In vitro and in vivo evaluation of customized polycaprolactone tricalcium phosphate scaffolds for bone tissue engineering

IN VITRO AND IN VIVO EVALUATION OF CUSTOMIZED
POLYCAPROLACTONE TRICALCIUM PHOSPHATE SCAFFOLDS
FOR BONE TISSUE ENGINEERING

ERVI SJU
(B.Eng.(Hons.), NUS)

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF ENGINEERING
DEPARTMENT OF MECHANICAL ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE

2010


PREFACE

The thesis is submitted for the degree of Master of Engineering in the Department of
Mechanical Engineering at the National University of Singapore under the
supervision of Professor Teoh Swee Hin and Dr Alvin Yeo. No part of this thesis has
been submitted for other degree at other university or institution. Parts of this thesis
have been published or presented in the following:

INTERNATIONAL JOURNAL PUBLICATION
A. Yeo, E. Sju, B. Rai, S.H. Teoh. Customizing the degradation and load-bearing
profile of 3D polycaprolactone-tricalcium phosphate scaffolds under enzymatic and
hydrolytic conditions. Journal of Biomedical Materials Research Part B: Applied
Biomaterials. (Published online: 10 June 2008).

CONFERENCE PAPERS
E. Sju, A. Yeo, B. Rai, S.H. Teoh. In vitro and in vivo degradation profile of

untreated, sodium hydroxide- and lipase-treated PCL-TCP scaffolds. International
Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering,
Singapore, 2008.

E. Sju, A. Yeo, B. Rai, S.H. Teoh. Enzymatic and hydrolytic degradation of poly(εcaprolactone)

tricalcium

phosphate

composite

scaffolds.

4th

International

Conference on Materials for Advanced Technologies (ICMAT), Singapore, 2007.

i


ACKNOWLEDGEMENTS

The author wishes to express her sincere gratitude and heartfelt appreciation to the
following people who have rendered generous support and technical assistance
leading toward the accomplishment of this project:


Professor Teoh Swee Hin (Department of Mechanical Engineering), supervisor,
for offering the privileged opportunity to work on this project and allowing the
author to join his team, for his expertise, kindness, and most of all, his patience.
His enthusiasm in research and continuous support have truly been a source of
inspiration and motivation for this project throughout.



Dr. Alvin Yeo (Department of Mechanical Engineering and National Dental
Centre), co-supervisor, for his patience and guidance on supervising the author
throughout the whole process. He has been an immense driving force behind this
project. One simply could not wish for a better or friendlier supervisor.



Dr. Bina Rai, mentor, for graciously sharing her knowledge and encouragement
in this project. Her kind assistance and time spent are greatly appreciated.



Dr. Zhang Zhiyong, Ms. Erin Teo Yi Ling and Mr. Mark Chong Seow Khoon,
PhD students, for their constructive feedbacks and for being excellent mentors.
They have gone out of their way to render assistance on many occasions.



Mr. Cheong Jia Jian, NUS alumnus, whom was unreserved in sharing his
knowledge and experience in this research field.



Mdm. Zhong Xiang Li (Materials Science Lab) for the use of the SEM (JEOL
JSM 5600LV) and the gold-sputtering machine.

ii




Dr. Zhang Yanzhong (Biomechanics Lab) and Ms. Eunice Tan Phay Sing
(NanoBiomechanics Lab) for the use of the Instron 3345 compressive
mechanical tester machine.



Dr. Jeremy Teo Choon Meng (DSO National Lab) and Ms. Lei Yang (Biosignal
and Instrumentation Lab) for the use of the Skyscan 1076 Micro-CT.



Dr. Lin Jian Hua and Ms. Juline Sim Siew Hong (PSB corporation) for their
assistance in Gel Permeation Chromatography.



Ms. Irene Kee (SingHealth Experimental Medicine Centre, Singapore General
Hospital) for her assistance in the animal handling and maintenance.



Ms. Han Tok Lin (Faculty of Dentistry, NUS) for her assistance in Histology.



