Uh~M
UNIVERSITI SAINS MALAYSIA
EVALUATION OF THE WOUND HEALING IN FULL
THICKNESS SKIN AUTOGRAFT WITH DIFFERENT DOSES OF TOPICAL STICHOPUS SPJ EXTRACT IN SPRAGUE DAWLEY
RATS
BY
DR HUSNAIDA BINTI ABDUL MANAN@SULONG
DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT OF THE MASTER OF SCIENCE IN
CLINICAL ANATOMY, UNIVERSITI SAINS MALAYSIA.
MARCH 2010
DECLARATION
This is to certify to the best of my knowledge, this dissertation is entirely the work of the candidate, H usnaida Abdul Manan@Sulong.
(Dr Mohd Asnizam bin Asari) Main Supervisor
Department of Anatomy School of Medical Sciences
Universiti Sains Malaysia)
11
ACKNOWLEDGEMENTS
Alhamdulillah, praise be to Allah, the Most Gracious ever Merciful. I would like to express my gratitude to all those who gave me the possibility to complete this thesis.
I am deeply indebted to the following individuals for all their help, support, interest and valuable hints during the preparation of this dissertation and during the course to pursue the Master of Clinical Anatomy in School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan.
1. My beloved parents and my sister Cik Husnani Abdul Manan were of great help in difficult times.
2. My employer, Universiti Sains Malaysia for the short term grant and financial support under the academic training scheme.
3. My supervisor Dr Mohd Asnizam bin Asari from Department of Anatomy whose help, suggestions and encouragement had helped throughout the research project and thesis writing.
4. My co-supervisors, Dr Shaifuzain bin Abdul Rahman, lecturer from Orthopedic Department and Dr Venkatesh R. Naik, a lecturer from Pathology Department, School of Medical Sciences, Universiti Sains Malaysia for their support and contribution of time for the research and completion of this dissertation.
5. My Dean and Deputy Deans of School of Medical Sciences, Universiti Sains Malaysia for permission and full cooperation for me to conduct this study.
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6. My head department, my colleagues and all staffs from the Department of Anatomy who supported me in my research work. Especially I am obliged to Dr Siti Nurma Hanim, Dr Fazlina, Dr Norhida and Tuan Hj. Shukri for their great support.
7. Dr Basaruddin, a statistician of Animal Ethic Committee (AEC) for his guidance and advice on sample size calculation.
8. En Koh Chun Haw and En Ahmad Kamaludin from Central Research Unit (CRL), School of Medical Sciences and En Nik Fakuruddin from Unit Kemudahan Makmal, School of Health Sciences for helping me in the preparation of research material in their lab.
9. All staffs from Laboratory Animal Research Unit, Universiti Sains Malaysia for their great support, advice and consultation in handling animals.
10. Last but not least to Prof. Syed Mohsin Syed Jamalullail, Dean of Research Biomedical and Health Sciences for his great effort in guiding me and share his ideas and giving me an opportunity to join his team to Pulau Perhentian and having a great experience dealing with the variety species of local sea cucumbers.
This research was funded by USM Short Term Grant (304/PPSP/6139067).
IV
TABLE OF CONTENT Page
DECLARATION
..
11ACKNOWLEDGEMENTS 111
TABLE OF CONTENT v
LIST OFT ABLES Xll
LIST OF FIGURES Xlll
LIST OF PLATES xvn
LIST OF ABBREVIATIONS AND SYMBOLS XIX
ABSTRAK XXI
ABSTRACT xxm
CHAPTERl: INTRODUCTION
v
1.1 Title 1
1.2 Overview 1
1.3 Benefit of the study 1
1.4 Justification of the study 3
1.5 Objectives 4
1.5.1 General objectives 4
1.5.2 Specific objectives 4
1.6 Research questions 5
1.7 Research hypothesis 5
CHAPTER 2: LITERATURE REVIEW
2.1 Traditional and Complementary Medicine 7
2.1.1 Overview 7
2.1.2 Roles of World Health Organization (WHO) 8
2.1.3 National Policy 9
2.2 Sea cucumbers 10
2.3 Gamat (Stichopus spl) 14
2.4 Studies on other sea cucumber species 18
2.5 Rat Skin Anatomy 20
2.5.1 Overview of Rat Skin 20
2.5.1.1 Epidermis 20
2.5 .1.2 Dermis 21
VI
.
