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Role of fibroblasts in soft tissue biomechanics Yip, Clare 2005

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ROLE OF FIBROBLASTS IN SOFT TISSUE BIOMECHANICS  by  C L A R E YIP B . A . S c , University of British Columbia, 2002 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF M A S T E R OF A P P L I E D SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES Chemical and Biological Engineering  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A June 2005 © Clare Yip, 2005  Abstract The aim of this project was to investigate the contribution o f fibroblasts on the mechanical properties o f the dermal connective tissue in rat skin.  The research  studied the effect o f soaking, strairiing and/or poison on the viability of the fibroblasts and the stress relaxation o f skin tissue.  These studies were carried out for skin soaked  in a physiological solution (Kreb's solution) with and without addition o f a metabolic poison, 2-Deoxy-D-Glucose. Thirty male rats were used in the study and four strips of two by five centimeters skin were removed from the back o f each rat.  Skin samples were either soaked in  Kreb's solution in the presence or absence o f poison, or simultaneously soaked and strained in Kreb's solution in the presence or absence of poison.  A l l tissues were  fixed and processed for apoptosis assay and light microscopy. The viability study showed that straining o f tissues soaked in Kreb's solution significantly increases the number of apoptotic cells, and it causes more apoptotic cells compared to just soaking in Kreb's solution. Straining the skin tissues causes the extracellular matrix and cytoplasmic contacts of the fibroblasts to become disconnected and allow the tissues to relax moreeasily.  However, straining o f tissues  soaked in Kreb's solution with 2-Deoxy-D-Glucose do not significantly increase the number o f apoptotic cells.  Thus, the morphological results did not support the  ii  hypothesis that 2-Deoxy-D-Glucose kills all o f the fibroblasts by apoptosis; however, it may kill the fibroblasts by inducing necrosis. Mechanical testing (straining) involved stress-relaxation for thirty seconds at a fixed strain (13%).  Samples strained in Kreb's solution are found to have higher  reduction in stress (i.e. stress relaxation) with more apoptotic fibroblasts and less non-apoptotic fibroblasts compared to samples strained in Kreb's solution with 2-Deoxy-D-Glucose.  The difference in stress relaxation between the two treatments  was found to correlate with the number o f non-apoptotic and apoptotic fibroblasts in the samples.  A direct linear relationship was observed between the stress relaxation  differences and the ratios of apoptotic cells for the two treatments.  Samples stretched  in Kreb's solution exhibited greater relaxation and had more apoptotic fibroblasts compared to those stretched in Poison.  There is an inverse relationship observed  between the stress relaxation differences and the ratios o f non-apoptotic cells for the two treatments.  Samples stretched in Kreb's solution exhibited greater relaxation, but  had less apoptotic fibroblasts compared to those stretched in Poison.  iii  Table of Contents Abstract  ii  Table o f Contents  iv  List o f Tables  vi  List of Figures..  vii  Acknowledgement  xiv  1. Introduction..  1  2. Literature Review  8  2.1 CELLS AND EXTRACELLULAR MATRIX  9  2.1.1 Cytoskeleton  U  2.1.2 Extracellular Matrix (ECM)  26  2.2  CELL INTERACTIONS  2.3  29  Ml^URl^IvlTOTOFBIOlvffiC^  36  2.3.1 In vivo tests  42  2.3.2 Invitro tests  43  2.3.3 Studies o f fibroblasts populated collagen lattice 2.4  48  CELL DEATH  52  2.4.1 The physical characteristics of a viable and an apoptotic fibroblast  56  2.4.2 2-Deoxy-D-Glucose as an antimicrobial agent that promotes apoptosis  58  2.4.3 Apoptosis assay methods  59  3. Materials and Methods  66  3.1 LOAD FRAME  6  3.1.1 Basic components of the load frame  7  67  3.1.2 PC-based data acquisition system and control components of the loadframe.  73  3.1.3 Linearity of the displacement and force transducers  75  3.2  LIGHT MICROSCOPE  76  3.3  EXPERIMENTAL PROCEDURES  77  3.3.1  Preliminary tests  77  3.3.2  Calibration of the load frame  80  3.3.3  Solution preparation  80  3.3.4 Harvesting tissue samples  •  3.3.5 Treatment groups 3.3.6 Biomechanical experiments 3.3.7 Processing tissue for light microscopy  82 83  ;  86 87  iv  3.3.8 Performing apoptosis test on processed tissue using Roche Scientific In Situ Cell Death Detection Kit 3.4  89  MORPHOMETRIC ANALYSIS  91  • 3.4.1 Cell counting  91  3.4.2 Statistical analyses  4.  Results 4.1  96  103  BlOMECHANICAL RESULTS  103  4.1.1 Stretching in Kreb's buffer  104  4.1.2 Stretching in Kreb's solution with 2-Deoxy-D-Glucose  106  4.1.3 Comparison of the tissues soaked and stretched in Kreb's solution with and without Poison 107 4.2  MORPHOLOGICAL ANALYSIS  110  4.2.1 Fibroblasts count for the four treatment groups  5.  Discussion 5.1  116  VIABILITY OF FIBROBLASTS  5.2  Ill  116  CORRELATION BETWEEN STRESS RELAXATION AND VIABILITY OF FIBROBLASTS IN TISSUES 124 5.2.1 Relation between reduction in stress and relative amounts of non-apoptotic and apoptotic  fibroblasts  125  5.2.2 Comparison between relaxation of tissues and ratios of relative amounts offibroblasts in tissues for the two treatments 5.3  SOURCES OF ERRORS  129 135  J. 3.1 Preparation of skin tissues 5.3.2 Load frame  135 136  5.3.3 Staining, cell counting and stress relaxation measurement  137  6.  Conclusions  139  7.  Future Works  141  8.  References  Appendix A :  143 Linearity and calibration o f the force and displacement transducers 148  Appendix B .  Records o f cell counting  150  v  List of Tables T A B L E 2.1  M E C H A N I C A L ASPECT OF FIBROBLASTS A N D E C M  C O M P O N E N T S ( C A R R A W A Y ET A L . , 1992) T A B L E 2.2  10  T H E FINDINGS, T Y P E S A N D SIZE O F S K I N S P E C I M E N S ,  E X P E R I M E N T A L CONDITIONS A N D S T R A I N R A T E S F O R S O M E IN VITRO TESTS T A B L E 3.1  45  INGREDIENTS OF T H E 1 L A L T E R E D K R E B ' S S O L U T I O N (G/L) 81  T A B L E 3.2  STRESS R E L A X A T I O N D I F F E R E N C E F O R TISSUES S O A K E D  A N D S T R E T C H E D IN K R E B ' S S O L U T I O N A N D IN K R E B ' S S O L U T I O N W I T H POISON T A B L E 3.3  ..97  P A I R E D T-TEST F O R E V A L U A T I N G T H E S I G N I F I C A N C E OF  T H E R E L A X A T I O N D I F F E R E N C E S B E T W E E N TISSUES S O A K E D A N D S T R E T C H E D IN K R E B ' S S O L U T I O N A N D IN K R E B ' S S O L U T I O N W I T H POISON...., T A B L E 4.1  101  T H E RATIOS O F T H E N U M B E R OF F I B R O B L A S T S IN TISSUES  S T R E T C H E D IN K R E B ' S S O L U T I O N V E R S U S T H O S E S T R E T C H E D IN K R E B ' S S O L U T I O N W I T H POISON R A T I O S F O R B O T H N O N - A P O P T O T I C A N D APOPTOTIC FIBROBLASTS T A B L E 5.1  123  T H E R E L A T I V E A M O U N T S OF NON-APOPTOTIC A N D  N O N - A P O P T O T I C F I B R O B L A S T S IN TISSUES S T R E T C H E D I N K R E B ' S S O L U T I O N A N D IN K R E B ' S S O L U T I O N W I T H POISON T A B L E 5.2  126  T H E RATIOS O F T H E R E L A T I V E A M O U N T S O F  N O N - A P O P T O T I C A N D A P O P T O T I C F I B R O B L A S T S OF T W O TREATMENTS  132  vi  List of Figures F I G U R E 1.1 F I G U R E 2.1  4  D I A G R A M OF SKIN T H E C Y T O S K E L E T O N . (A) S C H E M A T I C S E C T I O N OF A N  A N I M A L C E L L S H O W I N G T H E L O C A T I O N O F A C T I N F I L A M E N T S ; ( B) E L E C T R O N M I C R O G R A P H OF A R E G I O N OF A F I B R O B L A S T C Y T O P L A S M S H O W I N G T W O DISTINCT C Y T O S K E L E T A L R E G I O N . . . 13 F I G U R E 2.2  A C T I N F I L A M E N T S O F T H E C Y T O S K E L E T O N IN A N  EUKARYOTIC CELL F I G U R E 2.3  15  A TYPICAL ACTIN POLYMERIZATION C U R V E ILLUSTRATING  T H E T I M E C O U R S E OF A C T I N P O L Y M E R I Z A T I O N AS M E A S U R E D B Y VISCOSITY F I G U R E 2.4  ;  17  R A T E OF G R O W T H OF A C T I N F I L A M E N T S A T DIFFERENT  CONCENTRATIONS OF F R E E ACTIN F I L A M E N T S F I G U R E 2.5  18  R A T E O F G R O W T H O F B A R B E D E N D A N D P O I N T E D E N D OF  A C T I N FILAMENTS A T DIFFERENT CONCENTRATIONS OF F R E E MONOMERS F I G U R E 2.6  19  T H E A R R A N G E M E N T OF M I C R O T U B U L E S IN A C I L I U M O R  F L A G E L L U M . CROSS-SECTION T H R O U G H T H E CILIA ILLUSTRATES T H E I R 9+2 P A T T E R N O F M I C R O T U B U L E S F I G U R E 2.7  21  A N E L E C T R O N M I C R O G R A P H OF K E R A T I N I N T E R M E D I A T E  FILAMENTS  22  F I G U R E 2.8  F I L A M E N T CROSS-LINKS  23  F I G U R E 2.9  THE M E C H A N I C A L PROPERTIES OF ELASTIC, VISCOUS A N D  V I S C O E L A S T I C M A T E R I A L S A R E R E P R E S E N T E D B Y SPRINGS (ELASTIC M A T E R I A L S ) A N D DASHPOTS (VISCOUS MATERIALS). T H E G R A P H S H O W S T H E C H A N G E S IN L E N G T H S A S A F U N C T I O N OF  vii  T I M E W H E N T H E F I X E D STRESS IS A P P L I E D T O T H E M A T E R I A L I N A P E R I O D OF T I M E .  