Mr. Jackson Ong Sing Kiat and Dr. Chui Chee Kong (BIOMAT), Mr. Zhang
Jing (Biosignal and Instrumentation Lab), and fellow students at BIOMAT for
their support and encouragement throughout the fulfilling years.



To all, who have given contribution in one way or another.



And to all close friends, for being understanding during this challenging period.
Thank you for always being there during both good and bad times.



Last but not least, the author would like to thank her parents Mr. Sju Tjing
Kwang and Mrs. Tea Giok Tjian, and younger sister Ms. Lydia Sju, for their
constant love and support, without which this study would not have been
possible. Their undaunting confidence gave the author the strength to overcome
any difficulties. To them the author dedicates this thesis.

The author acknowledged the financial support by the following grants:


No. 016/06 from National Dental Centre (SingHealth), Singapore.



TDF/CD003/2006 from SingHealth (Talent Development Fund), Singapore.

iii


TABLE OF CONTENTS

PREFACE

i

ACKNOWLEDGEMENTS

ii

TABLE OF CONTENTS

iv

SUMMARY

ix

LIST OF TABLES

xi

LIST OF FIGURES

xii

LIST OF SYMBOLS

xviii

LIST OF ABBREVIATIONS

xx

CHAPTER 1: INTRODUCTION
1.1 BACKGROUND

1

1.1.1 Bone tissue engineering

1

1.1.2 Application in dentoalveolar defects

3

1.1.3 PCL-TCP scaffolds: Current drawbacks

4

1.2 RESEARCH OBJECTIVES

5

1.3 RESEARCH SCOPE

6

1.3.1 Part 1: Selective modification of PCL-TCP scaffolds
targeted for dentoalveolar reconstruction application

6

1.3.2 Part 2: Optimization of native and customized scaffolds
in vitro and their effects in initial bone healing

6

1.3.3 Part 3: Evaluation of PCL-TCP scaffolds in a clinically
relevant defect model

7

iv


CHAPTER 2: LITERATURE REVIEW
2.1 BONE PHYSIOLOGY

8

2.1.1 Composition

8

2.1.2 Morphology

11

2.2 POLY(ε-CAPROLACTONE)

13

2.2.1 Degradation of PCL polymer

14

2.2.1.1 Hydrolysis mechanism

16

2.2.1.2 Enzymatic degradation

17

2.3 TRICALCIUM PHOSPHATE (TCP)
2.3.1 Degradation mechanisms of calcium phosphate ceramics

18
19

2.3.1.1 Physicochemical degradation

19

2.3.1.2 Cellular degradation

21

2.3.1.3 Mechanical degradation

21

2.4 PCL-TCP SCAFFOLDS

22

2.4.1 Fabrication method of PCL-TCP scaffolds

24

CHAPTER 3: SELECTIVE MODIFICATION OF PCL-TCP SCAFFOLDS TARGETED
FOR DENTOALVEOLAR RECONSTRUCTION APPLICATION
3.1 INTRODUCTION

29

3.2 MATERIALS AND METHODS

30

3.2.1 Scaffold design and fabrication

30

3.2.2 Sterilization of scaffolds

30

3.2.3 Scaffold characterizations

31

3.2.3.1 Micro-computed tomography analysis

31

3.2.3.2 Gravimetric analysis

31

3.2.3.3 Compressive mechanical testing

32

3.2.3.4 Electron microscopy preparation and analysis

32

v

3.2.3.5 Molecular weight testing
3.2.4 Statistical analysis

33
33

3.3 RESULTS

34

3.3.1 Porosity measurements and 3D model analysis

34

3.3.2 Weight loss analysis

36

3.3.3 Compressive mechanical properties

37

3.3.4 Surface morphology analysis

38

3.3.5 Molecular weight analysis

40

3.4 DISCUSSION

40

3.5 CONCLUSION

43

CHAPTER 4: OPTIMIZATION OF NATIVE AND CUSTOMIZED SCAFFOLDS
IN VITRO AND THEIR EFFECTS IN INITIAL BONE HEALING
4.1 INTRODUCTION

