdominal retractors Renli Orthopaedic Inc. Tianjing, China
Steinmann nails Renli Orthopaedic Inc. Tianjing, China
Suction instruments frazier (20) Medical Instrument Inc. Shanghai, China
Suction instruments frazier (S) Medical Instrument Inc. Shanghai, China
Suture (J466H) Ethicon, NJ, USA
Suture (J304H) Ethicon, NJ, USA
Tissue forceps Medical Instrument Inc. Shanghai, China
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1.1.3 Animal drugs
Acepromazine (10mg) Reckitt & Colman, NSW, Australia
Aspirin (300mg) Reckitt & Colman, NSW, Australia
Buprenorphine Hydrochloride (0.3mg/ml) Reckitt & Colman, NSW, Australia
Diazepam (5mg/ml) Parke-Davis, Auckland,NZ
Halothane Zeneca, United Kingdom
Isofluorane (100mg/ml) Parke-Davis, Auckland, NZ
Ketamine Hydrochloride (100mg/ml) Parke-Davis, Auckland, NZ
Sodium Pentobarbitone (325mg/ml) Virbac, NSW, Australia
Xylazine (20mg/ml) Troy Laboratories, NSW, Australia
1.1.4 Chemical reagents
All chemical reagents used during the course of this experiment were of analytical grade
and were obtained through the following manufacturers.
Reagent Manufactory
Acetic Acid Merck, Victoria, Australia
Acetic Acid (3%) BDH, Poole, England
Agarose (molecular biology grade) Promega Corp., Madison, WI., USA
Ampicillin Sigma Chemical Co., St Louis, Mo., USA
Alcian Blue BDH, Poole, England
124
Aluminium Sulphate BDH, Poole, England
Aminopropyltriethoxy-saline Sigma, St Louis, USA
Bovine Serum Albumin (BSA) Boehringer, Manheim, Germany
Cacodylate BDH, Poole, England
Chloroform Merck, Victoria, Australia
Crystal Thymol BDH, Poole, England
D-MEM/F-12 Medium Gibco, New York, USA
Decon (2%) Decon Laboratories, England
Depex Mounting Medium Merck, Victoria, Australia
Dimethyldicarbonate Sigma, St Louis, USA
Eosin Merck, Victoria, Australia
Ethanol (100%) Merck, Victoria, Australia
Ethanol Spray Pharmacia & Upjohn, Australia
Ethylene Glycol BDH, Poole, England
Foetal Bovine Serum Trace, Sydney, Australia
Formic Acid Merck, Victoria, Australia
Glacial Acetic Acid APS Finechem, NSW, Australia
Glutaraldehyde (25%) TAAB, England, UK
Haematoxylin Sigma, Steinheim, Germany
Hydrochloric Acid Merck, Victoria, Australia
L- Ascorbic Acid Gibco, New York, USA
Magnesium Sulphate BDH, Poole, England
Neutral Red BDH, Poole, England
125
Osmium Tetroxide ProSciTech, Qld, Australia
Paraffin Wax Oxford Laboratories, USA
Paraformaldehyde Merck, Darmstadt, Germany
Penicillin-Streptomycin (2x) ICN Biomedicals, Ohio, USA
Safranin (0.5%) BDH, Poole, England
Sodium Chloride BDH, Poole, England
Sodium Iodate BDH, Poole, England
Sodium Bicarbonate BDH, Poole, England
Sodium Phosphate (dibasic) BDH, Poole, England
Sodium Phosphate (monobasic) BDH, Poole, England
Sterile DD Water Baxter, NSW, Australia
Sucrose APS Finechem, NSW, Australia
Super Dry Alcohol Merck, Victoria, Australia
Tannic Acid BDH, Poole, England
Trypan blue BDH, Poole, England
Trypsin-EDTA (0.5%) Gibco, New York, USA
Uranyl Acetate BDH, Poole, England
Xylene Merck, Victoria, Australia
1.1.5 Commercially purchased kids
RNeasy Mini Kit (RNA Extraction) QIAGEM Australia
RT-PCR Kit P r o m e g a U S A
126
Immunostaining Kit Dako Pty Ltd., Australia
1.1.6 PCR Primers
Type I Collagen (Rabbit) Prodigo, Sydney, Australia
Type II Collagen (Rabbit) Prodigo, Sydney, Australia
Type III Collagen (Rabbit) Prodigo, Sydney, Australia
Type III Collagen (Rabbit) Prodigo, Sydney, Australia
GAPDH (Rabbit) Prodigo, Sydney, Australia
Type I Collagen (Sheep) Prodigo, Sydney, Australia
Osteopontin (Sheep) Prodigo, Sydney, Australia
Osteonectin (Sheep) Prodigo, Sydney, Australia
GAPDH (Sheep) Prodigo, Sydney, Australia
1.1.7 Anitbodie
Table A1 Antibodies for immunohistochemistry
1°Ab Supplier Cat. No
Types Antigen Retrieval
1°Ab Dilution
/condition
Results
Coll Ⅰ MP
Biomedicals,
NSW, Australia
Clone I –
8H5
Monoclonal
antibody
30m 37℃ in
Trypsin-Digest
all 2(1:3)
1:1000
2hRT
/ON 4℃
In vivo
Present in
bone
matrix.