T H E A P P L I E D STRESS IS R E P R E S E N T E D B Y 2  ARROWS F I G U R E 2.10  26 THE FIVE C O M M O N C E L L JUNCTIONS INCLUDING  C O N T I N U O U S TIGHT J U N C T I O N S ( Z O N U L A O C C L U D E N S ) , A D H E S I O N BELTS (ZONULA ADHERENS), DESMOSOMES ( M A C U L A ADHERENS), G A P JUNCTIONS, A N D H E M I D E S M O S O M E S , P R O M O T E C E L L T O C E L L A N D C E L L TO M A T R I X INTERACTION FIGURE2.il  31  A D I A G R A M M A T I C R E P R E S E N T A T I O N OF T H E F O C A L  A D H E S I O N P L A Q U E S H O W I N G T H E M O L E C U L A R BASIS O F F O R C E TRANSMISSION ACROSS THE C E L L S U R F A C E F I G U R E 2.12  34  F O C A L A D H E S I O N S D E V E L O P W I T H I N 4 H O U R S IN C E L L S I N  H I G H (HD) A N D L O W (LD) C E L L D E N S I T Y M A T R I C E S IN P R E S E N C E O F G R O W T H F A C T O R (PDGF) A S S H O W N B Y T H E P R E S E N C E O F V I N C U L I N A N D A C T I N IN C E L L S F I G U R E 2.13  34  E F F E C T OF F U N C T I O N - B L O C K I N G A N T I B O D I E S O N F O R C E  GENERATION B Y D E R M A L FIBROBLASTS.  THE FORCE GENERATED  B Y CELLS WAS M E A S U R E D USING THE C U L T U R E FORCE M O N I T O R T H E F O R C E G E N E R A T E D B Y 6 x 106 C E L L S W A S M E A S U R E D O V E R A P E R I O D OF 20 H O U R S IN T H E A B S E N C E O F A N T I B O D Y (I), W I T H 1 uG-ML-1 M A B 484 (II) A N D 2 uG-ML-1 4B4 (III) F I G U R E 2.14 SKIN.  35  S T A T I C S T R E S S - S T R A I N C U R V E OF Y O U N G A N D A G E D P H A S E I C O R R E S P O N D S TO T H E S T R E T C H I N G O F M A I N L Y  E L A S T I C FIBERS.  P H A S E II I N V O L V E S T H E A L I G N I N G A N D  S T R A I G H T E N I N G O F C O L L A G E N FIBERS.  P H A S E III D E M O N S T R A T E S  L I N E A R STRESS A N D S T R A I N R E L A T I O N S H I P .  T H E POINT B E Y O N D  viii  P H A S E III IS T H E F R A C T U R E POINT, A POINT W H E R E C O L L A G E N FIBRILS B R E A K A P A R T F I G U R E 2.15  38  A R E C T A N G U L A R S K I N S A M P L E A1B1C1D1 ( S T A T E 1) C U T  F R O M A R A B B I T ' S S K I N U N D E R N O E X T E R N A L L O A D , I.E., T H E U N D E F O R M E D S T A T E (WITH R E S I D U A L STRESSES).  A0B0C0D0 IS  T H E STRESS F R E E S T A T E W H E N T H E S A M P L E A1B1C1D1 IS A L L O W E D T O FIND ITS N A T U R A L S T A T E , I.E. T H E S T A T E W I T H O U T R E S I D U A L STRESSES, C A L L E D T H E ZERO-STRESS S T A T E .  S T A T E 2 ( A B C D ) IS  T H E S T A T E IN T H E D E F O R M E D POSITION, I.E., U N D E R E X T E R N A L LOADS F I G U R E 2.16  42 S C A N N I N G E L E C T R O N M I C R O G R A P H S OF  1-MONTH-OLD  R A T S K I N (A) R E L A X E D ; (B) S T R E T C H E D ; 4 - M O N T H - O L D S K I N (C) R E L A X E D ; (D) S T R E T C H E D F I G U R E 2.17  FIBROBLASTS' M O R P H O L O G Y AFTER SKIN WAS  S T R E T C H E D IN A S I N U S O I D A L M A N N E R (20% STRAIN) F I G U R E 2.18  50  ILLUSTRATION OF THE M O R P H O L O G I C A L FEATURES OF  N E C R O S I S A N D APOPTOSIS F I G U R E 2.20  47  F I B R O B L A S T S ' M O R P H O L O G Y IN F L O A T I N G (LD) A N D  R E S T R A I N E D (HD) M A T R I X F I G U R E 2.19  47  54  A N APPARENTLY N O R M A L D E R M A L FIBROBLAST  O B S E R V E D IN A S K I N S A M P L E S O A K E D I N K R E B ' S B U F F E R F O R 90 MINUTES BEFORE FLXATION F I G U R E 2.21  T H E BIOCHEMISTRY OF D N A F R A G M E N T A T I O N A N D THE  A P P E A R A N C E OF T H E " D N A L A D D E R " F I G U R E 3.1A  57  61  B A S I C C O M P O N E N T S OF T H E L O A D F R A M E (TOP VIEW).  S T A I N L E S S S T E E L C L A M P S T O H O L D T H E TISSUE S A M P L E , F O R C E  ix  TRANSDUCER, P U L L E Y , SCREW, WHEEL, DISPLACEMENT T R A N S D U C E R A N D E X T E N D A B L E P R O B E TIP F I G U R E 3.1B  68  C L O S E UP F R O N T V I E W O F T H E C L A M P I N G P O R T I O N OF  THE LOAD FRAME  68  F I G U R E 3.1C  C L O S E U P V I E W OF T H E C L A M P I N G PORTION OF T H E L O A D  FRAME  69  F I G U R E 3.ID  O V E R V I E W OF THE FLUID CIRCULATION S Y S T E M .  THE  P R I N C I P A L C O M P O N E N T S OF T H E C I R C U L A T I O N S Y S T E M U S E D F O R T H E M A I N T A I N I N G T H E V I A B I L I T Y OF T H E TISSUES A R E P R E S E N T E D IN THIS FIGURE.  T H E B L U E LINE REPRESENTS THE F L O W OF  WATER; THE Y E L L O W LINE REPRESENTS T H E FLOW OF K R E B ' S S O L U T I O N ; T H E R E D L I N E R E P R E S E N T S T H E F L O W OF K R E B ' S S O L U T I O N W I T H POISON; T H E O R A N G E L I N E R E P R E S E N T S T H E F L O W OF W A S T E S O L U T I O N  71  F I G U R E 3.2  PC-BASED D A T A ACQUISITION S Y S T E M  74  F I G U R E 3.3  C L O S E U P V I E W OF T H E C A L I B R A T I O N SETUP  76  F I G U R E 3.4  T H E STRESS-STRAIN C U R V E S O F T H E T W O PIECES O F  TISSUES F R O M T H E F R O N T A L D O R S A L P A R T O F T H E B O D Y ( R E D CURVE) A N D THE R E A R DORSAL PART OF T H E B O D Y (BLUE CURVE). , F I G U R E 3.5  P R E C O N D I T I O N I N G OF T H E TISSUE.  78  THE LOADING AND  U N L O A D I N G C Y C L E S A R E R E P E A T E D T H R E E TIMES F O R PRECONDITIONING PURPOSE, A N D T H E F O U R T H C U R V E OF E A C H T E S T IS U S E D IN T H E B I O M E C H A N I C A L A N A L Y S I S F I G U R E 3.6 RAT  80  THE FOUR T R E A T M E N T GROUPS H A R V E S T E D F R O M E A C H 85  x  F I G U R E 3.7  T H E 10 S Q U A R E S B Y 10 S Q U A R E S O C U L A R G R I D U S E D F O R  ENCLOSING A FIXED A R E A FOR C E L L COUNTING  92  F I G U R E 3.8  BLOOD VESSELS  92  F I G U R E 3.9  FAT LOBULES  93  F I G U R E 3.10  A HAIR FOLLICLE  F I G U R E 3.11 A A N D B F I G U R E 3.12  A PROPER A R E A FOR C E L L COUNTING  95  T H E CHARACTERISTICS OF THE NON-APOPTOTIC  F I B R O B L A S T S IN A N E G A T I V E C O N T R O L F I G U R E 4.1  95  T Y P I C A L S T R E S S - S T R A I N C U R V E S F O R S A M P L E S S O A K E D IN  K R E B ' S S O L U T I O N A N D POISON, R E S P E C T I V E L Y F I G U R E 4.2A  94  T H E M O R P H O L O G Y O F F I B R O B L A S T S IN A POSITIVE  CONTROL F I G U R E 3.13  93  105  A V E R A G E STRESS R E L A X A T I O N F O R TISSUES S O A K E D IN  K R E B ' S S O L U T I O N W I T H A N D W I T H O U T T H E P R E S E N C E OF POISON. 108 F I G U R E 4.2B  A V E R A G E R E D U C T I O N I N STRESS F O R TISSUES S O A K E D I N  K R E B ' S S O L U T I O N W I T H A N D W I T H O U T POISON F I G U R E 4.3  108  A V E R A G E STRESS R E L A X A T I O N F O R T H E 20 S E L E C T E D  R A T S W I T H TISSUES S T R E T C H E D A N D S O A K E D IN K R E B ' S S O L U T I O N W I T H A N D W I T H O U T POISON F I G U R E 4.4A  T O T A L N U M B E R OF NON-APOPTOTIC A N D APOPTOTIC  F I B R O B L A S T S IN 20 S A M P L E S F O R F O U R T R E A T M E N T G R O U P S F I G U R E 4.4B  110  114  T H E A V E R A G E N U M B E R OF N O N - A P O P T O T I C A N D  A P O P T O T I C F I B R O B L A S T S IN 20 S A M P L E S F O R F O U R T R E A T M E N T GROUPS  114  xi  F I G U R E 4.4C  R E L A T I V E A M O U N T S OF N O N - A P O P T O T I C A N D A P O P T O T I C  F I B R O B L A S T S IN 20 S A M P L E S F O R F O U R T R E A T M E N T G R O U P S F I G U R E 5.1A  115  N U M B E R OF A P O P T O T I C A N D N O N - A P O P T O T I C  F I B R O B L A S T S F O R S A M P L E S S O A K E D A N D S T R E T C H E D IN K R E B ' S SOLUTION  -.  118  F I G U R E 5.1C R E L A T I V E A M O U N T S OF N O N - A P O P T O T I C A N D A P O P T O T I C F I B R O B L A S T S F O R S A M P L E S S O A K E D A N D S T R E T C H E D IN K R E B ' S SOLUTION F I G U R E 5.2A  119  N U M B E R OF N O N - A P O P T O T I C A N D A P O P T O T I C  F I B R O B L A S T S F O R S A M P L E S S O A K E D A N D S T R E T C H E D IN POISON. ; F I G U R E 5.2B  120 R A T I O OF N O N - A P O P T O T I C T O A P O P T O T I C F I B R O B L A S T S  F O R S A M P L E S S O A K E D A N D S T R E T C H E D I N POISON F I G U R E 5.2C  121  R E L A T I V E A M O U N T S OF N O N - A P O P T O T I C A N D A P O P T O T I C  F I B R O B L A S T S F O R S A M P L E S S O A K E D A N D S T R E T C H E D IN POISON. 121 F I G U R E 5.4  R E D U C T I O N IN STRESS F O R TISSUE S A M P L E S S T R E T C H E D IN  K R E B ' S S O L U T I O N A N D IN POISON V E R S U S T H E R E L A T I V E A M O U N T S OF N O N - A P O P T O T I C F I B R O B L A S T S F I G U R E 5.5  127  R E D U C T I O N IN STRESS F O R TISSUE S A M P L E S S T R E T C H E D IN  K R E B ' S S O L U T I O N A N D IN POISON V E R S U S T H E R E L A T I V E A M O U N T S OF APOPTOTIC F I B R O B L A S T S F I G U R E 5.6.  128  D I F F E R E N C E IN STRESS R E L A X A T I O N F O R TISSUES S O A K E D  A N D S T R E T C H E D IN K R E B ' S S O L U T I O N A N D IN K R E B ' S S O L U T I O N W I T H POISON V E R S U S T H E RATIO OF T H E R E L A T I V E A M O U N T S O F  xii  N O N - A P O P T O T I C F I B R O B L A S T S IN TISSUES S O A K E D A N D S T R E T C H E D IN K R E B ' S S O L U T I O N A N D IN POISON F I G U R E 5.7  130  D I F F E R E N C E IN STRESS R E L A X A T I O N F O R TISSUES S O A K E D  A N D S T R E T C H E D IN K R E B ' S S O L U T I O N A N D IN K R E B ' S S O L U T I O N W I T H POISON V E R S U S T H E R A T I O OF T H E R E L A T I V E A M O U N T S O F A P O P T O T I C F I B R O B L A S T S IN TISSUES S O A K E D A N D S T R E T C H E D I N K R E B ' S S O L U T I O N A N D IN POISON  133  xiii  Acknowledgement I would like to thank my supervisor Dr. Joel Bert for giving me an opportunity to be his student and his friend. throughout the project. happy and sad.  