44

4.1.1 In vitro degradation study

44

4.1.2 In vivo degradation study

45

4.2 MATERIALS AND METHODS

46

4.2.1 Scaffold design and fabrication

46

4.2.2 Sterilization of scaffolds

47

4.2.3 Animal husbandry

47

4.2.4 Scaffold implantation

48

4.2.5 Scaffold characterizations

49

4.2.5.1 Micro-computed tomography analysis

50

4.2.5.2 Gravimetric analysis

50

4.2.5.3 Compressive mechanical testing

50

4.2.5.4 Electron microscopy preparation and analysis

50

4.2.5.5 Molecular weight testing

50

vi


4.2.5.6 Histology preparation and analysis

50

4.2.6 Statistical analysis

51

4.3 RESULTS - In vitro degradation study

51

4.3.1 Porosity measurements and 3D model analysis

51

4.3.2 Weight loss analysis

57

4.3.3 Compressive mechanical properties

58

4.3.4 Surface morphology analysis

60

4.3.5 Molecular weight analysis

66

4.4 RESULTS - In vivo degradation study

67

4.4.1 Porosity measurements and 3D model analysis

67

4.4.2 Weight loss analysis

70

4.4.3 Compressive mechanical properties

71

4.4.4 Surface morphology analysis

72

4.4.5 Molecular weight analysis

75

4.4.6 Histology analysis

76

4.5 DISCUSSION

79

4.5.1 Comparison between in vitro and in vivo studies
4.6 CONCLUSION

82
83

CHAPTER 5: EVALUATION OF PCL-TCP SCAFFOLDS IN A CLINICALLY
RELEVANT DEFECT MODEL
5.1 INTRODUCTION

85

5.2 MATERIALS AND METHODS

88

5.2.1 Implant design and fabrication

88

5.2.2 Animal husbandry

89

5.2.3 Pre- and postoperative medication

90

5.2.4 Surgery 1 (Extraction and defect creation)

91

vii


5.2.5 Surgery 2 (Ridge augmentation)

93

5.2.6 Sacrifice

95

5.2.7 Micro-computed tomography analysis

96

5.3 RESULTS

96

5.3.1 Gross examinations

96

5.3.2 New bone formation

98

5.3.3 Ratio of bone volume fraction for PCL-TCP scaffolds
with respect to autografts

100

5.3.4 3D model analysis

101

5.3.5 Two-dimensional x-ray radiographs evaluation

102

5.4 DISCUSSION

104

5.5 CONCLUSION

109

CHAPTER 6: FINAL CONCLUSIONS AND RECOMMENDATIONS
6.1 FINAL CONCLUSIONS

110

6.2 RECOMMENDATIONS FOR FUTURE WORK

112

REFERENCES

114

APPENDICES

122

APPENDIX A – PART 1 STUDY

122

APPENDIX B – PART 2 STUDY

134

APPENDIX C – PART 3 STUDY

148

viii


SUMMARY

The research scope encompasses the degradation and load-bearing profile of 3D
bioresorable polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds under
enzymatic and hydrolytic conditions and subsequently to evaluate the efficacy of the
scaffolds in both small and large animal models. The purpose was to develop
scaffolds with desirable customized properties and increased degradation rates
suitable for application in dentoalveolar defects treatment. The scope of this thesis
ended with a large animal study, a stage just before preclinical trials.