Osteonectin Developmental
Studies
Hybridoma
Bank, IA, USA
MPIIIB10 Monoclonal
antibody
30m 37℃ in
Pepsin-Digest
all 3
1:100
2h RT
/ON 4℃
In vivo
Present in
osteoblasts
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1.1.8 Buffers and solutions
Alcian Blue (pH=2.5)
Alcian Blue 1 g
3% Acetic Acid 1 0 0 m l
Solution mixed and stored at room temperature
1.5% aqueous safranin O
Safranin O 1.5g
Distilled water 100ml
Solution mixed and stored at room temperature
0.2% alcoholic fast green
Fast green 0.2g
95% ethanol 100ml
Solution mixed and stored at room temperature
1% acetic acid
Glacial acetic acid 5ml
Distilled water 495ml
Solution mixed and used fresh
128
95% Alcohol
100% Ethanol 9 5 0 m l
The solution was made up to 1L in DEPC treated water, and stored at -20°C
70% Alcohol
100% Ethanol 700ml
The solution was made up to 1L in DEPC treated water, and stored at -20°C
Alcohol : Xylene (1:1)
Alcohol 1 Vo l u m e
Xylene 1 Vo l u m e
Solution is mixed by gentle inversion and stored at room temperature
2% 3-Aminopropyltriethoxy-silane in Acetone
3-aminopropyltriethoxy-silane 20ml
Solution was diluted with acetone to 1L and stored at room temperature
10mg/ml Bovine Serum Albumin
Bovine Serum Albumin Powder 5 m g
1X PBS 500μL
Solution vortexed and stored at -20°C
0.2M Cacodylate Buffer
Cacodylate 8 5 . 6 g
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Distilled water 2 0 0 0 m l
Solution mixed and stored at 4°C
Collagenase type II (4000u/ml stock)
Collagenase type II powder (284u/mg) 1 g
1X PBS 5 6 . 8 m l
Solution is mixed, divided into 2ml aliquots and stored at -20°C
Collagenase type II (416 units/ml working conc. I)
Stock type II collagenase (4000 units/ml) 0 . 4 m l
Serum-free DMEM-F/12 medium 2.1ml
Stock 0.5% tripsin-EDTA 2 . 5 m l
Solution is mixed, divided into 2ml aliquots and stored at -20°C
Collagenase type II (416 units/ml working conc. II)
Stock type II collagenase (4000 units/ml) 0 . 1 m l
Serum-free DMEM-F/12 medium 4 . 4 m l
Fetal bovine serum (FBS) 0 .5ml
Solution is mixed, divided into 2ml aliquots and stored at -20°C
2% Decon
Decon 2 0 m l / L
Solution is stored at room temperature
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DEPC
Dimethyldicarbonate is dissolved to 10% (v/v) in absolute ethanol, mixed with DDW
1:10, incubated at room temperature in fumehood overnight, then autoclaved and stored
at room temperature.
0.1% DEPC Water
2ml DEPC is made up to 20ml with absolute ethanol, DDW then added to make up to
2L. Solution is autoclaved and stored at room temperature.
1% Eosin
Eosin 2 0 g
95 % Alcohol 2 L
Eosin is dissolved and stored at 4°C
15% Formic Acid
Formic Acid 1 5 0 m l
The solution was made up to 1L with DEPC treated water and stored at room
temperature
Gill’s Haematoxylin
Ethylene Glycol 3 7 5 m l
Haematoxylin 6 . 0 g
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Sodium Iodate 0 . 6 g
Aluminium Sulphate 4 2 . 8 g
Glacial Acetic Acid 50ml
Dissolved in 1085ml of DDW and stored at room temperature
2.5% Glutaraldehyde
Sucrose 3 6 g
Phosphate Wash Buffer 6 0 0 m l
Glutaraldehyde (25%) 2 0 0 m l
Distilled Water 1 2 0 0 m l
Solution mixed well and stored at 4°C
Growth Medium (20% FBS)
αMEM 4 0 0 m l
Foetal Bovine Serum (FBS) 1 0 0 m l
L-Ascorbic Acid 0 . 5 m l
Penicillin-Streptomycin (2X) 2 . 5 m l
Solution mixed and stored at 4°C
1%Neutral Red Solution
Neutral Red 1 g
Distilled Water 1 0 0 m l
Solution mixed and stored at room temperature
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1% Osmium Tetroxide
Osmium Tetroxide 1 g
Distilled Water 50ml
Shaken for 1 minute then added to
Distilled Water 50ml
Solution mixed and stored at room temperature
4% Paraformaldehyde in PBS
Paraformaldehyde 1 6 g
PBS 3 0 0 m l
Heat, and add a few drops of NaOH until dissolved. Add 100ml of PBS and chill on ice.
0.4M Phosphate Buffer
Sodium Phosphate (monobasic) 6 2 . 4 g
Distilled Water 1 0 0 0 m l
Mix and add to
Sodium Phosphate (dibasic) 1 7 0 . 4 g
Distilled Water 3 0 0 0 m l
Solution mixed and stored at 4°C
Phosphate Buffered Saline (1X PBS)
Sodium Chloride 4 . 2 5 g
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Sodium Phosphate (monobasic) 1 . 9 2 5 g
Sodium Phosphate (dibasic) 5 . 0 9 g
Dissolve completely in Milli Q water to 500ml volume and autoclave
Phosphate Wash Buffer
Phosphate Buffer (0.4M) 1 0 0 m l
Distilled Water 2 0 0 m l
Mixed and stored at 4°C
Serum-Free Medium
DMEM-F/12 5 0 0 m l
L-Ascorbic Acid 0 . 5 m l
Penicillin-Streptomycin (2X) 2 . 5 m l
Mixed and stored at 4°C
Scott’s Tap Water
Sodium Bicarbonate 7 . 0 g
Magnesium Sulphate 4 0 g
Tap Water 2 L
Crystal Thymol A few crystals
Solution stored at 4°C
134
1.6M Sodium Chloride
4.68g of sodium chloride is dissolved in DDW to 50ml, autoclaved and stored at room
temperature
1% Tannic Acid
Tannic Acid 0 . 1 g
0.2M Cacodylate Buffer 10ml
Mixed and used fresh
Trypan Blue solution (0.4% w/v)
Trypan blue powder 0 . 0 4 m g
95% Ethanol 10ml
Solutions mixed and stored room temperature
0.05% Trypsin-EDTA
0.5% Trypsin-EDTA 10ml
Sterile PBS 90ml
Solution mixed, divided into 5ml aliquots and stored at -20°C
0.5% Uranyl Acetate
Uranyl Acetate 0 . 0 8 g
Distilled Water 20ml
135
Mixed with magnetic stirrer for 2 hours minimum, then add to 0.2M Cacodylate Buffer
180ml
1.1.9 Laboratory Instruments
Clyde-Apac BH2000 Series Clean Air fumehood
Emitech K850 Critical Point Dryer
Leica DC100 camera light microscope and software
MSE Centrifuge
Neubauer haemocytometer
Nikon Diaphot phase-contrast microscope
Phillips XL30 Scanning Electron Microscope
Polaron Equipment SEM Coating Unit E5100 (sputter coater)
Ratek orbital mixer incubator
Ratek waterbath
Reichert-Jung 2030 microtome
W & T Avery weighing machine
WTB Binder cell incubator
MIDGET 3 Anaesthetic apparatus Australia.