2.5 .1.3 Hypodermis 22
2.5 .1.4 Skin Appendages 23
2.5.2 Blood Supply and Lymphatic Drainage of Skin 24
2.5.3 Nerve Supply of the Skin 24
2.6 Skin Grafts 25
2.6.1 Overview of Skin Grafts 25
2.6.2 History of Skin Grafts 25
2.6.3 Classification of Skin Grafts 26
2.6.4 Indications of Skin Grafting in Clinical Setting 27
2.6.5 The Healing of Skin Grafts 29
2.6.5.1 Serum imbibition 29
2.6.5.2 Revascularization 30
2.6.5.3 Organization or Maturation 30
CHAPTER 3: METHODOLOGY
3.1 Experimental Animals and Ethical Consideration 31
3.2 Study design 31
3.3 Calculation of Sample Size 32
3.4 Duration of study 32
3.5 Preparation of Stichopus spl (Gamat) Extract 33
3.6 Preparation of Test Substances 33
3.6.1 Construction of Base 36
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3.6.2 Mixture of the Base and Test Substances 36
3.6.3 Packaging 38
3.6.4 Sterilization 38
3.6.5 Application of Topical Substances 41
3.7 Surgical procedures 43
3.7.1 Skin Harvesting and Application of Topical Substances 43
3.7.2 Skin Preservation 52
3.7.3 Preparation of Recipient Areas and Transplantation of 56 Skin Grafts
3.7.4 Post-operative Care and Monitoring 58
3.7.5 Animal Euthanasia and Graft Tissue Fixation 60
3.7.6 Preparations of Histological Sections 64
3. 7.6.1 Tissue Fixation 64
3.7.6.2 Tissue Processing 64
3.7.6.3 Tissue Embedding 65
3.7.6.4 Tissue Sectioning 65
3.7.6.5 Tissue (H&E) Staining Protocol 68
3.7.6.6 Permanent Mounting 69
3.8 Assessments of Skin Grafts 69
3.8.1 Macroscopic Assessment of Skin Grafts. 70
3.8.2 Microscopic Assessment of Skin Grafts. 70
3.8.2.1 Histopathological Assessment 70
3.8.2.1 Histomorphometric Assessment 79
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3.9 Data collection 3.10 Statistical Analysis 3.11 Study Flow Chart
CHAPTER 4: RESULTS
4.1 Introduction
4.2 General outcome of the study 4.2.1 Animal survival 4.2.2 Graft survival 4.3 Body weight changes
4.3.1 Post-harvesting body weight changes 4.3.2 Post-grafting body weight changes 4.4 Macroscopic Assessment of Skin Grafts 4.5 Microscopic Examination of Skin Grafts
4.5.1 Histopathological Assessment 4.5.2 Histomorphometric Assessment
CHAPTER 5: DISCUSSION
5.1 Introduction 5.2 Animals
5.2.1 Animal survival
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81 81 83
84 84 84 86 89 89 95 99 101 104 107
110 110 111
5.2.2 Graft survival 114
5.2.3 Body weight changes 114
5.3 Assessment of Skin Grafts 117
5.3.1 Macroscopic Assessment of Skin Grafts 117
5.3.1.1 Graft adherence 117
5.3.1.2 Graft color 118
5.3.1.3 Graft pliability 119
5.3.2 Microscopic Assessments of Skin Grafts 119
5.3.2.1 Histopathological Assessment 119
5.3.2.1 Neutophils 120
5.3.2.2 Tissue macrophages 124
5.3.2.3 Fibroblasts 125
5.3.2.4 New blood vessels 125
5.3.2.2 Histomorphometric Assessment 126
5.3.2.1 Graft Mobility 127
5.3.2.2 Hair Growth Effect 127
5.3.2.3 Surgical sutures 128
5.4 Limitation 129
5.5 Conclusion 129
5.6 Recommendation 130
REFERENCES
132X
APPENDICES
Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Appendix G Appendix H
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Table 2.1 Table 2.2
Table 2.3 Table 3.1 Table 3.2 Table 3.3 Table 4.1 Table4.2 Table 4.3 Table 4.4 Table 4.5 Table4.6 Table 4.7
LIST OF TABLES
Phylogenetic information of Sticopus spl.