Initially, the PCL-TCP scaffolds were degraded in either sodium hydroxide or lipase
solution for 0, 12, 24, 36, 48, 60, 72, 84, 96, and 108 hours. Samples were
recovered at each time point and the following properties of the scaffolds were
measured: porosity, 3D structure, weight loss, compressive strength and modulus,
surface morphology, polymer molecular weight, and histology. In the second part of
the study, in vitro and in vivo degradation behaviours of these treated scaffolds were
investigated. PCL-TCP scaffolds were monitored after immersion in standard culture
medium for 0, 6, 12, 18 and 24 weeks in vitro. In vivo degradation of the scaffolds
was performed by implanting these scaffolds subcutaneously at the back of rats for
12 and 24 weeks. Upon retrieval, analyses similar to those described above were
performed. Lastly, another in vivo study was conducted whereby PCL-TCP scaffolds
and sheets were evaluated as defect fillers and barrier membranes respectively for
novel guided bone regeneration technique in the reconstruction of localized

ix

dentoalveolar defects in a micropig model for up to 6 months. The possibility of the
PCL-TCP scaffold for use as a bone substitute was compared to the current gold
standard of using autogenous bone.

The first objective of the study was achieved with scaffolds of approximately 85%
porosity obtained after 96 hours of treatment in 3M NaOH and 12 hours in 0.1%
lipase. These pre-treated scaffolds demonstrated favourable mechanical strength,
structure, and surface morphology. Secondly, the in vivo degradation profile of
porous PCL-TCP scaffolds are comparable with the obtained in vitro profile. Further,
the degradation rate of the lipase-treated scaffolds was noted to be the highest. This
is followed by NaOH-treated scaffolds and native untreated scaffolds. Overall, the
data suggest that NaOH-treated scaffolds demonstrate the best degradation profile
and physical properties for dentoalveolar reconstruction applications. They possess
the potential to degrade in a controlled and predictable fashion and still display
favourable mechanical strength within a desired time period for new bone formation
to occur. Lastly, healing was uneventful in all micropigs showed that the PCL-TCP
scaffolds exhibited good biocompatibility. Across the tested treatment options, defect
sites augmented with autografts and collagen membranes showed the most
promising results with greater bone formation detected as compared to PCL-TCP
scaffolds and collagen membranes which were about 64% efficient. The collagen
membranes were found to offer the advantage of a reduced frequency of soft tissue
dehiscence in comparison to PCL-TCP sheets. More improvements are needed to
increase the efficiency of the PCL-TCP scaffolds in bone healing as they could ruled
out the need for harvesting grafts.

x

LIST OF TABLES

Table 3.1

Mw, Mn, and PDI of NaOH-treated and lipase-treated PCL-TCP
Scaffolds.

40

Table 4.1

Mw, Mn, and PDI of native, NaOH-treated, and lipase-treated
PCL-TCP Scaffolds in vitro.

66

Table 4.2

Mw, Mn, and PDI of native, NaOH-treated, and lipase-treated
PCL-TCP Scaffolds in vivo.

75

Table 5.1

Number of sites with soft tissue dehiscence for the implanted
autograft, collagen membranes, PCL-TCP scaffolds, and PCLTCP sheets.

98

xi


LIST OF FIGURES

Figure 1.1

Schematic diagram of research scope.

6

Figure 1.2

Schematic diagram of part 1 and part 2 study.

7

Figure 2.1

Composition of bone.

9

Figure 2.2

The assembly of collagen fibrils and fibers and bone mineral
crystals.

9

Figure 2.3

Microscopic structure of cortical and cancellous bone.

11

Figure 2.4

The hierarchical structure of bone from macrostructure to subnanostructure.

12

Figure 2.5

Chemical structure of PCL (as circled).

13

Figure 2.6

The degradation rate of PGA, PLA, and PCL.

16

Figure 2.7

The chemical structure of TCP.

19

Figure 2.8

Schematic diagram of the FDM process.

25

Figure 2.9

Sequence of the data preparation for FDM model fabrication.

26

Figure 3.1

Centrifuge tubes.

29

Figure 3.2

Illustration of scaffold with 0/60/120º lay-down pattern.

30

Figure 3.3

5x5x3mm PCL-TCP scaffold.

31

Figure 3.4

Porosity measurements of NaOH-treated and lipase-treated
PCL-TCP Scaffolds over time.

34

Figure 3.5

3D model of original scaffold (of 75% porosity) at 0 hour:
(L) top view, (R) tilted view.