Sephatech Microcentrifuge
Philips, Maximus M80 (Germany) radiographic machine
Lorao M IV mammographys machine (4-000-0014 Danbury CT06810 USA)
SkyScan-1072 High-resolution desk-top micro-CT system (Skyscan N.V., Aartselaar,
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Belgium)
1.1.10 Software
DESCRIPTION SUPPLIER
Adobe® Photoshop® CS2 Version 9 Adobe Systems Inc., USA.
ANT 3D Creator, Version 2.4 Skyscan N.V., Aartselaar, Belgium.
CT Analyser (CTAn) Version 1.5.0.2 Skyscan N.V., Aartselaar, Belgium.
CTVol Realistic 3D-Visualisation, Version 1.9.4.1 Skyscan N.V., Aartselaar, Belgium.
Dataviewer Version 1.2.2 Skyscan N.V., Aartselaar, Belgium.
Microsoft ® Office Word 2003 Microsoft Corporation, USA.
NRecon Reconstruction Software Skyscan N.V., Aartselaar, Belgium.
SIMI Motion Simi Reality Motion, Unterschleissheim, Germany
SPSS 12.01 for Windows SPSS Inc, Chicago, Illinois, USA.
TVIEW Version 1.5.0.0 Skyscan N.V., Aartselaar, Belgium.
1.2 General Methods
1.2.1 Animal anesthesia
1.2.1.1 For rabbit
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Upon removal from their holding cage and weighing, rabbits were administered a
muscle relaxant/ analgesic (Xylazine) and general anaesthetic (Ketamine) to render the
animals unconscious and reversibly block any neural excitation that might cause the
animal pain or discomfort during surgery. Injections were administered intra-muscularly
into the hind leg muscles of the rabbit. The xylazine/ ketamine mix was administered in
three steps: (1) a fixed dose of xylazine was given as a premed to make the animal
drowsy and relieve anxiety, (2) ketamine was given to render the animal unconscious,
and (3) xylazine was given to aid distribution of the anaesthetic and relax the leg
muscles for compliant surgery. Doses of both compounds were given as per body
weight requirements formulated by departmental and animal house protocols.
1.2.1.2 For sheep
The sheep were housed in the Large Animal Facility for more than 10 days prior to
surgery to allow for acclimatization. Anaesthesia was performed by Dr Martin Cake, a
veterinarian from Murdoch University with more than 10 years of experience with
sheep anesthesia and orthopaedic surgery. Dr Cake also attended every session of the
surgery in the operating suite to assist and advise throughout the entire procedure.
The sheep were premedicated with Acepromazine (0.05mg/kg) combined with
Buprenorphine (6-10mcg/kg IM) approximately 20 minutes prior to induction.
Anaesthesia was induced with a mixture of Diazepam and Ketamine used at a ratio of
1:1 (diazepam 5mg/ml and ketamine 100mg/ml; totaled approximately 1ml per 10kg
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IV). The sheep were then intubated and placed under Isofluorane (2% Isofluorane, O2
1L/min) throughout the duration of the procedure. Vital signs were monitored
throughout the entire operation. No complications were observed due to anesthesia.
1.2.2 Surgical procedures
1.2.2.1 For rabbit
Once the animals were unconscious, the dorsal aspect of the lumbar spine and iliac crest
were shaved. To stabilize the rabbit in the prone position, a towel roll was placed under
the abdomen and between the thighs. The surgical field was then prepared and draped
using strict sterile technique (Fig A1.A). All surgical personnel were required to wear
sterile gloves, gowns, caps and face masks to avoid potential sources of infection.
The spinal level to be fused, L5–L6, was then identified by palpation. A line drawn
from the most cranial aspect of one iliac crest to the other, the intercrestal line, would
generally pass between the L6 and L7 spinous processes (Fig. A1.E). A second method
to verify the correct operative level was based on the anatomy of the lumbosacral
spinous processes. Specifically, there was a much wider interspinous distance at L6–L7
than there was at L5–L6, L7–S1 or between the sacral processes (Fig. A1.E).
Using both techniques of localization, the L5–L6 level can be correctly identified in the
vast majority of animals. Errors can occur, however, because of the presence of osseous
anomalies of the lumbosacral vertebrae. A preoperative anteroposterior radiograph was
139
advisable. In the presence of an abnormality, which alters the typical number of lumbar
motion segments, the animal should be excluded or the spinal level just cranial to the
intercrestal line can be used.
A dorsal midline skin incision measuring approximately 6 cm in length was centered
over the L5–L6 level (Fig. A1.G). A full-thickness flap of skin and subcutaneous tissue
was developed and retracted to one side. Approximately 2 cm lateral to the midline at
the L5–L6 level, a 4- to 6-cm longitudinal incision was made through the lumbar fascia.
Through this fascial incision, the iliocostalis muscle was divided exposing the
underlying longissimus muscle (Fig. A1.B).
To reach the transverse processes, blunt dissection was performed along the lateral
border of the longissimus muscle. Exposure of the posterolateral fusion site was
accomplished by elevating the iliocostalis muscle in a lateral direction off the transverse
processes and intertransverse ligament. Dorsomedial retraction of the longissimus
muscle was required to expose the medial aspect of the transverse processes and the
pars interarticularis. A small self-retaining retractor will maintain exposure of the two
transverse processes and the intertransverse ligament (Fig. A1.C).