I thanked him for being patient with me and guiding me  I also like to thank him for listening to my stories when I was  He taught me how to live a full life and how important it is to chase  after my dreams even i f there were doubts and obstacles.  His detenriination,  optimism, and his efforts in all aspects of his life have fascinated me, and because o f him, I have changed the way I viewed life and learned to cherish everything that I have even more.  Even though we had many problems in this project and even doubts about  whether the project could be carried on, his teachings have always been in my mind to allow me to struggle and overcome the problems and successfully completed the project. I would also like to thank my other supervisor, Dr. Ken Pinder for supporting me when I felt lost and helpless and guiding me to the end of the project.  I thanked him  for his valuable time that he spent in teaching me and discussing with me the engineering prospect of the project. patient when there were obstacles.  He taught and reminded me to be strong and H e shared his remarkable life experiences with me  and taught me how life is never easy, but the problems will eventually be resolved i f I remained determined. years.  His teachings will always be with me, guiding me to the future  I also thank my supervisor, Dr. Goran Fernlund, for his continuous support and  encouragement throughout the project.  He taught me the mechanical aspect of the  project and gave me lots of valuable comments.  He always made sure that I focused  on my objectives and helped me when I encountered problems.  I would also like to  thank Dr. David Walker with whom it has been a great pleasure to discover the world of biology.  I thanked him for being patient with me and teaching me techniques in  handling rats.  His knowledge of microscopy and biological tissues fascinated me and  motivated me to further pursue my work in tissues in the future years. I would like to thank Vlady Pavlova for all the tissue sectioning she has done for me and for teaching me the technical procedures of sectioning.  It was a great pleasure  to be her friend as she always listens to my stories and shares her life experiences with me. I would like to thank the professionals in Waxit Company for performing the apoptosis tests and allowing me to take micrographs of the tissue sections.  xiv  I am grateful for the financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC). I would also like to thank Yu-min for providing me valuable ideas in designing my experimental setup and helping me to put together and organize my thesis. I thanked him for encouraging me and giving me confidence throughout my studies. His working attitude and optimism changed my living attitude, and because of this, I learned to overcome obstacles courageously. Last but not least, I would like to thank all my family members from the bottom of my heart for their continuous support and encouragement throughout my studies. I thanked God for giving me this wonderful family. grandma for their support and love.  I thanked my grandpa and  I thanked my father (Alus) for inspiring me with  his engineering knowledge and valuable teachings about life and his great effort in supporting the family, and I thanked my mother (Amy) for her continuous caring and concerns about my future.  I am so grateful to have my sisters, Peggy, Jennie, Elsa,  Bonnie and Grace, and my beloved brother, Morgan, to be with me throughout these years.  I enjoyed every happy and sad moment that we have spent together. Without  their company and support, I would not be who I am today.  I thanked Jennie for  giving me confidence, encouraging me to achieve my goals and teaching me to believe in myself.  I thanked Peggy for her tremendous support to the family these few years,  Elsa for her care and support, and Bonnie for being there for me when I need someone to talk to when I am happy and sad. Lastly, 1 would like to thank Grace for always keeping me in company and sharing life experiences with me, especially the humorous aspects of life, and because of her presence, I learned to be a good sister.  xv  1. Introduction  Skin is the largest and the heaviest organ o f the human body, being about 1.7 m in area and approximately 5.5% of the body mass (Gartner et al., 2000).  2  It covers the  entire body and serves as a waterproof barrier that protects the internal organs and tissues from external conditions, including mechanical trauma, pressure, cuts, shear, and friction. protection.  It also absorbs ultraviolet radiation for vitamin D3 production and As an important homeostatic monitor, skin contains sweat glands that are  important in thermoregulation of the body.  It also contains sensory receptors and  nerve endings that allow the body to sense touch, changes in temperature and vibration. Skin is composed of two layers, the epidermis and the dermis. is shown in Figure 1.1. the epidermis.  A diagram o f skin  Skin can be either thick or thin, depending on the thickness o f  Thick skin, found on soles and palm, is smooth and non-hairy.  skin, on the other hand, is hairy, and is found on the remainder of the body.  Thin  The  epidermis is the thin outer layer of the skin that is 75 to 150 micrometers thick (thin skin) (Chaudhry et al., 1998).  It is composed o f stratified squamous keratinized  epithelial cells interspersed with three additional cell types, including melanocytes, Langerhans' cells, and Merkers cells.  Keratinocytes are the most abundant cells and  are found in the outer layer o f the epidermis called the stratum comeum (Potts et al.,  1  1983).  They are responsible for the production of keratin.  Melanocytes are  responsible for the production o f melanin, which is crucial for protecting the body from ultraviolet radiation.  Langerhans' cells are derived from bone marrow, and they  function as antigen-presenting cells that process and present antigens to the epidermal lymphocytes.  Merkel's cells, which are found mostly in thick skin, are believed to be  mechanoreceptors, the receptors that sense mechanical stimuli.  The second layer, the  dermis, is dense irregular collagenous connective tissue that is about 1 to 4 millimeters thick (Gartner et al., 2000). and fibroblasts.  It is composed primarily o f extracellular matrix ( E C M )  The extracellular matrix contains Type I collagen fibers (soluble and  insoluble) and elastic fibers embedded in a gel-like matrix, known as ground substance.  Fibroblasts are the most abundant cells in dermis (Figure 2).  The  primary function of these cells is the production o f collagen fibers, elastic fibers and ground substance.  They also organize and maintain connective tissue during  development and in response to injury (Grinnell, 2003).  Moreover, it has been shown  that fibroblasts are connected to their neighboring fibroblasts by specific junctions, and at the same time, they are connected to components o f the extracellular matrix ( E C M ) by cytoplasmic extensions (Grinnell, 2000).  As shown in Figure 1.1, hair follicles,  sebaceous glands, sweat glands, vessels and nerves are other components that can be found in the dermis.  2  Cell-to cell and cell-to-matrix interactions have been an area o f interest for the past 25 years.  In skin, these interactions play a role in cell communication, cell  migration, cell differentiation, cell growth, and physical organization o f cells in tissue. They are mediated by specific receptors and binding proteins that provide direct connection between adjacent cells and their extracellular matrix, allowing the cells to perceive and rapidly respond to life-threatening conditions, including temperature, source of nutrition, acidity and external mechanical stress (Beningo et al., 2001). The biomechanical properties o f human and animal skin tissues have been studied in both in vivo and in vitro studies to examine the changes they undergo caused by aging, moisture content, temperature and external mechanical stress.  Skin tissue is  continually exposed to external mechanical forces or stresses. Due to its viscoelastic properties, including elasticity and tensile strength, skin tissues are able to protect the body against injury and mechanical trauma, move in response to tension and compression and play a significant role in wound healing process.  Furthermore,  many experiments have demonstrated that natural tension exists in skin, due to the fact that fibroblasts in skin tissues are. able to generate and exert mtrinsic contractile forces to the E C M through cell adhesions (Jenkins et al., 1999).  3  • c-bi tod >«rc  -  51a i u  si**  Figure 1.1  Diagram of skin (Gartner et al., 2000).  Therefore, all additional external compression, bending or tensile forces are exerted on an already existing cellular force system, known as Cell Tensegrity. The natural force system varies between body sites, and is believed to decrease slowly with age. Maintaining this tensional homeostasis in skin is especially important in normal tissue organization and proper cellular function. Recently, fibroblast activity, including cell migration and attachment, has been hypothesized as a potential monitor that influences the mechanical properties o f skin. Figure 1.2 shows a normal dermal fibroblasts observed in rat skin.  Studies on  fibroblast responses (mechanical and biochemical) to mechanical stimulation are important in understanding how tensional homeostasis is maintained in tissue (Brown et al., 1998; Prajapati et al., 2000).  