Compositions of heavy metals in the crude water extract of sea cucumber Stichopus sp 1.
Clinical Classification of Skin Grafts.
Microscopic Parameters Used for Histopathological Assessment of Full Thickness Skin Healing.
Page 17 17 28 72 Descriptions of the Individual Microscopic Parameter Scores. 73 Criteria Used for Morphological Identification of Fibroblasts 74 and Inflammatory Cells.
Comparison of post harvesting body weight changes within 90 group variables.
Comparison of post harvesting body weight changes between 91 group variables.
Comparison of post harvesting body weight changes within 93 group.
Comparison of post grafting body weight changes within 96 group variables.
Comparison of post-grafting body weight changes between 97 group variables
Comparison of median score for microscopic parameters 105 between groups
Comparison of median score for graft plane separation I 08 between groups
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LIST OF FIGURES Page Figure 2.1 A sea cucumber mouth end is ringed by five feeding 11
tentacles and used in gathering food particles.
Figure 2.2 A special respiratory structure (orange in color indicated 11 by black arrows), called the respiratory tree derived from a
branch of sea cucumber lower digestive tract.
Figure 2.3 Sea cucumbers have many different means of defense 13 from predators. Some species eject sticky threads
(Cuvierian tubules indicated by black arrows) from the anus.
Figure 3.1 Steps for preparation of topical test substance. 35 Figure 3.2 Mixture of the base and Stichopus spl extract granules. 37 Figure 3.3 The mixtures were labeled with sticker in color coding. 39 Figure 3.4 All prepared syringes were sealed in sterile wrappers 39
(Sterilization Flat Reel, Westfield Medical Limited, WR050.
Figure 3.5 The radiation sterilization was implemented using Gamma 40 Cell irradiator equipment.
Figure 3.6 A) Allocation of topical substance on surgical sites at Day 42 0.
B) Allocation of topical substance on surgical sites at Day 42 5.
Figure 3.7 A) and B) A 2 em x 2 em square was centrally outlined 44 by using an applicator dipped in iodine solution within a
template
Figure 3.8 A measured landmark used for the determination of the 46 position of the template for skin harvesting.
Figure 3.9 The freed recipient skin was picked up with a tissue 47 forceps while the panniculus carnosus muscle was brushed
back down gently with saline-soaked gauze sponges held in mosquito forceps.
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LIST OF FIGURES Page Figure 3.1 0 Each bandage was sutured at different sites to avoid 49
displacement of the bandages and from easily being removed by the animals once they recovered from the anesthesia.
Figure 3.11 Elizabethan collar used to prevent biting of bandage by the 49 rat was applied once the animals were able to tolerate
orally and active.
Figure 3.12 Administration of subcutaneous prophylactic fluid 51 therapy.
Figure 3.13 The graft was folded half longitudinally with the dermal 53 surfaces in contact.
Figure 3.14 The graft was rolled in 'Swiss-roll' fashion with the tulle 53 grass.
Figure 3.15 The rolled graft was then wrapped in plain gauze (4 em x 54 4 em size) moistened in normal saline solution.
Figure 3.16 The graft was transferred in a sterile Petri dish which later 54 sealed with Para film to prevent graft dehydration.
Figure 3.17 The Petri dish with the graft inside it was then properly 55 placed in a sterilized and labeled transparent plastic zipper
bag in a small domestic type refrigerator.
Figure 3.18 Grafts were approximated to the recipient beds with eight 57 interrupted sutures using 5.0 silk reverse cutting needles.