To minimize bleeding during and after surgery, the dorsal branch of the segmental artery
was cauterized as it passes with the posterior ramus through the operative field. As a
means of limiting hemorrhage, it was also helpful to pack the wound with gauze upon
completing the first surgical approach. After exposure and packing of the contralateral
fusion site, retractors were replaced on the initial side to begin the decortication process.
140
It was advisable to simultaneously palpate the left and right fusion sites to verify that
the same level has been exposed on both sides of the spine.
Decortication of the fusion site was performed with a motorized burr. To avoid L5
ventral root injury (with resultant femoral nerve dysfunction), great care needs to be
exercised while decorticating the medial aspect of the transverse processes and the
lateral edge of the pars interarticularis. The fifth lumbar root was vulnerable to injury as
it exits the L5–L6 intervertebral foramen immediately dorsal to the plane of the
intertransverse ligament and transverse processes (Boden, Schimandle et al. 1995;
Valdes, Palumbo et al. 2004). The lumbar plexus was also vulnerable to injury as its
component nerves pass just ventral to the intertransverse ligament making it essential to
preserve the integrity of this ligament during the exposure and decortication process.
The grafting materials (autograft, ISBG, OP-1, ISBG+OP-1) were then placed into the
fusion bed, the graft material under analysis should be placed over the intertransverse
ligament such that it spans the distance between the L5 and L6 transverse processes and
contacts the lateral edge of the pars interarticularis (Fig. A1.C).
Arthrodesis using autogenous bone from the ilium was often implemented as a control
group in spinal fusion research using the NZW rabbit model. Working through the same
dorsal skin incision, the cranial and lateral surface of the middle two-thirds of the iliac
crest was exposed in a subperiosteal plane. This central part of the iliac wing contains
the greatest amount of cancellous bone and can be localized by palpation of the medial
iliac spine. The recommended quantity of graft, 2.5 to 3.0 cc3 per side of the spine,
generally requires exposure of both iliac. During graft harvest, it was critical to avoid
141
the caudal aspect of the posterior third of the iliac wing. Dissection in this area can
traumatize the neurovascular structures that pass through the sciatic notch leading to
serious hemorrhage and/or sciatic nerve palsy.
After placement of graft material into the fusion bed, a running 3-0 vicryl stitch was
used to reapproximate the muscle aponeurosis and then the more superficial layer of
lumbodorsal fascia. Before completing closure of the subcutaneous layer with
interrupted 2-0 vicryl stitches, it was advisable to anchor this tissue to the underlying
fascia to prevent seroma formation. The skin was closed with staples, and antibiotic
ointment was spread over the incision. A funnel-shaped cervical collar was applied to
prevent the rabbit from biting the wound (Fig. A1. D).
A B C
D E
F G
Fig A1. Dorsal view of the rabbit lumbosacral region (A) and posteroanterior radiograph of the normal lumbar spine (B) (Valdes, Palumbo et al. 2004). Palpable bony landmarks include the L5–S1 spinous processes, the iliac crests (IC) and the greater trochanters (GT). The intercrestal line (ICL), and a dorsal midline skin incision was made from L4 to L7 (C). Surgical procedure: The surgical field is then prepared and draped using strict sterile technique (D). The transverse processes of L5-L6 were exposed and the dorsal aspects transverse processes were decorticated(B). Graft materials were then implanted in between the transverse processes (F). The wounds were closed by layers (G).
142
143
1.2.2.2 For sheep
The sheep were housed in the Large Animal Facility for more than 10 days prior to
surgery to allow for acclimatization. Anaesthesia was performed by Dr Martin Cake, a
veterinarian from Murdoch University with more than 10 years of experience with
sheep anesthesia and orthopaedic surgery. Dr Cake also attended every session of the
surgery in the operating suite to assist and advise throughout the entire procedure.
Under intubated anaesthesia (2% Isofluorane, O2 1L/min), the sheep was positioned on
its right lateral side. Left side approach was preferred as it reduces surgical exposure of
venous structures (especially the azygous vein). The operative area was routinely
prepared, draped, and sterilized in a standard surgical fashion. Landmarks for skin
incision were identified: the left side tips of the transverse lumbar processes, iliac crest,
costal arch and the anterior superior iliac spine. Skin incision was designed one finger’s
breadth ventral (anterior) to the tips of the left transverse process along the long axis of
the spine, starting from the iliac crest to the costal arch cranially for explosion from T13
to L5 (Fig. A2). Depending on the number and level of surgical lumbar segments,
incision can be used partly or fully. After dividing subcutaneous tissue, the lateral
borders of abdominal muscles, including external oblique abdominal muscle, internal
oblique abdominal muscle and transverse abdominal muscle were identified in
succession. The edges of these three layers of abdominal muscles were located at the
posteo-lateral abdominal wall, and were very close to the tips of lumbar transverse
144
processes. Abdominal muscles connect paralumbar muscles with three layers of
translucent and weak fascias. Intact abdominal muscles were pulled ventrally by
dissecting their semi-lucent fascia layers, and thus abdominal muscles were preserved
(Fig. A3 A). Then the retroperitoneal space was accessed. Further blunt dissection of the
retroperitoneal space between extraperitoneal adipose layer and major psoas was
required to expose the lumbar spine. There was a potential interval between psoas major
and retroperitoneal adipose layer, which was easy to be separated bloodlessly. And thus,
peritonea and ureter could be protected well with the retroperitoneal adipose layer. The
abodominal aorta was exposed after abdominal content was retracted ventrally.
Segmental vessels were distributed transversely at the surface of the vertebral column.
On the left side of the lumbar column, segmental arteries were located on the
transvertebral discs levels relatively with less variation, which could be easily identified
from their roots from aorta. Segmental veins were located on the 1/3 superior levels of
vertebral bodies with more variations in number and location. Ligation of the segmental
vessels optimises hemostatis and safe retraction. After identification of lumbar levels by
palpation, psoas attachment on relative vertebral bodies was elevated. A Deaver was
placed over the retroperitoneal adipose layer to protect the abdominal contents, ureter
and main vessels, and under the vertebral body (thus retracting the abdomen ventrally)
to expose the lumbar structures. A Richardson retractor was placed over left psoas major
to retract the para-vertebral muscles dorsally (Fig A3 B). Care was taken to preserve the
abdominal vessels, ureter, sympathetic chain, and genitofemoral nerve.