35

Figure 3.6

3D model of scaffolds after 96 hours immersion in 3M NaOH:
(L) top view, and (R) tilted view.

35

Figure 3.7

3D model of scaffolds after 12 hours immersion in 0.1%
lipase: (L) top view, and (R) tilted view.

36

xii

Figure 3.8

Comparison of weight loss between NaOH-treated and
lipase-treated PCL-TCP scaffolds.

36

Figure 3.9

Compressive strength of PCL-TCP scaffolds when treated
with NaOH and lipase at pre-determined time intervals.

37

Figure 3.10

Compressive modulus of PCL-TCP scaffolds when treated
with NaOH and lipase at pre-determined time intervals.
Electron micrographs of original scaffold (of porosity 75%) at 0
hour: (L) overall view, and (R) higher-magnification view.

38

Figure 3.12

Electron micrographs of scaffold after 96 hours immersion in
3M NaOH: (L) overall view, and (R) higher-magnification
view.

39

Figure 3.13

Electron micrographs of scaffold after 12 hours immersion in
0.1% lipase: (L) overall view, and (R) higher-magnification
view.

39

Figure 4.1

Native (left), NaOH-treated (middle), and lipase-treated (right)
scaffolds.

45

Figure 4.2

Rat at the start of experiment (left) and at the end after 6
months (right).

46

Figure 4.3

50x50x3mm PCL-TCP scaffold.

46

Figure 4.4

5x5x3mm PCL-TCP scaffold.

47

Figure 4.5

Rat cages.

47

Figure 4.6

Rat shaved and scrubbed with iodine.

48

Figure 4.7

Scaffolds’ positioning.

48

Figure 4.8

Incision made (left), implanted scaffold (left, inset), scaffold
positions (right).

49

Figure 4.9

Sacrifice of rats.

49

Figure 4.10

Removal of scaffolds.

49

Figure 4.11

Porosity measurements of native, NaOH-treated, and lipasetreated PCL-TCP scaffolds after immersion in DMEM for 6,
12, and 18 weeks.

52

Figure 4.12

3D model of native scaffold (of 85% porosity) at week 0:
(L) top view, and (R) tilted view.

52

Figure 3.11

xiii

38


Figure 4.13

3D model of native scaffold after 6 weeks immersion in
DMEM: (L) top view, and (R) tilted view.

53

Figure 4.14

3D model of NaOH-treated scaffold after 6 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

53

Figure 4.15

3D model of lipase-treated scaffold after 6 weeks immersion in
DMEM: (L) top view, and (R) tilted view.

53

Figure 4.16

3D model of native scaffold after 12 weeks immersion in
DMEM: (L) top view, and (R) tilted view.

54

Figure 4.17

3D model of NaOH-treated scaffold after 12 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

54

Figure 4.18

3D model of lipase-treated scaffold after 12 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

54

Figure 4.19

3D model of native scaffold after 18 weeks immersion in
DMEM: (L) top view, and (R) tilted view.

55

Figure 4.20

3D model of NaOH-treated scaffold after 18 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

55

Figure 4.21

3D model of lipase-treated scaffold after 18 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

55

Figure 4.22

3D model of native scaffold after 24 weeks immersion in
DMEM: (L) top view, and (R) tilted view.

56

Figure 4.23

3D model of NaOH-treated scaffold after 24 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

56

Figure 4.24

3D model of lipase-treated scaffold after 24 weeks immersion
in DMEM: (L) top view, and (R) tilted view.

56

Figure 4.25

Weight loss of PCL-TCP Scaffolds In vitro.

58

Figure 4.26

Relative compressive strength of PCL-TCP Scaffolds In vitro.

59

Figure 4.27

Relative compressive modulus of PCL-TCP Scaffolds In vitro.

59

Figure 4.28

Electron micrographs taken after 6 weeks immersion in
DMEM for: (a,b) native, (c,d) NaOH-treated, and (e,f) lipasetreated scaffolds. (L) overall view, and (R) highermagnification view.