Figure 3.19 The cage was raised about 30° to 40° at one end (near the 59 hind limb) to increase relatively circulation to brain as the
respiration more or less affected by lack expansion of the thorax after the application of the bandages and also in view of Xylazine effect which is muscular relaxation.
Figure 3.20 At Day 12, all rats were euthanized usmg lethal 61 intraperitoneal pentobarbital sodium injection (Nembutal)
(200mglkg).
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LIST OF FIGURES Page Figure 3 .21 An extra care was taken when removing the bandages 61
especially the part that covering the bandage. The bandage may adhere to the graft especially when the paraffin wet gauze was getting dried over time.
Figure 3.22 The skin graft together with its surrounding normal skin 62 (about three millimeter) as well as its underlying
connective tissue including the panniculus ca~·nosus
muscle layer was excised.
Figure 3.23 A) and B) Excised graft tissue sample 62 Figure 3.24 The purpose of placing the tissues into this cassette is to 63
prevent curling of the skin when fixed in 1 0% formalin.
Figure 3.25 The fixative solution was prepared before hand in a 63 labeled tissue container at least fifteen to twenty times
greater than the tissue volume to prevent poor tissue fixation.
Figure 3.26 A schematic diagram showing the alignment and the 67 orientation of dissected skin graft tissues in their
respective paraffin block.
Figure 4.1 Evidence of lung infection that was noted during post 85 mortem evaluation. Note the multiple white patchy
consolidations seen on lung surface.
Figure 4.2 Percentage of survived grafts for microscopic analyses 87 Figure 4.3 Representative photomicrographs showing the sequence of
events (A to D) of a graft necrosis: A) Loosened bitten bandages will B) expose of graft to environment and contamination and C) frequent manipulations of bandages causing increased in graft mobility and thus D) graft tissue necrosts.
Figure 4.4 The graph showed significant decreased in body weight 88 changes within group from Day 0 to Day 4
Figure 4.5 The graph showed no significant body weight changes 98 between and within group variables
XV
LIST OF FIGURES Page Figure 4.6 Skin grafts for macroscopic assessments: Control group I 00
(A): The graft was soft and smooth but loosely adhered to graft bed. Low dose treated group (B): The graft was tightly adhered except at the suture sites. The pliability of the graft was soft and smooth. Medium dose treated group (C) The graft was loosely adhered and the graft was minimally hardened. High dose treated group (D) The graft was tightly adhered and pliability of the graft maintained soft and smooth.
Figure A. I Each animal was acclimatized to reduce animal stress and familiarized them to human contact and daily procedures (e.g. documenting body weight of the animals).
Figure A.2 Single polypropylene cages were used to house each animal throughout the study period.
Figure A.3 Animals were kept in a same room throughout the study period.
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Plate 3.1
Plate 3.2
Plate 3.3
Plate 3.4
Plate 4.1
Plate 4.2
Plate 4.3
LIST OF PLATES
Four representative photomicrographs of 4J..Lm thickness sections that showing (A) Neutrophils, (B) Macrophage, (C) New Blood vessels and (D) Fibroblast.
(Bar= 1 OOJ..Lm)(Magnification: 1 OOOX; H&E).
Schematic mosaic image of graft tissue section that demonstrating systematic random sampling procedure to obtain views (numbered squares) for histopathological assessment considering the lateral and central regions of grafts at depth of superficial to deep dermis.
Schematic mosaic image of graft tissue section that demonstrating views (numbered squares) obtained from systematic random sampling procedure for histomorphometric assessment considering the lateral and central regions of skin grafts.
A Representative photomicrograph of 4J..Lm thickness section. Three measurements of 1 OOJ..Lm intervals were taken from each view for histomorphometric plane
separation measurement. (Bar= 1 00 J..Lm). (Magnification:
200X)(H&E)
Representative photomicrographs of 4J..Lm thickness taken from (A) Control group, (B) Low dose treated group, C) Medium dose treated group and (D) High dose treated group. Each photomicrograph showed well preserved epidermis (Ep) and dermis (d) regions with normal hair follicles (HF). The infiltration of inflammatory cells (dark small dots pointed by black arrows) into the epidermis and superficial dermis was prominent in the first there groups.