145
Two segments of anterior lumbar fusion procedures were carried out following adequate
explosion. Following ligation of the segmental vessels, the intervertebral discs of L3-L4
and L4-L5 were removed, together with the cartilage layers of the vertebral body to the
layer where subchondral bone was exposed. Fusion materials were then implanted into
the empty disc space with appropriate impact. The surgical segments were fixed with
bone staples (2 staples per segment) to enhance fusion. The abdominal cavity was
carefully checked for any iatrogenic injury prior to closing. Wounds were closed to both
the fascial and subcutaneous layers.
B
A
Fig A2. (A) Skin incision of less invasive approach, showing the animal position and landmarks. Skin incision is designed parallel with dorsal mid line, located at the ends of left transverse processes of lumbar vertebrae. From the iliac crest, it can be extended to the costal arch according the segments that need to be exposed. (B) Illustration of cross section at the sheep’s L4 lumbar vertebrae, 1) transverse process, 2) psoas major muscle, 3) ureter, 4) psoas minor muscle, 5) abdominal aorta, 6) L4 vertebral body, 7) inferior vena cava, 8) peritoneum, 9) external abdominal oblique muscle, 10) internal abdominal oblique muscle, 11) transverse abdominus muscle, 12) rectus abdominus; skin incision of less invasive approach (white arrow) is closer to lumbar rather than skin incision of conventional retroperitoneal approach (black arrow).
146
A
B
C
Fig A3. (A) Exposure of fascia plane after skin and subcutaneous tissue dissection. F: semi-lucent fascia between the lateral edges of abdominal muscles (E: external abdominal oblique muscle, I: internal abdominal oblique muscle, T: transverse abdominus muscle) and L: paralumbar muscles. (B) Retroperitoneal exposure of L4 and L5 lumbar vertebrae through the fascia plane between the abdominal muscles and para-vertebral musculature; (C) Illustration of lumbar vertebrae exposure with psoas major muscle retracted dorsally and a Deaver placed over the retroperitoneal adipose layer to protect the peritoneum, ureter and abdominal vessels.
147
148
1.2.3 Postoperative animal care
1.2.3.1 For rabbit
For the first hour after surgery, pulse and respiratory rate were monitored; supplemental
fluids were administered intravenously as needed. Buprenorphine (0.03 mg/kg) was
administered by means of intramuscular injection every 12 hours during the first 3
postoperative days for pain control. The general condition of the rabbit was monitored
twice each day for the first 3 days after surgery, followed by once a day for the next 7
days, and three times per week thereafter. Any signs of wound infection or hindlimb
paresis were to be reported to the veterinarian and primary researcher. Rabbits were
killed with a sedating dose of intramuscular injection of xylazine (2.5mg/kg) followed
by a lethal dose of intravenous pentobarbital at 6 weeks postoperatively.
1.2.3.2 For sheep
Postoperatively, the sheep were housed in floor pens for 5-7 days without any
restrictions to mobility, diet, or use of orthotic devices. All sheep received
prophylactic Amoxycillin (20mg/kg IM) daily for 5 days, and postoperative analgesia
was achieved with intramuscular analgesic injections of Buprenorphine
(0.005~0.01mg/kg IM 4~6 hourly) for 5 days.
149
1.2.4 Manual palpation
Two independent observers manually evaluated the animal lumbar spines in a blinded
fashion once specimens were retrieved en bloc. Vertebrae were manipulated with forc
small enough to avoid producing gross trauma but great enough to evaluate for gross
intervertebral motion. Each motion segment was graded solid or not solid. Only levels
graded solid were considered fused. Specimens were determined to be fused when no
significant motion was noted by either observer.
1.2.5 Radiography
All lumbar spines were examined by anterior-posterior (AP) (66kV, 6.4 mA, 17 ms) and
lateral (58kV, 6.4 mAs, 17 ms) plain radiographs (Philips, Maximus M80, Germany)
when lumbar samples were harvested. Radiographs were assessed by two independent
observers blindly.
1.2.6 Micro-CT
Fusion masses were scanned using micro-CT X-ray tomography (SkyScan-1076 in vivo
micro-CT system, Skyscan N.V., Aartselaar, Belgium). The lumbar fusion masses were
imaged using an X-ray tube voltage of 74kV and current 140 μA, with a 1 mm
aluminium filter. The scanning angular rotation was 180°and the angular increment 0.
150
8°. Datasets were reconstructed using the cone beam reconstruction software (NRecon
version 1.4.4) based on the Feldkamp algorithm and segmented into binary images
using adaptive local thresholding. Each whole fusion mass was selected, excluding
transverse processes, as volume of interest, and parameters associated with bone
formation, including tissue volume, bone volume, tissue surface, bone surface,
intersection surface, were calculated using the CT analyser software (Skyscan). Three
dimensional images were generated using the Cone-Beam Reconstruction and 3D
realistic visualisation software (Skyscan).
1.2.7 Biomechanics
1.2.7.1 For rabbit lumbar
Biomechanical testing was performed by three-point flexion-bending test using the
Biaxial Material Testing Machine System (Zwick Z010, Zwick Inc., Ulm, Germany;
Test-expert, Zwick Inc., Ulm, Germany). Osteo-ligamentous L4-L7 specimens were
dissected of all soft tissues with the exception of ligaments and joint capsules. The L4
vertebrae was secured into the machine basement with bone cement while four pure
movements (flexion and extension, left and right lateral bending) was applied to the L7
vertebrae. L5 and L6 were marked with two 0.062-inch K-wires fixed through the
sagittal and transverse planes at each vertebral body. The loading protocol involved the
application of pure movements in a stepwise fashion to a maximum of 0.27 Nm. Each
step (0.00, 0.09, 0.18 and 0.27 Nm) was sequentially applied for 30 seconds, with a still
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photograph taken at the end of each loading step (Image J 1.36; Wayne Rasband,
National Institutes of Health, USA) to detect the range of motion (ROM) of L5-L6 by
measuring the change in the angle between the K-wires.