Many recent experiments and models have  4  focused on the in vitro morphological changes and mechanical role o f migrating fibroblasts on both low cell density (10 cells /mL) and high cell density (10 cells/mL) s  collagen matrices.  6  The fibroblasts are usually harvested from rat skin due to its close  resemblance to human skin in both compositions and texture to human skin. Morphologically, fibroblasts that are harvested from skin are round in shape, but in a collagen matrix, they spread through the gel, develop adhesions with adjacent cells and the E C M , and become stellate or bipolar in form.  Mechanical experiments have  illustrated the importance of cell-to-cell and cell-to-matrix adhesions in maintaining tensional homeostatic condition (endogenous equilibrium o f tension) of fibroblasts in collagen gel (Brown et al., 1998; Grinnel et al., 2003; Prajapati et al., 2000). Fibroblast motility in a collagen matrix affected the cell tensegrity in the matrix and stimulated the formation o f cell adhesions with the elements of the E C M .  As cells  adhesions with other cells and the E C M increase in number and mature, cell movements ceases, and cells begin to anchor themselves to the collagen matrix (Grinnel et al., 2003; Tamariz et a l , 2002).  However, the dermis is dramatically more  complex than these models, and these model systems cannot replicate the mechanical responses and signaling that occur as a result o f dynamic and adaptive interactions when fibroblasts in the dermis are completely surrounded by a matrix.  The precise  5  mechanisms of fibroblast mechanical linkages with each other and the E C M in vivo have yet to be investigated in skin tissue. The objective of this thesis is to determine the contribution o f fibroblasts to the mechanical properties of the connective tissue in rat skin, specifically the dermis.  In a  previous study, Martel et al. 2001 showed that mechanical disruption of the fibroblast contacts with the extracellular matrix and with each other affected tissue mechanics and induced apoptosis or programmed cell death in some fibroblasts.  However, the  current studies were carried out on strips o f fresh skin soaked in a physiological solution (Kreb's buffer) in the presence or absence o f a metabolic Poison, 2-Deoxy-D-Glucose, to remove the fibroblasts from skin pharmacologically rather than mechanically before mechanical properties o f the tissue are measured. The project consists o f two parts:  a viability study and a biomechanical study.  For the viability study, using the In Situ Cell Death Detection Kit, POD, we investigated the effects of Kreb's buffer, sketching and exposure to 2-Deoxy-D-Glucose on fibroblast's viability in the dermis o f the skin.  Using  enzymatic labeling of D N A strand breaks and observations with a light microscope, non-viable or apoptotic fibroblasts can be accurately distinguished from the viable cells.  In the biomechanical study, the force and displacement resulting from loading  6  and unloading o f skin samples soaked in Kreb's buffer with (section 4.1.2) or without the 2-Deoxy-Glucose (section 4.1.1) was recorded.  7  2. Literature Review Cell-to-cell and cell-to-matrix interactions in connective tissues have been a subject o f increasing interest during the last decade.  A detailed description o f the  structural components o f the cells and the E C M and their functions and the mechanism by which cells connect to each other and to elements of the extracellular matrix is presented in this chapter.  Since there is already an extensive literature on the  mechanical properties o f skin, the specific skin properties evaluated and the results obtained in these recent in vivo and in vitro studies are reviewed in this part of the thesis. A major challenge for most in vitro studies o f skin is maintaining the skin samples under physiological conditions, which involve temperature, p H , and moisture content control, and air and nutrient supply. Moreover, most findings in these studies only considered the importance o f collagen and elastic network and neglected the potential role that fibroblasts might play in influencing the mechanical properties o f skin.  Recently, many  studies have recognized and evaluated the importance o f fibroblasts as monitors of tissue tensegrity.  The findings, relative merits and drawbacks o f these fibroblasts' studies are  also discussed.  Finally, this chapter ends with a description of cell death, mainly  apoptosis, a metabolic Poison, 2-Deoxy-D-Glucose, that acts on the fibroblast structure and its interactions with E C M , and a description o f the assay methods that will be used to determine the viability of the fibroblasts.  8  2.1 Cells and Extracellular Matrix Connective tissue, including dermis, is made up fibroblasts and extracellular matrix.  Each fibroblast has a cytoskeleton, the skeletal network o f a cell, and it is  surrounded by the extracellular matrix (ECM), the outside supporting and anchoring materials.  The structural components of the cytoskeleton and E C M and their  functions are described in this section in detail. In terms o f Cell Tensegrity, the mechanical components of the tensional force system in most connective tissues may involve those of the cytoskeletal network o f fibroblasts and the extracellular matrix. Their contribution to the tissue tensegrity is shown in Table 2.1.  9  Table 2.1  Mechanical aspect offibroblastsand ECM components (Carraway et al., 1992).  Fibroblast's Components  E C M Components  Actin filaments  Collagen fibers  •  generate endogenous force for  •  regdaring cell shape and  insoluble collagen •  extension  resist endogenous force and provide flexibility and strength  • soluble collagen •  viscous material that dissipates the energy of deformation stored in stressed tissue  Microtubules •  transmission of endogenous force  •  sustain compression and bending force  Intermediate filaments •  resist endogenous force  Elastic fibers •  provide elasticity and recoiling mechanism for skin after an applied load  2.1.1 Cytoskeleton Cytoskeleton is a 3-dimensional intracellular meshwork of protein filaments extending throughout the cytoplasm o f a fibroblast (Janmey et al., 1998).  It is the cell  residue located outside the nucleus and the membrane-bounded organelles after removing the soluble components of the cell.  It provides mechanical support to the  cell and helps to maintain and change its shape.  It also anchors organelles and even  cytoplasmic enzymes in the cytoplasm. From a biochemical standpoint, the cytoskeleton coordinates and permits cell attachment and movement, which involve the growth and the shrinkage of protein polymers, and linkage with other proteins, especially motor proteins.  Cell  movements include both movement o f the entire cell and movement of organelles within the cell.  For instance, the cytoskeleton provides specific "tracks" for  transporting vesicles, secretory granules that deliver newly manufactured proteins, to travel from the Golgi apparatus, an organelle responsible for the modification and packaging of the proteins, to the plasma membrane domains (Bittar et al., 1996). Components of the cytoskeleton can form locomotive structures, such as cilia and flagella, o f the cells.  They also enable other cell movements, including muscle  contraction, mitosis, and phagocytosis.  Mitosis is a process of cell division in  eukaryotic cells that equally allocate replicated, chromosomes to each o f the daughter  11  cells, and phagocytosis is the cellular uptake o f macromolecules and particulate substances by plasma membrane that surrounds the substance (Campbell, 1996). The plasma membrane o f a cell is a double layer of phospholipids that has transmembrane proteins which control the passage o f materials across the membrane (Campbell, 1996).  The membrane contains cell surface receptors that recognize  specific substrate, such as signaling and protein molecules, and only allow these substrates to be transported across the membrane.  Cytoskeletal filaments can interact  with these cell surface receptors and convey these signals from the environment to the cell's nucleus.  In this aspect, cytoskeleton acts.as an extensive network of wires that  physically links the nucleus to the external environment.  This allows the cell to  initiate nuclear signals, which promote cellular responses, such as cell movements, that involve structural reorganization ofthe cytoskeleton. The cytoskeleton o f a fibroblast is a highly integrated structural network, which is composed o f three major classes o f filamentous polymers: actin filaments (microfilament), microtubules, and intermediate filaments (Figure 2.1).  These  filaments form a continuous and dynamic connection between nearly all cellular organelles and allow spatial organization o f the cellular structures in the cytoplasm. Also, they promote changes in the cell locomotion and shapes as a result of alteration  12  ofthe cytoskeletal dynamics, involving polymerization, depolymerizaton, contraction, and elongation of the protein filaments (Bronner et al., 1989).  <«>)  (b)  I  I  1  jim  Figure 2.1 The cytoskeleton. (a) Schematic section of an animal cell showing the location of actin filaments; ( b) Electron micrograph of a region of a fibroblast cytoplasm showing two distinct cytoskeletal region (Bray, 2001).  