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UNIVERSITI PUTRA MALAYSIA

STRUCTURAL AND ULTRASTRUCTURAL STUDIES OF TISSUE ENGINEERED CORNEA

SITI SALEHA BINTI MASRUDIN

FPSK(m) 2010 15

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STRUCTURAL AND ULTRASTRUCTURAL STUDIES OF TISSUE ENGINEERED CORNEA

SITI SALEHA BINTI MASRUDIN

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2010

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SITI SALEHA MASRUDIN

MASTER OF SCIENCE

2010

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STRUCTURAL AND ULTRASTRUCTURAL STUDIES OF TISSUE ENGINEERED CORNEA

By

SITI SALEHA BINTI MASRUDIN

Thesis Submitted to the School of Graduate Studies, Universiti Putra

Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

December 2010

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I would like to dedicate this thesis to my parents and family for their

unconditional love and sacrifices, and forbearing with me during the writing

of this thesis and bring joy and happiness to my heart

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

STRUCTURAL AND ULTRASTRUCTURAL STUDIES OF TISSUE ENGINEERED CORNEA

By

SITI SALEHA BINTI MASRUDIN

December 2010

Chairman : Professor Fauziah Othman, PhD

Faculty : Faculty of Medicine and Health Sciences

This study was carried out to evaluate corneal organisation and regeneration

after transplantation of bilayer in vitro cornea construct (BICC) into the New

Zealand White Strain rabbit’s eye. Studies were conducted to investigate the

structural and ultrastructural features after corneal regeneration 90 days

post-transplantation. The epithelial cells and keratocytes were isolated from

the limbus of the rabbit and then cultured in vitro in 5 mL tissue culture

flasks. BICC and fibrin without seeded cell construct (FWCC) were produced

by mixing the epithelial cells and keratocytes with rabbit fibrin and calcium

chloride (CaCl2) and maintained in culture media. The cornea was subjected

to lamellar keratectomy before BICC and FWCC were implanted into the

defect area. The transplanted corneas were harvested after 90 days post-

transplantation for microscopic analysis and immunolabelling studies for

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cytokeratin 3. Slit lamp microscopic analysis revealed that engineered cornea

(EC) showed good corneal regeneration with no significant difference in

cornea transparency to normal cornea (NC). However, for fibrin cornea (FC),

the cornea was opaque compared to NC. In addition, the defect cornea (DC),

which was cornea without implantation after lamellar keratectomy, showed

a transparent cornea similar to NC. Morphometric analysis of the corneal

thickness was done by using Least Significant Difference (LSD) test and

Analysis of Co-Variance, showed that EC was capable of regenerative similar

thickness functional cornea as NC when compared to FC and DC (p<0.05).

Scanning electron microscope (SEM) analysis demonstrated that epithelial

surface of EC showed significantly similar features to NC compared to FC

and DC (p<0.05). Transmission electron microscope (TEM) analysis showed

that the basal lamina development of EC was similar to NC with the

establishment of cell junction compared to FC and DC. Furthermore, the EC

showed a compact stromal organization with homogenous collagen fibrils

diameter similar to NC (p<0.05). However, FC and DC showed a loose

stromal organization with heterogenous fibrils diameter, with FC fibrils

diameter were bigger than that of NC; while for DC, the fibrils diameter was

smaller than NC (p<0.05). Confocal microscopy analysis confirmed that the

regenerated epithelial cells in all groups were corneal epithelial cells by using

corneal differentiation marker, cytokeratin 3 (CK3). As a conclusion, the EC

demonstrated excellent regenerative ability of cornea and better wound

healing.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

KAJIAN TERHADAP STRUKTUR DAN STRUKTUR ULTRA TISU KORNEA YANG TELAH DIJURUTERAKAN

Oleh

SITI SALEHA BINTI MASRUDIN

Disember 2010

Pengerusi : Professor Fauziah Othman, PhD

Fakulti : Fakulti Perubatan dan Sains Kesihatan

Kajian ini telah dijalankan bagi menilai organisasi kornea dan penjanaan

semula selepas pemindahan dwilapisan konstruk kornea in vitro (BICC)

kepada mata arnab strain New Zealand White. Kajian ini telah dijalankan

bagi menyiasat ciri-ciri struktur dan ultrastruktur selepas penjanaan semula

kornea 90 hari selepas implantasi. Sel epitelium dan keratosit telah

diasingkan daripada limbus arnab dan kemudian dikultur secara in vitro

dalam 5 mL kelalang kultur tisu. BICC dan fibrin konstruk tanpa sel (FWCC)

telah dihasilkan dengan mencampur sel epitelium dan keratosit dengan

fibrin arnab dan kalsium klorida (CaCl2) dan disimpan di dalam media

kultur. Kornea arnab melalui proses keratektomi lamela sebelum BICC dan

FWCC ditransplan ke dalam kawasan dicacatkan. Selepas 90 hari, kornea

yang telah ditransplan, dituai untuk analisis mikroskopi dan kajian

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perlabelan immuno untuk cytokeratin 3 (CK3). Analisis ‘mikroskop lampu

celahan’ mendedahkan bahawa kornea yang dijuruterakan (EC)