1.2.7.2 For sheep lumbar
To assess the stiffness of surgical ovine lumbar segments, non-destructive
biomechanical testing was performed using a four-point flexion-bending test model
(Cottrell, van der Meulen et al. 2006). All posterior structures and surrounding muscles
and ligaments were removed from intact lumbar vertebral bodies L3 to L5, keeping the
annulus fibrosus intact. End vertebrae were potted in stainless steel tubes and loaded in
a four point bending setup, with an outer span of 138 mm and an inner span of 100 mm.
Four planes of motion: flexion, extension, right and left lateral bending were applied
using Instron Materials Testing Machine System (Instron, 5566, MA, USA). The
loading protocol was designed in a stepwise fashion using an initial loading of 0.0095
Nm, and sequentially increased to 0.162, 0.323 and 0.475 Nm using a loading time of
10 sec, followed by release for 5 sec. To monitor the motions of segments, two
reflecting balls were fixed on each vertebral body and tracked by video camera. The
range of motion (ROM) and neutral zone (NZ) were then analyzed using the software of
SIMI Motion (Simi Reality Motion Systems GmbH, Unterschleissheim, Germany).
ROM was defined as the segmental displacement at the end of each loading, and NZ
was defined as the segmental displacement after all the load was released.
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1.2.8 Histology
Histologic analysis weas performed to evaluate the maturity of bone newly induced.
This included an assessment of callus constituents: bone, cartilage, and fibrous tissue.
Immediately after biomechanical testing, the L5–L6 spine segments were isolated and
divided along the midsagittal plane.
Samples were fixed in 10% phosphate-buffered formalin for at least 24-48 hours,
followed by decalcification with 10% EDTA for 4-8 weeks according to the sizes of
samples, except in cases of no decalcification for slides for von Kossa staining.
All samples were processed in a Miles Scientific, Tissue Tek vacuum infiltration
processor overnight at 37°C, using the following protocol: 70% ethanol for 1 hour, 95%
ethanol for 1 hour, 100% ethanol twice for 1 hour each, 100% ethanol twice for 30
minutes, chloroform twice for 2 hours, chloroform once for 1 hour, finally, molten wax
at 60°C, twice for 2 hours. The processed membranes were then embedded in paraffin
wax.
The samples embedded in paraffin blocks were then sectioned to a thickness of 5μm
using a Reichert-Jung 2030 microtome. The sections were floated on a water bath at
40°C and picked up onto silanated slides. The slides were then dried overnight at 37°C.
153
After drying the slides, the specimens were dewaxed. The paraffin was dissolved by
treatment in 3 solutions of 100% xylene (RNase free) for 3 minutes each. The tissue was
then rehydrated by successive immersion in the following solutions for 3 minutes each:
100% ethanol, 95% ethanol, two changes of 70% ethanol and finally DEPC-treated
water. At this point, the sections from each mouse were stained with H & E.
1.2.8.1 H &E staining
Sections from rabbits were stained using H & E for routine histological examination.
Following dewaxing and rehydration, slides were immersed in Gills Haematoxylin for 3
minutes and rinsed in tap water to remove excess stain. The slides were then
immersed in Scotts tap water for 2 minutes and rinsed again in running tap water for 3
minutes. Sections were placed in 70% ethanol and 90% ethanol for 3 minutes each and
counterstained in 1% eosin for 1 minute. Dehydration was then carried out by
immersing in the following solutions in succession for 3 minutes each: 100% ethanol
(three solutions) and absolute alcohol/xylene (1:1). The sections were cleared in three
changes of xylene for 3 minutes each. The slides were mounted in DePeX and left to
dry overnight.
1.2.8.2 Saffanin O staining
The slides were depataffinized in 3 changes of xylene, 5min each, and rinsed in 2
changes of 100% ethanol, 1 min each to remove the xylene. Then, the sections were
154
hydrated in 2 changes of 95% ethanol, 1 min each before hydration with 3 changes of
distilled water, 1 min each. The slides were put in 1.5% safranin O for 40 min, and then
rinsed in 3changes of distilled water, 6 dips each. Care was taken to immerse the entire
slide and not leave it in water for excessive amounts of time, which would wash out
the safranin O. The slides were dipped in 0.2% alcoholic fast green for 30s, then were
dipped in fresh 1% acetic acid solution for 8 dips. Then the slides were dipped quickly
in distilled water for 6dips. The slides were dipped in 95% ethanol for 6 dips and then
were dehydrated in 2 changes of 100% ethanol for 8 dips each. Then, one slide was
rinsed in xylene for 1 min and then the slide was checked for the staining under the
microscope. If the stain was good, the next step would be carried out; if it was not good,
the slide was rinsed in 100% ethanol to remove xylene and the stain or water rinsing
was repeated. Finally, the stained slides were mounted with cover slips.
1.2.8.3 Von Kossa staining
Dewaxed sections were incubated with 5% silver nitrate solution under ultraviolet light
for 60 minutes. Then un-reacted silver was removed with 5% sodium thiosulfate for 5
minutes, followed by counter staining of H& E.
1.2.9 Immunohistochemistry
All the slides were de-waxed by the following steps: xylene 3 x 2 minutes, dehydrated
through 3 x 100% Ethanol; 1 x 95% Ethanol; 1 x 70% Ethanol 2 minutes each, and
155
washed with double distilled water for 20 minutes. From the first [?] step onwards, the
sections had to be kept wet all the time. The endogenous peroxidase is blocked with
3% H2O2 in Milli Q water for 5 minutes. Sections are rinsed with 3 changes of water,
and then the last time with TBS. Excess TBS is blotted from the slide with tissue and
circle sections with hydrophobic marker pen (PAP pen). An appropriate antigen
retrieval solution is applied which depends on 1°Ab (see 1°Ab list). An appropriate
amount of solution is placed to cover sections in a wax ring and slides are placed in a
humid tray and in a 37℃ oven. They are given a quick rinse in 3 changes of TBS.