(Bar= 1 OJ..Lm) (Magnification: 40X, H&E).
Representative photomicrographs of 4J..Lm thickness taken from the control group illustrating marked infiltration of neutrophil cells (Neu) into the superficial dermis region as pointed by the arrows. HF= Hair follicle. (Magnification:
lOOX, H&E)
Four representative photomicrographs of 4J..Lm thickness taken from Control (A), Low dose treated (B), Medium dose treated (C) to high dose treated groups (D) showing the viability of the epidermal cells. Ep=epidermis.
(Bar= 1 OJ..Lm)(Magnification: I OOOX; H&E).
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Page 75
78
80
80
102
103
106
Plate 4.4
LIST OF PLATES
Representative photomicrographs of 4J..lm thicknesses taken from the (A) Control group and (B) Medium group. Both at magnification of 40X. (H&E) (C) Control group and (D) Medium group both at magnification I OOX (H&E). Plane separation (PS) was obvious in the control group as pointed by the black arrows whereas the plane separation in the medium group was not obvious. (Bar-I OOOJ..Lm).
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Page 109
p.m em eta/.
g H&E
kg
mg
ml mm w/w rpm
Gy MV
0
<
>
IQR T/CM WHO
LIST OF ABBREVIATIONS AND SYMBOLS
percent
Degree Celsius micrometer centimeter
And others (Latin: et alii) gram
Hematoxylin and Eosin kilogram
milligram milliliter millimeter
weight per weight revolution per minute Gray
Megavolts degree less than More than
interquartile range
Traditional and Complementary Medicine World Health Organization
XIX
SCE sp PPSP PPSK
LARUSM
AEC IACUC CRL SPSS PS FTSG STSG MOH
Stichopus spl Extract species
Pusat Pengajian Sains Perubatan Pusat Pengajian Sains Kesihatan
Laboratory Animal Research Unit Universiti Sains Malaysia
Animal Ethic Committee
Institutional Animal Care and Use Committee Central Research Lab
Statistical Package for Social Sciences Power and Sample Size Software Full Thickness Skin Graf
Split Thickness Skin Graft Ministry of Health
XX
ABSTRAK
EVALUASI KE ATAS PEMULIHAN LUKA TERARUH AUTOGRAF KULIT KETEBALAN PENUH DENGAN DOS-DOS BERBEZA EKSTRAK TOPIKAL STICHOPUS SPJ KE ATAS TIKUS SPRAGUE DAWLEY.
Pengenalan: Gamat merujuk kepada beberapa jenis timun laut yang seringkali diambil oleh rakyat di Malaysia sebagai ubatan tradisional. Dengan keupayaan gamat dalam penyembuhan luka yang telah diketahui setakat ini kesan topikal ekstrak gamat jenis Stichopus spl ke atas pemulihan autograf boleh dibuktikan dan dosnya yang efektif boleh ditentukan. Objektif: Kesan topikal ekstrak gamat jenis Stichopus spl pada kepekatan yang berbeza (iaitu 5, 10 dan 20 peratus berat per berat) diperhatikan ke atas pemulihan luka teraruh autograf kulit ketebalan penuh tikus betina Sprague Dawley secara makroskopik dan mikroskopik. Kaedalr kajian: Kajian ini adalah uji klinik secara rawak (randomized control trial) dengan kerangka selari (parallel design).