menunjukkan penjanaan semula kornea yang baik dengan transparensi

kornea menyerupai kornea normal (NC). Namun, untuk kornea fibrin (FC),

kornea adalah legap berbanding NC. Seperkara lagi, kornea yang dicacatkan

(DC), iaitu kornea tanpa pengimplanan selepas keratektomi lamela,

menunjukkan kornea telus dan menyerupai NC tanpa sebarang ciri-ciri

nekrotik. Analisis morfometri bagi ketebalan kornea telah dibuat dengan

menggunakan ujian perbezaan minima bererti (LSD) dan ANCOVA,

menunjukkan bahawa EC mampu meregenerasi dengan baik untuk

menghasilkan ketebalan yang serupa dengan NC berbanding dengan FC dan

DC (p<0.05). Analisis mikroskop elektron imbasan (SEM) pada permukaan

epitelium EC membuktikan ia mempunyai ciri-ciri signifikan yang

menyerupai NC berbanding dengan FC dan DC (p<0.05). Analisis daripada

mikroskop elektron pancaran (TEM) menunjukan bahawa pembangunan

basal lamina dalam EC menyerupai NC dengan penubuhan simpang sel

hemidesmosom, lamina lusida, lamina densa dan rangkaian fibril berlabuh

berbanding FC dan DC. Tambahan pula, organisasi stroma EC menunjukkan

satu persamaan dengan NC yang mempunyai satu persamaan susunan

corak tenun reraga bungkusan kolagen yang padat, dan kolagen fibril EC

mempunyai garis pusat yang seragam dan secara signifikan menyerupai NC

(p<0.05). Namun begitu, kornea FC dan DC menunjukkan satu susunan

yang longgar dan menunjukkan garis pusat yang berbeza dan pelbagai, di

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mana garis pusat fibril FC lebih besar apabila dibandingkan dengan NC,

manakala untuk DC; garis pusat fibril ternyata lebih kecil daripada NC

(p<0.05). Analisis mikroskop konfokal mengesahkan sel epitelium yang telah

diregenerasikan dalam semua kumpulan adalah sel epitelium kornea dengan

menggunakan penanda pembezaan kornea, cytokeratin 3 (CK3).

Kesimpulannya, kejuruteraan semula kornea dan kemajuan penyembuhan

adalah sangat baik serta mampu memberikan harapan baru kepada kaedah

yang sedia ada.

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ACKNOWLEDGEMENTS

All praise to Allah S.W.T., the Almighty God and the Lord of the Universe.

Firstly, I am grateful to my supervisor, Prof. Dr. Fauziah Othman, who

inspired me throughout my study, financially, physically and spiritually.

Without her continuous support and interest, I will not be able to complete

this research. I am also grateful to the support and mentoring of my co-

supervisors, Prof. Dr. Asmah Rahmat and Prof. Dr. Ruszymah Haji Idrus for

their guidance and encouragement on me throughout this project.

Secondly, I would like to thank my research collaborator Dr. Norzana Abdul

Ghafar, for her cooperation and valuable advice during sample preparation

for this study. I would also like to acknowledge the support of my lab mates

and friends, especially Anushia, Zolkapli, Norhayati, Syafini, Phang,

Maslina, Lee Fong and Zarida for providing me with happiness especially in

times of sorrow. Not forgetting the staff of Microscopy Unit, Institute of

Bioscience, Universiti Putra Malaysia for their excellence services and help

during microscopy imaging.

I would like to thank my parents and siblings for their understanding,

support, and unconditional love. Their love and support have been and will

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continue to be my inspiration, and I am so blessed to have such a caring and

supporting family to enable me to complete this research.

Last but not the least; I would like to thank Ministry of Science and

Technology (MOSTI) for sponsoring the research project.

Thank you.

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I certify that a Thesis Examination Committee has met on 29 December 2010 to conduct the final examination of Siti Saleha Binti Masrudin on her thesis entitled ‘Structural and Ultrastructural Studies of Tissue Engineered Cornea’ in accordance with the Universities and Universiti College Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee recommends that the student be awarded the Master of Science. Members of the Thesis Examination Committee were as follows: Patimah binti Ismail, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Saidi bin Moin, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Sabariah binti Abdul Rahman, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Prof Dr John Paul Evangel Judson, PhD Professor Faculty of Medicine and Health International Medical University (External Examiner)

________________________ SHAMSUDDIN SULAIMAN, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 24 March 2011

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Fauziah Othman, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairperson) Asmah Rahmat, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) Ruszymah Haji Idrus, PhD Professor Faculty of Medicine Universiti Kebangsaan Malaysia (Member)