Non-specific staining is blocked with 10% FBS (fetal bovine serum) in 1% BSA in TBS
with 0.1% TritonX100 for 30 minutes. The serum is shaken off without rinsing, then
sections are covered with 1°Ab diluted in antibody diluent(see 1°Ab list). Slides with
antibody are placed in a humid tray with the lid on and put in a fridge for overnight
incubation. They are washed 3 times, 10 minutes each in wash buffer. Sections are
incubated in secondary antibodies ‘link solution’ for 20 minutes at RT. They are washed
3 times, 10 minutes each in wash buffer. Sections are incubated in ‘streptavidin-HRP’
from the LSABTM 2 kit for 20 minutes at RT. They are washed 3 times, 5minutes each
in wash buffer and the last wash in TBS. The section is incubated in Dako Liquid
DAB in the dark until a brown colour has developed, 6-8 minutes. They are washed in
several changes of MilliQ water. Stain is countered with Gill’s hematoxylin for 30
seconds before mounting in Depex.
1.2.10 Bone marrow aspiration and isolation
156
Approximately 15 ml of bone marrow per ovine was harvested from the iliac crest. To
prevent peripheral blood contaminating, multiple aspirations were carried out with
variations of depths and punch points, and no more than 2ml bone marrow was
harvested from each point (Muschler, Boehm et al. 1997), using bone marrow aspiration
needles (Angiotech, DBMNI 1501, Gainesvlile, FL) connected with 20-ml heparinised
syringes. To remove red blood cells, the buffy coat was pipetted after bone marrow was
centrifuged at 1500 r/m for 10 minutes, and was centrifuged with ficoll-paque plus
(Amersham Pharmacia Biotech, Uppsala, Sweden) at 1500 r/m for 10 minutes. Thus,
enriched bone marrow cells were pipetted for in vitro population expansion and in vivo
implantation.
1.2.11 Cells co-culture with scaffolds
Enriched BMCs were cultured with a complete culture medium composed of alpha
minimum essential medium (α-MEM), supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM
L-glutamine (all from ThermoTrace, Melbourne, Australia). The re-suspended cells
(1×105cells/ml) were plated in 75cm2 culture flasks and incubated at 37°C, 90%
humidity and 5% CO2. After 48h incubation, non-adherent cells were discarded, and
adherent cells were washed with serum free medium twice and fresh complete medium
was added into the flasks. The medium was changed every three days. The cells were
then passaged into two 75cm2 flasks when the cell population reached 80% confluent.
The confluent monolayer of the cells was collected after the second passage for seeding
157
in NBCS. The cells were then loaded into the NBCS in the density of 1.5x104 / scaffold
(approximate 0.01g), and spun down at 500 r/m for 5minutes to enhance the attachment
of the cells on to the scaffolds. The cells loaded scaffolds were cultured in a 48-well
plate with complete medium supplemented with 2.0×10-4M ascorbic acid, 7×10-3M
beta-glycerophosphate and 1.0×10-8M dexamethasone. The medium was changed
every 2 days. Scaffolds with the cells were sampled at 1, 2, 3, or 4 weeks after
co-culture commenced.
1.2.12 Scanning electron micrograph (SEM)
Samples were dehydrated using a series of ethanol solutions at increasing
concentrations, then coated with 5 nm thick platinum (SEM coating unit, E 5100,
Polaron equipment LTD). Coated samples were viewed under a scanning electron
microscope (XL-30, Philips) at a low voltage (15 kV). Images were saved as TIFF
formate.
1.2.13 RNA extraction
Total cellular RNA was extracted from rabbit intertransverse process fusion tissues in
accordance with the instructions in the RNA extraction kit (RNeasy Mini Kit QIAGEN
Australia) as follows: Thirty-milligram fusion mass tissue is removed from
intertransverse process area and placed in a sterile and liquid nitrogen cooled mortar.
Fusion mass tissue was then ground thoroughly to powder in liquid- nitrogen with a
158
sterile pestle. Subsequently, the tissue powder and liquid nitrogen were decanted into an
RNase-free 10ml tube and lysed in 1ml Buffer RLT (provided in kit, ensure that 10μl
β-ME is added to 1ml Buffer RLT). In order to homogenize the sample the lysate was
pipetted directly onto a QIAshredder spin column placed in a 2ml collection tube
(provided in kit) and centrifuged for 5 min at maximum speed in a microcentrifuge
(Sephatech). When the centrifuge finished, the supernatant (lysate) was carefully
transferred to a new 2ml microcentrifuge tube by pipetting. After mixing the lysate with
700μL 70% ethanol, the mixture was immediately and equally distributed into two
RNeasy mini columns placed in 2ml collection tubes (provided in kit) and centrifuged
for 15s at 15,000rpm. The flow-through was discarded. (At this stage, the total RNA and
few contaminants are dissolved in RNeasy silica-gel membrane which is manufactured
inside the RNeasy mini columns.) To remove contaminants, the tubes were washed and
spun with the following solutions (provided in kit): add 700μL Buffer RW1 to the
RNeasy column, close the tube gently, and centrifuge for 15s, 15,000rpm, to wash the
column, discard the flow-through and collection tube. Transfer the RNeasy column into
2ml collection tube (supplied). Pipet 500μL Buffer RPE onto the RNeasy column. Close
the tube gently, and centrifuge for 15s 15,000rpm to wash the column. Discard the
flow-through.( Buffer RPE is supplied as a concentrate, before using for the first time,
all 4 volumes of 96%-100% ethanol. Add another 500μL Buffer RPE to the RNeasy
column, and centrifuge for 2 min 15,000rpm to dry the RNeasy silica-gel membrane.