Mechanically, the cytoskeletal network of a cell can produce tensile forces that are 50 to 10,000 times greater than the gravitational force to which it is exposed (ie. 1 udyn) (Frangoes, 1993).  These tensile forces help to hold cells and tissues together, and  they are exerted on the extracellular attachments as well as neighboring cells.  Table  2.1 lists the mechanical roles that are played by the cytoskeletal components of the fibroblasts.  13  Out of the three kinds o f cytoskeletal proteins, actin is the major structural component of the cell skeleton and is recognized as one o f the most abundant proteins found in cells. Actin filaments, also called microfilaments, are located in the peripheral region o f the cell.  These thin filaments are made up o f actin, the  archetype of cytoskeletal proteins (Figure. 2.2).  Actin molecule, also called G-actin,  is a non-filamentous form o f microfilament.  It is a globular protein composed o f a  single polypeptide chain o f 375 amino acids.  Actin was first extracted and purified  from skeletal muscle, where it formed thin filaments of sacromeres (Bittar et al., 1996). Sacromeres are the contractile subunits of the muscle made up of parallel arrangement of thick myosin and thin actin filaments (Campbell, 1996).  The structures that are  built from the actin molecules participate in movements, including phagocytosis, cell crawling and muscle contraction, o f every known eukaryotic cell (Bray, 2001). actin molecules can be arranged into three forms o f structures:  parallel contractile  bundles, 2-dimensional network and 3-dimensional dense weave. also called F-actin, is the filamentous form o f microfilaments. diameter of approximately 6 to 10 nm. structure with 2 non-identical ends:  The  Actin filament,  The filaments have a  They are a tightly wound helical polar  a pointed end and a barbed end.  The pointed  end is where dissociation o f protein polymer occurs, and the barbed end is where the net association o f protein polymers occurs (Bray, 2001).  14  J lOOnm  Figure 2.2 Actin filaments of the cytoskeleton in an eukaryotic cell (Bray, 2001).  Actin filaments are especially important in muscle contraction, which involves the shortening of the sacromeres.  The thick protein filaments, called myosin, slide along  the thin actin filaments to allow contraction of the sacromeres.  Actin filaments are  also responsible for generating intracellular or endogenous force that is essential for cell shape, extension and movement (Table 2.1).  They interconnect with extracellular  matrix at specific sites o f the focal adhesion plaque, to provide the essential pathway for the signal transmission between the cell and the environment. In order to perform their cellular functions, actin molecules must polymerize to form filaments.  The cofactors that are essential in activating the polymerization o f  the actin molecules are A T P and calcium ions (Ca ). These cofactors bind to the 2+  specific binding sites of the actin molecules and fill up the spaces between the actin 15  molecules.  These extensive connections between the actin molecules permit strong  and stabilized actin filaments to be formed.  The two major types of actin filaments  that can be formed are polymeric filament and filamentous filament (Bray, 2001). Polymeric filaments are formed when globular actin monomers or G-actin associates with other monomers through their binding sites.  Filamentous filament, F-actin, is a  double stranded helix in which each actin monomer associates with four other actin monomers, creating the strongest link along each of the two strands. polymerization involves two stages as shown in Figure 2.3.  Actin  The first stage, called the  lag phase, is slower than the second stage, and it lasts for seconds to minutes depending on the initial concentration of actin monomers.  It involves the association  of actin monomers into nucleating seeds. This seed acts as a nucleus that allows more monomers to bind to each other.  The second stage involves successive addition o f  monomers to the ends of the growing actin filaments. the concentration of the monomers will fall.  A s the actin filaments grow,  The filaments will eventually stop  growing when the concentration of these filaments reaches a stable value, known as the critical concentration, at which a low concentration of actin monomers is at equilibrium with the actin filaments.  The critical concentration is also defined as the  ratio between rate of dissociation and association o f actin monomers.  16  a c t i n  f i l a m e n t s  (plus a c t i n  m  o  critical  c o n c e n t r a t i o n  n  j l S t a n  a  o  m  e  a o <  r  s  e r f  acttn m  g p  h  o  a  n  o  m  e  n  s  )  e  p o l y m e r i s a t i o n  Figure 2.3 A typical actin polymerization curve illustrating the time course of actin polymerization as measured by viscosity (Bray, 2001).  In the presence o f A T P and calcium ions, actin filaments can grow at their free ends by a reversible assembly o f actin monomers (Bray, 2001).  For instance, when a solution  of a high concentration of actin monomers is exposed to the growth cofactors, polymerization of the filaments will occur rapidly. critical concentration of monomers is reached.  The growth will cease when the  However, a mixture o f pure actin  filament with no monomers will dissolve initially into actin monomers, resulting in a negative initial rate of polymerization.  The concentration o f the actin monomers rises  until it reaches the critical concentration, at which net polymerization occurs again (Figure 2.4).  During polymerization, the barbed end and the pointed end o f each  filament grow at different rates (Figure 2.5). association of monomers.  Barbed end is the end with rapid  Its rate of dissociation or  koff  and rate of association or  ko  n  17  are 1/s and SuJVf^s , respectively, resulting in a critical concentration of 0.2uM (Bray, 2001).  Pointed end is the end with slow association of monomers.  dissociation or kofr and rate of association or kon are 0.2/s and 0.1  Its rate of  uM'V , 1  respectively,  resulting in a high critical concentration o f 2 u M (Bray, 2001). During polymerization, actin monomers continue to add to the growing filament until the monomers concentration is less than 2 u M .  At this concentration, the pointed end o f  the filament stops growing and begins to slirink, but the barbed end continues to grow. The monomers that fall off from the pointed end will be added to the barbed end. This movement o f actin monomers is known as treadmilling.  NET DEPOLYMERIZATION  \  \  NET POIYMERIZATION  Figure 2.4 Rate of growth of actin filaments at different concentrations of free actin filaments (Bray, 2001).  18-  critical concentration of barbed end  critical concentration of pointed end  concentration of free actin monomers  ^  Figure 2.5 Rate of growth of barbed end and pointed end of actin filaments at different concentrations of free monomers (Bray, 2001).  Microtubules are located near the nucleus in the central region ofthe cell. They are the thickest filaments o f the three types, and they are interspersed by organelles and granular materials of the cell.  Microtubules are straight and flexible  rods, measuring about 25 nm in diameter and 200 nm to 25 urn in length (Campbell, 1996).  These hollow rods are polymers o f tubulin, of which there are two closely  related kinds, a-tubulin and p-tubulin (Bittar et al., 1996).  A microtubule is  constructed by adding tubulin molecules to its ends. It can also disassemble into tubulin molecules and assembled again into filaments at another location o f the cell. Microtubules are functionally interconnected with both actin filaments and intermediate filaments.  They are responsible for the transmission o f forces that are  generated by the actin filaments throughout the cell.  Due to their flexibility and  hollow cylindrical form, microtubules can sustain external compression and bending  19  force (Table 2.1).  They can reinforce cell shape and support the cell.  They also  dominate intracellular movement, since they act as pathways along which organelles can travel,  hi terms o f cell locomotion, specific arrangement o f microtubules is  responsible for the beating o f the motile structures, such as flagella and cilia, which exist in some eukaryotic cells. Each cilia and flagella has nine doublets o f microtubules arranged in a ring, with two single microtubules in the center of the ring (Figure 2.6).  There are arms, made up of motile proteins called dynein, which extend  from each doublet of microtubules.  When cilia or a flagella move, the arms perform  bending movements caused by changes in the shape o f the dynein (Jockusch et al., 1995).  During the conformational changes of the dynein, A T P , an  adenme-conlaining nucleoside triphosphate that stores energy for cells to use, provides the energy for these changes (Campbell, 1996).  20  Figure 2.6 The arrangement of microtubules in a cilium or flagellum. Cross-section through the cilia illustrates their 9+2 pattern of microtubules (Bray, 2001).  Intermediate filaments are found in the central region of the cell, and they are interconnected with microtubules.  They are irregular, flexible ropes that are made up  of subunits that belong to a family o f proteins called keratin (Figure 2.7).  The  diameter of each filament is about 8 to 12 nm, which is larger than the diameter of the microfilaments but smaller than that of microtubules (Campbell, 1996). also disassemble and assemble in different locations of the cell.  