62

xiv


Figure 4.29

Electron micrographs taken after 12 weeks immersion in
DMEM for: (a,b) native, (c,d) NaOH-treated, and (e,f) lipasetreated scaffolds. (L) overall view, and (R) highermagnification view.

63

Figure 4.30

Electron micrographs taken after 18 weeks immersion in
DMEM for: (a,b) native, (c,d) NaOH-treated, and (e,f) lipasetreated scaffolds. (L) overall view, and (R) highermagnification view.

64

Figure 4.31

Electron micrographs taken after 24 weeks immersion in
DMEM for: (a,b) native, (c,d) NaOH-treated, and (e,f) lipasetreated scaffolds. (L) overall view, and (R) highermagnification view.

65

Figure 4.32

Electron micrographs of native scaffold (of 85% porosity) at
week 0: (L) overall view, and (R) higher-magnification view.

66

Figure 4.33

Porosity of PCL-TCP Scaffolds In vivo.

67

Figure 4.34

3D model of native scaffold after 3 months implantation:
(L) top view, and (R) tilted view.

68

Figure 4.35

3D model of NaOH-treated scaffold after 3 months
implantation: (L) top view, and (R) tilted view.

68

Figure 4.36

3D model of lipase-treated scaffold after 3 months
implantation: (L) top view, and (R) tilted view.

68

Figure 4.37

3D model of native scaffold after 6 months implantation:
(L) top view, and (R) tilted view.

69

Figure 4.38

3D model of NaOH-treated scaffold after 6 months
implantation: (L) top view, and (R) tilted view.

69

Figure 4.39

3D model of lipase-treated scaffold after 6 months
implantation: (L) top view, and (R) tilted view.

69

Figure 4.40

Weight loss of PCL-TCP Scaffolds In vivo.

70

Figure 4.41

Relative compressive strength of PCL-TCP Scaffolds In vivo.

71

Figure 4.42

Relative compressive modulus of PCL-TCP Scaffolds In vivo.

72

Figure 4.43

Electron micrographs taken after 3 months implantation:
(a,b) native, (c,d) NaOH-treated, and (e,f) lipase-treated
scaffolds. (L) overall view, and (R) higher-magnification view.

73

xv


Figure 4.44

Electron micrographs taken after 6 months implantation:
(a,b) native, (c,d) NaOH-treated, and (e,f) lipase-treated
scaffolds. (L) overall view, and (R) higher-magnification view.

74

Figure 4.45

H&E stain of native scaffolds after 3 months implantation.

76

Figure 4.46

H&E stain of native scaffolds after 6 months implantation.

76

Figure 4.47

H&E stain of NaOH-treated scaffolds after 3 months
implantation.

77

Figure 4.48

H&E stain of NaOH-treated scaffolds after 6 months
implantation.

77

Figure 4.49

H&E stain of lipase-treated scaffolds after 3 months
implantation.

78

Figure 4.50

H&E stain of lipase-treated scaffolds after 6 months
implantation.

78

Figure 5.1

Timeline for the complete micropig study.

87

Figure 5.2

15x10x8mm PCL-TCP scaffold (left) and 25x25x1mm PCLTCP sheet (right).

88

Figure 5.3

Bioresorbable collagen membrane from BioGide (left) and
temperature-controlled hot water bath (right).

89

Figure 5.4

Micropig housing facility at SEMC, SGH (left) and weighing of
micropig prior to the experiment (right).

90

Figure 5.5

Removal of all premolars and first molar (left), and the
extraction sites (right).

92

Figure 5.6

The flaps were re-approximated with Vicryl sutures (left), and
the defect sites were closed (right).

92

Figure 5.7

Schematic illustrations of the four tested grafting procedures.

93

Figure 5.8

Placement of PCL-TCP scaffolds and autografts (left),
followed by PCL-TCP sheets and collagen membranes (right).

94

Figure 5.9

Micropig under euthanasia (left), and the mandible was block
resected using an oscillating autopsy saw (right).