Transfer the RNeasy column to a new 1.5ml collection tube (supplied) pipet 30μL
RNase-free water directly onto the RNeasy-gel membrane, and centrifuge for 1min at
15,000rpm to elute. The final yield was 30μl and the total RNA was stored at -80°C.
159
1.2.14 Reverse transcription for cDNA
In the first strand synthesis reaction, reverse transcriptase is used to make the
complementary DNA (cDNA) strand of a desired RNA target sequence. The template
RNA is usually heated prior to initiation of cDNA synthesis, in order to denature any
secondary structures which could impede the ability of the reverse transcriptase to
make a full length copy of the template RNA. Upon generation of cDNA, specific
sequences can be targeted and amplified in a polymerase chain reaction (Refer to 3.2.3
d).
For the purpose of this investigation mRNA from fusion mass tissue was reverse
transcribed using the RETROscript™ first strand synthesis kit (Ambion). Briefly, on ice,
a reaction mix was generated in a thin walled, RNAse-treated, PCR tube containing 4μl
of template mRNA, 8μl of dNTP mix (5mM), 1μl of oligo (dT) first strand primers and
7μl of DEPC-treated double distilled water. The mixture was combined, spun briefly
and then heated at 75°C for 3 minutes to disrupt secondary structures. After heating, the
reaction mix was returned to ice whereby 8μl of 5X RT-PCR buffer, 2μl of placental
RNase inhibitor, 2μl of Molney Murine Leukemia Virus (M-MLV) Reverse
transcriptase and 7μl of DEPC-treated double distilled water was added to give a final
reaction volume of 20μl. Again, the mixture was combined and spun briefly, then
incubated at 42°C for 1 hour in a thermal cycler (Perkins-Elmer Gene Amp 2400).
160
Finally, the tube was heated for a further 10 minutes at 92°C to inactivate the reverse
transcriptase. Upon completion, the reaction sample was stored at -20°C and ready for
Polymerase Chain Reaction (PCR).
1.2.15 Standard PCR
The polymerase chain reaction (PCR) is a powerful method for the enrichment of
specific target sequences. By carrying out repeated cycles of annealing, synthesis, and
denaturation in the presence of a thermostable DNA polymersase [Thermus aquaticus
DNA polymerase (Taq polymerase)], sequences bounded by a pair of short, sequence
specific primers can be amplified more than a millionfold.
For PCR isolation of type Ⅰ, Ⅱ, Ⅲ, Ⅹ collagen, osteopontin, osteocalcin and the
house keeping GAPDH gene, reaction mixtures containing 2μl of dNTP mix (5mM);
0.5 unit of Taq polymerase; 0.5μl of forward and reverse primers (Table 3.1); 2.5μl of
10× PCR buffer; 2μl magnesium chloride; 15μl DEPC-treated double distilled water;
and approximately 2μl of target cDNA in a final reaction volume of 25μl. Each reaction
was mixed and spun briefly before being placed into a thermal cycler (Perkins-Elmer
2400). The standard PCR profiles comprised 5 min of heat inactivation at 94°C;
denaturation at 94°C for 45 sec, an annealing temperature (varied with each primer,
60-62°C) and extension of 72°C for 45 sec, with 30 repeated cycles. Reaction mixtures
were subjected to a final extension at 72°C for 10 mins and stored at 4°C for further
experiments.
161
1.2.16 Agarose gel electrophoresis
Agarose gel electrophoresis is a routine technique in molecular biology for the
separation of molecules based on their size. Depending on the size of molecules being
studied, a quantity of agarose powder is melted in 1X TAE buffer to obtain desired gel
concentration. In this experiment, 50 ml of 1X TAE buffer was used for casting 7 x 10
cm2 gel; while 100 ml of 1X TAE buffer was used for casting 10X10 cm2 gel. For the
analysis of larger fragments a low concentration of agarose was desired thus 1.2% gel
were cast. Initially, 0.6 g of agarose powder was melted in 50 ml of 1X TAE buffer for
1~ 2 minutes in a microwave oven. Upon dissolution of the aragose in the buffer, the
molten gel was allowed to cool for 2-3 minutes at room temperature. After the cooling,
1 μl/50 ml of solution of ethdium bromide was added to the molten gel solution. Care
was taken in handling this solution since it is very toxic, and the addition of this
chemical was carried out within a fume hood. The solution was then poured into a
perspex gel tray taped at the open ends. A well forming comb was positioned at one end
of the gel tray, with the well teeth raised approximately 2mm from the base. The gel was
left to solidify for 15 minutes. Upon hardening, the comb was removed and the gel slab
was placed in a horizontal electroporesis tank (Biorad) and submerged in
electrophoresis buffer (1X TAE). PCR products mixed with 6X blue-orange loading
buffer were pipetted into the wells and subjected to the applied electric field across the
gel. The range of voltage used for gel electrophoresis was 80 to 110 volts.
162
Electrophoresed DNA products were visualised on a UV transilluminator (IBI) and a
permanent record of agarose gels was obtained by photographic means using a Polaroid
CU-5 land camera (USA) with 667 black and white Polaroid film.
1.2.17 Preparation of NBCS
NBCS was produced from organic human bone particles made by the Perth Bone and
Tissue Bank (Patent No. WO/2003/020840), according to the standard of the Australian
Goods Manufacturing Practice for Blood & Tissues of the Therapeutic Goods
Administration (TGA). In brief, bone powder was pre-washed with saline at 40-45°C
for 5 min, and then treated with 0.6 N HCl and 2.0 M CaCl for up to 12 hours at 4°C.
Bone was then washed with 0.5 M EDTA for 4 hours at 4°C, followed by sterilized
milliQ water at 55°C for 4 hours. Lastly, NBCS was sterilized by gamma irradiation (25
kGy) before use.
1.2.18 Statistical analysis
Average values are presented as mean ± standard deviation. Fusion rates as
determined by manual palpation and radiographic analysis were evaluated using
Fisher’s exact test. Comparisons of biomechanical testing of spines in each group were
made using one-way analysis of variance (ANOVA). Significance for all tests was
defined as P < 0.05.
APPENDIX B
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