__________________________________ HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or other institution.

_______________________________ SITI SALEHA BINTI MASRUDIN

Date: 29 December 2010

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TABLE OF CONTENTS Page

ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL x DECLARATION xii LIST OF TABLES xvii LIST OF FIGURES xviii LIST OF ABBREVIATION xxi CHAPTER 1 INTRODUCTION 1 1.1 Problem Statemement 2 1.2 General Objective 3 1.3 Specific Objective 3 2 LITERATURE REVIEW 2.1 Tissue Engineering 5 2.2 Tissue Culture 8 2.3 Cornea 9 2.3.1 Corneal Surface 11 2.3.2 Corneal Epithelium 12 2.3.3 Epithelial-Stroma Junction 15 2.3.4 Corneal Stroma 18 2.3.5 Descemets’ Membrane 19 2.3.6 Corneal Endothelium 20 2.4 Cell-cell junction 22 2.4.1 Hemidesmosome 22 2.4.2 Desmosomes 23 2.4.3 Tight Junction 25 2.5 Corneal Stem Cells 26 2.5.1 Cytokeratin 3/12 31 2.6 Cornea Transparency 34 2.7 Cornea Injury 36 2.8 Cornea Transplant 38 2.9 Cornea in Tissue Engineering 39 2.10 Biomaterial 41 3 MATERIALS AND METHODS 3.1 Experimental Design 43 3.2 Isolation of Epithelial Cells and Keratocytes 45 3.3 Cultivation of Epithelial Cells and Keratocytes 46 3.4 Formation of Biomaterial from Rabbit Plasma 47

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3.5 Formation of Fibrin without Seeded Cell Construct

47

3.6 Formation of Autologous Bilayered In vitro Cornea Construct

48

3.7 Autologous Transplantation of Bilayered In Vitro Cornea Construct (BICC) and Fibrin without Seeded Cell Construct (FWCC) in Lamellar Keratectomy Rabbits

48

3.8 Sample Collection 50 3.9 Light Microscopy Analysis and Transmission

Electron Microscopy Analysis 51

3.9.1 Primary Fixation and Post-Fixation 51 3.9.2 Dehydration 52 3.9.3 Preparation of Resin Mixture 53 3.9.4 Infiltration, Embedding and

Polymerization 53

3.9.5 Preparation of Glass Knives 54 3.9.6 Semithin sectioning 55 3.9.7 Quantitative Histologic Examination 55 3.9.8 Ultrathin Sectioning 56 3.9.9 Qualitative Histologic Examination 57 3.9.10 Quantification of Collagen Fibre

Diameter in Stroma Layer of Cornea Transplanted with Bilayered In Vitro Cornea Construct (BICC) and Fibrin without Seeded Cell Construct (FWCC) Post-Transplantation

58

3.10 Scanning Electron Microscopy Analysis 58 3.10.1 Primary Fixation and Post-Fixation 59 3.10.2 Dehydration 59 3.10.3 Critical Point Drying, Mounting and

Coating 59

3.10.4 Morphometric Analysis 60 3.10.5 Quantification of Microvilli on the

Surface of Corneal Epithelium 60

3.11 Immunolabelling of Cornea Transplanted with Bilayered In Vitro Cornea Construct (BICC) and Fibrin without Seeded Cell Construct (FWCC) Post-Transplantation

63

3.11.1 Tissue Preparation 63 3.11.2 Slide Preparation 64 3.11.3 Primary Antibody Reaction 64 3.11.4 Secondary Antibody Reaction 65

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4 RESULTS 4.1 Macrosopic Observation of Pre-Harvested

Cornea 67

4.2 Light Microscopy Analysis of Post-Transplanted Bilayered In Vitro Cornea Construct (BICC) and Fibrin without Seeded Cell Construct (FWCC)

71

4.2.1 Histological Analysia of Post-Transplanted Corneal Full Thickness

71

4.2.2 Epithelial Layer Formation Post-Transplantation

74

4.2.3 Stroma Layer Formation Post-Transplantation

77

4.2.4 Morphology of Descemets’ Membrane and Endothelial Layer Post-Transplantation

79

4.3 Scanning Electron Microscopy Analysis of Post-Transplanted Bilayered In Vitro Cornea Construct (BICC) and Fibrin without Seeded Cell Construct (FWCC)

80

4.3.1 Post-Transplanted Cornea Full Layer Thickness

80

4.3.2 Morphology of Post-Transplated Anterior Epithelial Surface

82

4.3.3 Structural Morphology of Epithelial Layer and Basement Membrane

89

4.3.4 Morphology of Stroma Layer 91 4.3.5 Morphology of Descemets’

Membrane and Endothelial Layer 93

4.4 Transmission Electron Microscopy Analysis of Post-Transplanted Bilayered In Vitro Cornea Construct (BICC) and Fibrin without Seeded Cell Construct (FWCC)

94

4.4.1 Ultrastructural Morphology of Corneal Epithelial Layer

94

4.4.2 Desmosome Integrity 104 4.4.3 Ultratsructural Features of Epithelial-

Stroma Junction of Basal Lamina 106

4.4.4 Ultrastructural Features of Stroma Layer

112

4.4.5 Ultrastructural Features of Descemets’ Membrane and Endothelial Layer

119

4.5 Immunolabelling of Cornea Post-Transplanted with Cytokeratin 3/12 Protein

121

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5 DISCUSSION 124 6 CONCLUSION AND RECOMMENDATIONS FOR

FUTURE RESEARCH 148

REFERENCES 151 APPENDICES 170 BIODATA OF STUDENT 194 LIST OF PUBLICATIONS 195