95

Figure 5.10

The recovered segment of mandible (left), the site after
removal (right).

95

Figure 5.11

The recovered segment of the mandible of a micropig.

97

xvi


Figure 5.12

Soft tissue dehiscence observed for the majority of grafts
covered with PCL-TCP sheets.

97

Figure 5.13

Bone volume fraction detected after 6 months of implantation
of autografts and PCL-TCP scaffolds for individual micropigs.

99

Figure 5.14

The average values of bone volume fraction detected after 6
months of implantation of autografts and PCL-TCP scaffolds.

100

Figure 5.15

The ratio of bone volume fraction for PCL-TCP scaffolds with
respect to autografts for individual micropigs.

101

Figure 5.16

PCL-TCP scaffold treated site: overview (left) and crosssection (right).

102

Figure 5.17

Autograft-treated site: overview (left) and cross-section (right).

102

Figure 5.18

X-ray image of a micropig’s left mandible treated with
autograft (posterior) and PCL-TCP scaffold (anterior), and
covered with collagen membrane.

103

Figure 5.19

X-ray image of a micropig’s right mandible treated with PCLTCP scaffold (posterior) and autograft (anterior), and covered
with collagen membrane.

103

Figure 5.20

X-ray image of a micropig’s left mandible treated with
autograft (posterior) and PCL-TCP scaffold (anterior), and
covered with collagen membrane.

104

xvii


LIST OF SYMBOLS

ºC

Celcius

CaCl2

Calcium Chloride

CO2

Carbondioxide

H&E

Hematoxylin & Eosin

H2O

Water

KCl

Potassium Chloride

KH2PO4

Potassium Dihydrogen Phosphate

kN

Kilonewton

kV

Kilovolt

mm

Milimeter

Mn

Number-average Molecular weight

MPa

Mega-pascal

Mw

Weight-average Molecular weight

NaCl

Sodium Chloride

Na2HPO4

Sodium Hydrogen Phosphate

NaOH

Sodium Hydroxide

O2

Oxygen

P

Probability

rpm

Revolution per minute

Tg

Glass transition temperature

xviii


Tm

Melting point

W0

Initial dry weight

Wdry

Dry weight at time t

μA

Microampere

μm

Micrometer

xix


LIST OF ABBREVIATIONS

3D

Three Dimensional

ABG

Autogenous Bone Graft

BMP

Bone Morphogenetic Protein

BV

Bone Volume

BVF

Bone Volume Fraction

CAD

Computer-aided design

CT

Computed Tomography

DMEM

Dulbecco’s modified Eagle’s medium

ECM

Extracellular matrix

FDA

US Food and Drug Administration

FDM

Fused Deposition Modeling

GA

Gravimetric Analysis

GBR

Guided Bone Regeneration

GPC

Gel Permeation Chromatography

IM

Intramuscular

ISO

International Standards Organization

IV

Intravenous

Lipase PS

Pseudomonas Lipase

Micro-CT

Micro-computed Tomography

PBS

Phosphate Buffered Saline

xx


PCL

Poly(ε-caprolactone)

PDI

Polydispersity Index

PGA

Poly(glycolic acid)

PLA

Poly(lactic acid)

QS

QuickSlice

rhBMP-2

Recombinant human Bone Morphogenetic Protein-2

RP

Rapid Prototyping

SD

Standard Deviation

SEM

Scanning Electron Microscope

SEMC

SingHealth Experimental Medicine Centre

SFF

Solid Free-form fabrication

SGH

Singapore General Hospital

STL

Stereolithography

TCP

Tricalcium Phosphate

THF

Tetrahydrofuran

TV

Tissue Volume

xxi


CHAPTER 1: INTRODUCTION

1.1

BACKGROUND

This section aims to provide background information regarding bone tissue
engineering strategy and the application in implant dentistry, as well as the current
drawback of PCL-TCP scaffolds in dentoalveolar defects treatment that lead the
author to pursue this research. Detailed research objectives and research scope are
discussed in the next and last sections respectively.