They can  Intermediate  filaments are responsible for mamtaining cell shape due to its ability to resist endogenous force generated by actin filaments (Table 2.1). positioning o f some organelles, such as nucleus.  They are involved in the  The nucleus is embedded in a  network o f intermediate filaments, and it is anchored near the center of the cell by these filaments.  Moreover, intermediate filaments maintain the shape of the nucleus  21  by lining the interior o f the nuclear envelope, the membrane that encloses the nucleus and controls materials going into and out of the nucleus (Campbell, 1996).  Figure 2.7 An electron micrograph of keratin intermediate filaments (Coulombe et al., 2002).  The three main types o f cytoskeletal filaments do not have structural form or strength i f they exist by themselves.  A n integrated cytoskeleton is constructed when  accessory proteins connect the three types of cytoskeletal filaments together and to other parts o f the cell (Figure 2.8).  The accessory proteins form infinite side-arms  and cross-bridges that project from the major cytoskeletal filaments, connecting one filament to the next (Bray, 2001). The filaments are bound along their lengths by these accessory side-arms, allowing them to become thicker, more stable and rigid supporting structures. filaments.  Therefore, the accessory proteins add enormous strength to the  For instance, they act as flexible links that connect the simple components,  such as actin filaments, to build rigid structures that can sustain large bending and compressive forces.  Accessory proteins are especially important in monitoring the 22  assembly o f bundles of filaments, which permit changes in cell shape, and the generation o f intracellular movements.  Each major type of filaments has its own  associated accessory proteins. (A)  Figure 2.8  Filament cross-links (Bray, 2001).  The two principal types o f microtubule-associated proteins (MAPs) are fibrous microtubule-associated proteins and force producing associated proteins (Bittar et al., 1996).  Fibrous microtubule-associated proteins, including MAP2 and tau, are  responsible for controlling the assembly and bundling o f microtubules (Bittar et al., 1996).  Force-producing M A P s , such as kinesin, dynein and dynamin, are involved in  microtubule-dependent movement, which include the beating actions of the cilia and flagella (Bittar et al., 1996).  The cyclic movements of these locomotive structures are  23  caused by the conformational changes in the shape ofthe force-producing protein molecules.  Force-producing M A P s are also involved in the intracellular transporting  processes, in which motor protein molecules attach to the organelles, such as vesicles, and allow the organelles to walk along the microtubules of the cytoskeleton. There are more than 50 actin-binding proteins that regulate the assembly and disassembly of actin filaments or the cross-linking o f actin filaments.  The major  types of actin-binding proteins include profilins, barbed-end-cappingproteins and pointed-end-capping protein (Bronner et al., 1989).  Profilins bind to actin monomers  and inhibit polymerization of actin filaments. Barbed-end-capping proteins, such as gelsolin and villin, bind to specific end of an actin filament and inhibit addition of actin molecules.  Pointed-end-capping proteins, such as acumentin, spectrin and 3-actinin,  aid in regulating the length of the actin filaments. The two major types o f intermediate-filament-associated proteins (IFAPs) are vimentin and desmin (Bronner et al., 1989).  They are both involved in the formation  of intermediate filaments. Each type of these protein monomers has the ability to self-assemble into polymers and to assemble with other intermediate filament-associated proteins to form mixed polymers. In the biomechanical standpoint, the cross-linking networks of cytoskeletal filaments of cytoskeleton exhibits properties of both viscous solutions and elastic  24  solids.  The viscoelastic behavior of the cytoskeleton allows its deformations to be  dependent on time.  The elastic mechanical properties, which are time-independent,  are represented by springs, and the viscous mechanical properties are represented by dashpots (Figure 2.9).  Elastic materials consist of molecules that connect to each  other with large number o f strong cross-linking bonds.  When a fixed stress is applied  to an elastic material, its deformations are shared among the cross-linking bonds, resulting in only small changes in its length.  The energy of deformation is then stored  in the molecules and bonds of the elastic material.  If the stress is removed from the  material, it will return to its original shape. O n the other hand, viscous fluids are made of molecules that are not strongly connected to each other. When a fixed stress is applied to a viscous fluid, a flow will result, and its flow properties can be measured by viscosity. The flow of the fluid aids in dissipating the energy o f deformation that is stored in the molecules and bonds.  However, i f the stress is removed from this  fluid, it will not return to its original form.  The behavior of the cytoskeleton not only  depends on time, but also depends on the applied stress. it will show a series of deformations.  When a force is applied to it,  Initially, it will behave like an elastic material,  since the force will be shared among the bonds between its molecules.  As time  elapses, some of these molecules will detach and rotate, causing a flow relative to each other.  If the stress is removed from the material, it will tend to resume its original  25  state as the molecules become cross-linked by the bonds again.  However, it will  never return to its original shape, and its deformation in length will reach a new plateau (Figure 2.9).  The ability of the cytoskeleton o f a fibroblast to dissipate the energy o f  deformation is crucial to minimize the level o f cell damage that is caused by any external stress.  ^  spring  lime  {USTlC  VISCOUS  VISCOtlASTIC  Figure 2.9 The mechanical properties of elastic, viscous and viscoelastic materials are represented by springs (elastic materials) and dashpots (viscous materials). The graph shows the changes in lengths as a function of time when the fixed stress is applied to the material in a period of time. The applied stress is represented by 2 arrows (Bray, 2001).  2.1.2 Extracellular Matrix ( E C M ) Located outside all eukaryotic cells, extracellular matrix provides support and anchorage for cells. Cells must be properly anchored in order to grow, differentiate and proliferate.  Extracellular matrix allows cells to adhere to specific external sites,  called ligands, which enables the cell to receive stimuli from the environment.  The  cell can then accommodate to the stimuli by modulating the intracellular organization  26  o f actin filaments.  This allows the cell to generate responses.  For example, a cell  can generate movements and escape from life-threatening stimuli and can move quickly towards favorable stimuli.  Extracellular matrix can regulate cell movement  by either triggering polymerization o f actin into fibrous bundles or depolymerization of actin filaments into monomers.  In addition to cell locomotion, external stimuli that  travel from the extracellular matrix to the cell can also affect the activity o f genes in the nucleus, especially in the production of messenger R N A .  Messenger R N A  delivers the genetic information from the nucleus to the cytoplasm and specifies the primary structure of a specific protein during protein synthesis (Campbell, 1996). The changes in the production of messenger R N A will cause changes in the primary structure of the newly produced protein molecules, leading to the generation o f cellular responses. Extracellular matrix consists o f ground substance and fibers.  In skin, the  extracellular matrix is consisted of Type I collagen fibers, and elastic fibers embedded in ground substance, known as dermatan sulphate.  The major components o f  dermatan sulphate are proteoglycan and structural proteins.  The precursors of these  components are secreted by cells, mainly fibroblasts, into the extracellular matrix. The most abundant protein fibers are collagen fibers.  Tropocollagen, about 280 nm  in length, is the precursor of collagen that is produced and secreted into the  27  extracellular matrix by fibroblast, chondroblasts and osteoblast.  Fibroblast is a type  o f cell in connective tissue that secretes the protein ingredients o f the extracellular matrix; chondroblasts is a cell that arises from the embryonic connective tissue, called the mesenchyma, and forms cartilage; osteoblast is a cell which arises from a fibroblast and is responsible for the production of bone (Campbell, 1996). Tropocollagen molecules can aggregate to form fibrils, and these fibrils can then form bundles o f collagen fibers. Collagen fibers make up 70% of dry weight o f skin (Chaudhry et al., 1998).  They resist endogenous force and provide connective tissue with flexibility  and tensile strength (Table 2.1).  Another type of important fibers are elastin, and they  make up 4% o f dry weight o f skin.  Elastic fibers are made o f cross-linking proteins,  known as desmosine and isodesmosine (Silver et al., 2001).  