Haiwan-haiwan telah dibahagikan secara rawak kepada kumpulan kawalan, kumpulan rawat dos rendah (5 %), kumpulan rawat dos tengah (10 %) dan kumpulan rawat dos tinggi (20 %). Grafkulit berukuran 2 em x 2 em diambil dan disimpan pada suhu antara 6-8°Celcius selama lima hari. Sebanyak 0.5 ml ekstrak gamat dan/atau bes control diratakan pada lantai luka graf pada hari kosong dan hari kelima. Graf kulit kemudian ditransplantasikan pada lantai luka graf pada hari ke lima. Tujuh hari selepas transplantasi, graf diperhatikan secara makroskopik dan mikroskopik. Keputusan:
Pemerhatian makroskopik menunjukkan tiada perbezaan ketara antara parameter yang
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diuji (perlekatan graf, wama graf dan kelenturan graf: p>O.OS masing-masing) antara kumpulan rawat gamat dengan kumpulan kawalan. Pemerhatian mikroskopik semikuantitatif menunjukkan tiada perbezaan ketara dalam infiltrasi sel-sel inflamasi (neutrofil, makrofaj) dan proliferasi sel fibroblas dan salur darah baru graf antara kesemua kumpulan rawat tikus dengan kumpulan kawalan: p>O.OS).
Walaubagaimanapun, terdapat perbezaan yang ketara terhadap anjakan satah graf (plane separation) antara kumpulan kawalan dengan kumpulan tikus raw at dos tengah:
p<O.OOS. Dalam setiap kumpulan tikus, ukuran berat badan menunjukkan perbezaan yang ketara dari hari kosong ke hari kelima. Tiada perbezaan yang ketara dalam setiap kumpulan tikus dilihat pada hari keenam ke hari kedua belas. Kesimpulan: Aplikasi ekstak gamat jenis Stich opus spl (I 0 %) dilihat mempercepatkan penerimaan graf kulit oleh lantai graf seperti yang boleh dilihat pada pengurangan anjakan satah berikutan aplikasi ekstrak tersebut dalam kajian ini. Maka, ekstrak gamat Stichopus spl didapati ada mempunyai kesan terhadap penyembuhan luka graf kulit tikus betina Sprague Dawley.
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ABSTRACT
EVALUATION OF THE WOUND HEALING IN FULL THICKNESS SKIN AUTOGRAFT WITH DIFFERENT DOSES OF TOPICAL ST/CHOPUS SPJ EXTRACT IN SPRAGUE DAWLEY RATS
Introduction: Gamat refers to a collection of sea cucumbers most commonly consumed as traditional remedies by Malaysians. With its known healing properties to date, the effect of topical gamat extract could be beneficial in the healing process of the skin grafts and the effective dose could be determined. Objectives: The aim of this study was to investigate the effect of topical gamat from the species Stichopus spl at different concentrations (5 %, 10 % and 20 % w/w) on the healing process of skin auto grafts in female Sprague Dawley rats using macroscopic and microscopic parameters respectively. Method: This study was a randomized control trial with parallel design.
Animals were randomly divided into control, low dose treated (5 %), medium dose treated (10 %) and high dose treated (20 %) groups. Dorsal full thickness skin sheets (2cm x 2cm) were harvested and were preserved between 6° to go Celsius for 5 days. 0.5 ml of topical substances (control and gamat extracts) was uniformly applied onto the graft wound beds at Day zero and Day five post-harvesting. Skin grafts were auto- transplanted at Day five and fixed with 5-0 silk interrupted sutures. Seven days post graft transplantation, the grafts were assessed macroscopically and microscopically.
Results: The animal mortality rate observed in this study is 27.6 %. Macroscopic assessment showed that there were no significant difference, in term of graft adherence,
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graft color and graft pliability between all gamat-treated groups and the controls.
Semiquantitative microscopic assessment revealed that there were no significant difference in the infiltration of inflammatory cells (neutrophils, macrophages) and proliferation of fibroblasts and new blood vessels of the grafts between gamat-treated animals and the controls. However, there was a significant difference in term of graft plane separation between the controls and the medium dose treated group (10 %) (P<O.OOS). In all groups, the body weight showed significantly difference with declining pattern from Day zero to Day fivr post-harvesting (P<O.OS). There were no significant differences within groups seen from Day six to Day twelve. Cmrclusion: Application of Stich opus sp 1 extract (1 0 % w/w) seemed to hasten the acceptance of the skin graft by the wound bed, as demonstrated by the reduction of the plane separation following the application of the extract. Therefore, Stichopus spl extract does have an effect on the healing process of skin auto grafts in Sprague Dawley rats.
XXIV