1.1.1 Bone tissue engineering
Loss of human tissues or organs is a devastating problem that can affect individuals
of all ages. Bone, a complex living tissue that provides internal support for all higher
vertebrates, is currently heralded as the most commonly replaced organ of the body.
In fact, with over 1.3 million bone repair procedures performed per year in the United
States alone [Chim, 2006], the ability to come up with an innovative and effective
defects treatment to satisfy the major clinical need has indeed been a great

challenge for many researchers.

Historically, autogenous or allogenic bone grafts have been used for treatment in
bone defects. Often, the bone repair mechanism fails as a result of magnitude,
infection or other causes. Autogenous bone grafts are those made of tissue obtained
from the patient who receives the graft, while allogenic bone grafts are those made
of tissue from a human donor, usually post-mortem. However, these techniques

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have some drawbacks. Harvesting of autogenous bone grafts induces additional
trauma and morbidity, increase operation times, and are often limited in supply. At
the site of bone transplantation, the risks of wound infection, necrosis, and resorption,
representing up to 30% of transplanted material have also been experienced [Betz,
2002; Horch, 2006]. Allogenic bone grafts present risks of possible disease
transmission and problems of religious implications [Hutmacher, 2005; Celil, 2006].
These limitations have then instigated new research aiming to provide a bone graft
engineered in the laboratory and readily available. The ultimate goal of this approach
was the regeneration rather than just the repair of skeletal tissue, and this treatment
strategy was later coined as “bone tissue engineering”.

A key component in tissue engineering for bone regeneration is the scaffold that
serves as a 3D template for initial cell interactions and the formation of boneextracellular matrix to provide structural support to the newly formed tissue. The
porous scaffold provides the necessary support for cells to attach, proliferate, and
maintain their differentiated function. The ability of the scaffold to be metabolized by
the body allows it to be gradually replaced by cells to form functional tissues [Pollok,
1996]. A well-designed scaffold for bone tissue engineering then plays an important
role in facilitating bone healing. To do so effectively, several qualities of an effective
scaffold material must be satisfied. Ideally, a scaffold should possess the following

properties: (1) a 3D structure with an increased porosity and a highly interconnected
pore network for cellular or vascular ingrowth and transport of nutrients and
metabolic waste; (2) biocompatibility and bioresorbability with controlled degradation
and resorption rates to match tissue replacement; (3) suitable surface properties for
cell adhesion, proliferation, and differentiation; and (4) sufficient mechanical

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properties to match those of the tissues at the site of implantation [Hutmacher, 2001].
The latter is extremely crucial in skeletal tissue such as bone and cartilage where
certain mechanical properties are required. These scaffolds serve as temporary
load-bearing devices that provide adequate strength and help maintain space for
new bone formation to occur [Hutmacher, 2000; Rezwan, 2006; Zhou, 2007].

1.1.2 Application in dentoalveolar defects
In implant dentistry, clinical situations involving major defects or deformities as the
result of trauma or diseases are often faced. The outcome is a compromised and
deficient alveolar ridge, which is often extended and non-contained and frequently
requiring extensive guided bone regeneration (GBR) procedures. In the dentoalveolar skeleton, an inadequate bone volume always creates problems in the
prosthetic and esthetic reconstruction of partially and completely edentulous
situations. In an era where implant borne tooth restorations have became the
standard of care for the replacement of missing teeth, the quantity and quality of the
available bony ridge is critical in determining whether ridge augmentation is required
prior to dental implant placement [Adell, 1990; Jemt, 1993]. This will not only
determine the outcome of a favorable ridge shape and the contour of the overlying
soft tissue, but also the optimal three-dimensional placement of the dental implant.
This is where the role of scaffolds come into the picture as they may eliminate the
need for an extensive bone harvesting procedure from a donor site. However in
facing a complex biological system as the human body, the requirements of scaffold

materials for bone tissue engineering in dentoalveolar application can be extremely
challenging.

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