As described in Table  2.1, they form a scattered, delicate network o f small fibers between the collagen fibres that contribute to the elasticity or the recoiling mechanism o f skin after a stress or deformation has been applied (Reihsner et al., 1995).  Both elastin and collagen fibers  are embedded in ground substance, the gelatinous matrix of proteins. substance makes up 20% o f dry weight o f skin (Chaudhry et al, 1998).  Ground Dry weight  constitutes approximately 24% of total weight o f skin, and the remainder (76%) o f the skin is water (Chaudhry et al., 1998).  Ground substance consists o f a network o f  proteoglycans, unbranched polysaccharide chains, which are bond to a protein core.  28  They are glycoproteins that are rich in carbohydrate.  They can serve as  transmembrane links between the cytoskeleton and the extracellular matrix. Structural glycoproteins are composed o f protein chains that bond to branched polysaccharides.  The major structural protein is fibronectin, which allows cells to  attach to their extracellular matrix.  Fibronectin bind to the receptor proteins, known  as integrins, which are embedded in the plasma membrane o f the cell.  Integrins  connect the inner peripheral side ofthe cell to the actin filaments o f the cytoskeleton. They can help to initiate cytoskeletal responses that allow cells to cope with changes in the extracellular matrix by transmitting stimuli from the extracellular matrix to the cytoskeleton.  These stimuli can trigger the production of chemical signals that affect  the activities in the nucleus, such as messenger R N A production.  2.2  Cell interactions  Many intensive studies in the past decade have shown that both cell-to-cell and cell-to-matrix interactions play a role in cell communication, cell differentiation, cell growth, and physical organization o f cells in tissue.  These interactions involve  specific binding proteins that provide direct connection between the cytoskeletons o f the adjacent cells, mostly fibroblasts, and their extracellular matrix, allowing the cells to perceive and rapidly respond to life-threatening conditions.  29  In skin, the five common cell junctions that permit cell to cell and cell to matrix  interactions are continuous tight junctions (zonula occludens), adhesion belts (zonula adherens), desmosomes (macula adherens), gapjunctions, and hemidesmosomes (Figure 2.10).  Continuous tight junctions are made up o f integral membrane proteins  that cause fusion o f cell membrane by sealing epithelial cells together.  Adhesion  belts contain contractile microfilament bundles that control cell shapes and promote cell-to-cell adhesion.  Desmosomes and hemidesmosomes, on the other hand, are the  most abundant cell junctions that are found in the epidermal layer o f skin.  In the  mechanical aspect, desmosomes increase the strength of skin cell adhesion and resist abrasion, and they have bundles of tonofilaments or cytokeratin filaments that are responsible for transmitting stress throughout the cells.  In skin, gap junctions are  very important to cell-to^cell communication. They consist of narrow channels found between the membranes o f two adjacent cells.  These channels are made up o f  proteins called connexins that allow passage o f important inorganic ions, proteins, signals and hormone molecules from cell to cell.  They are especially important for  embryonic development, cell locomotion and tissue fluid balance.  30  Figure 2.10 The five common cell junctions including continuous tight junctions (zonula occludens), adhesion belts (zonula adherens), desmosomes (macula adherens), gap junctions, and hemidesmosomes, promote cell to cell and cell to matrix interaction (Dirby et al., 1998).  31  In the dermis, the macromolecules found in the E C M , including proteoglycans, glycoproteins and hyaluronan, interact to form large supramolecular structures.  Cells  of dermis, mostly fibroblasts, not only interact with each other, but also interact with elements of E C M . Almost 80 years ago, Paul Weiss demonstrate the dynamic interaction between cells and their physical environment by studying the varying stellate or bipolar morphology o f fibroblasts when they are cultured in a blood plasma clot (Grinnell, 2003).  The adhesion between fibroblasts and extracellular matrix  ( E C M ) are mediated by specific surface receptors, known as integrins (Frangos et al., 1993).  These transmembrane receptors recognize a common sequence, arg-gly-asp  (RGD), which is found within many different E C M proteins.  They are heterodimers  consisted of one a and one B subunit, both o f which embed and span the plasma membrane.  Both subunits possess a large extracellular domain to form the binding  region for E C M proteins and a membrane-spanning region to interact with the actin cytoskeleton via binding to the cytoskeletal proteins (Jenkins et al., 1999).  a subunits  can determine the binding specificity o f the receptor to extracellular proteins by associating with specific P subunits.  Recently, 16 a- and 8 p- subunits have been  identified, and these subunits combine into at least 20 different mtegrins (Clark et al., 1995; Horwitz, 1997).  When the cells attach to their E C M , the cell-surface integrin  receptors cluster at specific sites, known as focal adhesions.  They interact with both  32  the molecules of the extracellular matrix (i.e. fibronectin) and the actin filaments of the cell's cytoskeleton, forming the organized focal adhesion plaque (Figure 2.11).  The  relationship that is established between the extracellular environment and cell interior via integrins is very close, since disruption of actin filaments with cytochalasin (actin-disrupting poison) leads to a parallel disruption of the fibronectin matrix on the outside (Schoenwaelder et al., 1999).  O n the outside of the cell, focal adhesion  plaque provides anchorage to its extracellular matrix or to other cells. of the cell, it provides sites of attachment for the cytoskeleton.  On the inside  In between the  integrins and the actin filaments are some cytoskeletal-associated proteins, including vinculin, a-actinin and talin, which are found in the adhesion complex.  These  proteins form a molecular link between the integrins and the actin filaments (Figure 2.12).  In addition, studies carried out on dermal fibroblasts have shown that integrins  (ie. p i integrins receptors) play an important role in the generation of mtrinsic tensional forces by fibroblasts, since function-blocking antibodies directed against integrins can significantly reduced the tensional forces generated by the fibroblasts in three-dimensional matrix (Figure 2.13).  33  Figure 2.11 A diagrammatic representation of the focal adhesion plaque showing the molecular basis of force transmission across the cell surface (Frangos, 1993).  BSA  LPA  PDCF  Figure 2.12 Focal adhesions develop within 4 hours in cells in high (HD) and low (LD) cell density matrices in presence of growth factor (PDGF) as shown by the presence of vinculin and actin in cells (Tamariz et al., 2002).  34  0  6  10 Time  F i g u r e 2.13  15  20  (Hour*)  Effect of function-blocking antibodies on force generation by  d e r m a l fibroblasts. force monitor.  T h e force generated b y cells w a s m e a s u r e d u s i n g the c u l t u r e  T h e f o r c e g e n e r a t e d b y 6 x 106 c e l l s w a s m e a s u r e d o v e r a p e r i o d  o f 20 h o u r s i n t h e a b s e n c e o f a n t i b o d y (I), w i t h 1 u g - m l - 1 m A b 484 (II) a n d 2 u g - m l - 1 4B4 (III) ( J e n k i n s e t a l . , 1999).  Focal adhesions not only physically link adjacent cells to each other or to components of E C M , but also promote signal conduction between the ligands outside the cell and the components of the cytoplasm, known as the outside-in signaling.  In  addition to outside-in signaling, integrins in focal adhesion also respond to signals coming from inside the cell, known as the inside-outside signaling.  These  inside-outside signaling can alter the affinity of an integrin for a specific ligand or change the strength with which the integrins bind.  The integrin signal transduction  can directly affect gene expression, cell division, cell differentiation, and cell locomotion (Hancock, 1997).  Without focal adhesion and attachment to extracellular  matrix, the cells round up and lose their connection with the environment, causing cell death.  35  From a mechanical aspect, focal adhesion plaque o f a cell serves as a pathway by which tensile forces are transmitted across the cell surface.  The viability o f a cell is  determined by its ability to detect and resist these external forces.  When a cell  generates tensile forces, the extracellular matrix anchors will resist the forces and produce feedback that can help to regulate cell functions.  When the cell is exposed to  external force, the adhesion complex can initiate the production o f intracellular messengers, or can directly mediate nuclear signals. factors then initiate second signals.  Cell surface receptors for growth  Collectively, these signals are integrated by the  cell to generate various responses, including changes in cell shape, cell growth, locomotion, attachment, protein synthesis, gene expression, proliferation, and differentiation.  2.3 Measurement of bioraechanical properties of skin  Due to the importance of the mechanical properties o f skin, measurements o f these properties using different testing techniques and loading machines have been performed. The most common properties that are studied are stress-strain relationships during uniaxial elongation o f skin (Belkoff et al., 1991; Lank et al., 1974; Oxlund et al., 1988; Silver et al., 2001), creep behaviour, i.e., measurement o f tissue elongation during  36  constant load (Lanir et al., 1974; Vogel, 1977), stress relaxation, i.e., measurement of the force on a tissue under constant deformation ( Wan Abas, 1995; Vogel, 1985) and measurement of hysteresis resulting from cyclic loading and unloading (Vogel, 1997, 1983 and 1981). Skin is a viscoelastic material that exhibits a non-linear stress-strain behaviour. Stress (a) is the force per unit area, and it measures the load that a tissue bears.  Strain  (e), on the other hand, measures the lengthening or shortening of a tissue with respect to its initial length in response to stress. sample length.  It is the change in length divided by the initial  Skin does not behave like Hookean materials (ie, materials that obey  Hooke's law, a (stress) = E (modulus of elasticity) * e (strain), in that they deform elastically with force increasing linearly with applied deformation) (Foutz et a l , 1992). When skin is exposed to a mechanical load, its mechanical response involves both a viscous component associated with energy dissipation and an elastic component associated with energy storage.  This complex mechanical response is mainly related to  the structure and properties of the collagen, elastic fibers and the proteoglycans that are found in the dermis (Silver et al., 2001).  Two typical static stress-strain curves for  young and aged skin are shown in Figure 2.14.  37  Strain (Length)  Figure 2.14 Static stress-strain curve of young and aged skin. Phase I corresponds to the stretching of mainly elastic fibers. Phase II involves the aligning and straightening of collagen fibers. Phase III demonstrates linear stress and strain relationship. The point beyond phase in is the fracture point, a point where collagen fibrils break apart (Dirby et al., 1998).  These curves are characterized by a low-stiffness region at small strains followed by a dramatic increase in stiffness as the strain becomes large (Bischoff et al., 2000). stress-strain behavior of skin is composed of 3 phases.  The  In phase I (up to strains of about  0.3), the collagen fibers in skin are not aligned, and they offer little resistance to deformation.  This is the zone o f increasing strain with minimal elevation o f stress,  which mainly corresponds with the stretching o f elastic fibers and insoluble collagen fibers.  The elastic behavior o f skin is important in ensuring shape recovery after  deformation.  This initial part o f the stiess-strain curve, also known as the low-stiffhess  region, is the physiological range in which the skin usually is exposed to.  Thus, most  researchers have focused their studies on this part o f the curve o f small forces and  38  strains.  Between higher strains o f about 0.3 and 0.6, phase II describes the increase in  stress needed to produce further strain as a result o f collagen fibers aligning and straightening parallel to the maximum stretch direction (Dirby et al., 1998).  The  viscous sliding o f collagen fibrils during alignment with the force directions allows energy applied to the skin to be partially dissipated.  These changes in the collagen  fibril orientation during deformation o f the dermis are critical to maintaining the large extensibility and increasing the stiffness of human skin (Manschot et al., 1986). At even higher strain, all collagen fibers become aligned and increase in length due to the stretching of collagen fibers.  At phase III, the stress-strain relationship becomes linear.  The slope o f the straight region of the curve is known as the Young's modulus or a modulus o f elasticity and is a measure o f "stiffness" of the tissue (Alexander et al., 1999).  Stiffness is important for evaluating a specimen's loading response and  resistance of the tissue fibers to damage from impacts (i.e., collisions and falls) (Foutz et al., 1992).  The stiffness in human skin ranges from about 15 to about 150 N / m m  (Escoffier et al., 1989).  2  Scanning electron micrographs of 4-month-old rat skin in both  relaxed and stretched (ie, linear region) state are shown in Figure 8 (Belkoff et al., 1991). Finally, in the yield and failure region (strains o f about 0.6 or above), the collagen fibrils break apart until the rupture o f the skin tissue occurs. load and strain that the skin tissue can resist.  This fracture point is the ultimate  Tensile strength is calculated by dividing  39  ultimate load by the cross sectional area (Vogel, 1997).  It is a measurement which  determines the load that the material can withstand per unit area (Lanir et al., 1974). Fung et al. (1993) demonstrated that i f a tissue specimen is subjected to repeated loading and unloading at a constant rate of elongation, the stress-strain curve shifts to the right along with an increase in the low strain phase (i.e. phase I in Figure 2.14) (Fung, 1993).  The stress-strain curve shifts to the right in the first three cycles  of loading pattern and then becomes repeatable without any shifting (Lanir et a l , 1974).  In the first few cycles o f loading, the structural organization of the tissue  specimen changes until it reaches a steady state where no further change occurs. Then the specimen is said to have been preconditioned.  Preconditioning is usually  performed in order to obtain reproducible experimental results. Many biomechanical studies involve comparing the skin tissues under stress with control skin tissues with no stress to evaluate the effect of mechanical loads on the morphology o f skin tissues.  Chaudhry et al. (1998) realized that stresses, known as  residual stresses, remain when a tissue specimen is under no external load (Fung, 1993).  When a skin sample is taken from the body, it becomes elliptical in shape.  This shows that residual stresses in skin are released when it is allowed to find its natural position even under no external load. Figure 2.15 shows a rectangular skin sample cut from a rabbit's skin under no external load (i.e. the undeformed state with  40  residual stress) and a stress free state when the sample is allowed to find its natural state (i.e. zero-stress state with no residual stress).  It is crucial that one takes residual  stresses into account, since stress concentration and stress gradient were reduced compared to the very high stress concentration and stress gradients when residual stresses were not taken into account.  Therefore, zero stress state of a tissue specimen  should be used as the control or reference state and compare to its deformed state to evaluate the effects o f external loads on the tissue. Many past biomechanical experiments performed on skin tissues have been carried out in both in vivo and in vitro conditions. In addition to human skin samples, those from pigs, rats and rabbits have also been used because o f their close resemblance to human skin.  These studies mainly illustrated the contribution o f E C M components  to the mechanical aspect o f skin.  Recently, the potential role o f fibroblasts in regulating  skin tissue tensegrity and wound closure has also been examined by in vitro studies. The current investigation will focus on the findings and the drawbacks of the various techniques used in these studies.  41  After cut  Before c u t  Loaded s t a t e  A,  Figure 2.15 A rectangular skin sample A1B1C1D1 (state 1) cut from a rabbit's skin under no external load, i.e., the undeformed state (with residual stresses). A0B0C0D0 is the stress free state when the sample A1B1C1D1 is allowed to find its natural state, i.e. the state without residual stresses, called the zero-stress state. State 2 (ABCD) is the state in the deformed position, i.e., under external loads (Chaudhry et al., 1998).  2.3.1 In vivo tests In vivo tests are used to examine how skin reacts to external forces of stretch, shear, compression and torsion when it is still on the animal body.  The most common  in vivo studies are the compression and shearing tests and the elastic wave propagation tests.  Compression and shearing tests study the changes in collagen fibril diameter  and density and dermal and epidermal thickness.  Sander et al. (2001) reported that  compression and shearing caused collagen fibril diameter to increase and density to decrease for all layers o f pig dermis, but caused no changes in dermal and epidermal thickness.  Elastic wave propagation tests involves applying waves to wet or dry skin  via a vibration apparatus and measure the propagation velocities and their dissipation rate in skin over a low (0-600 Hz) and high frequency (600-1000 Hz).  Under low  42  frequency, propagation velocities provided information on the mechanical properties of the epidermis, which had low propagation velocities and stiffness due to its greater ability in absorbing water; under high frequency, propagation velocities provided information on the mechanical properties of the dermis, which had high propagation velocities and stiffness due to its lower ability in absorbing water (Potts et al., 1983).  2.3.2 In vitro tests To evaluate the mechanical roles of skin components, in vitro tests are used.  In vitro  tests involve removing skin samples from the animal body, paring off subcutaneous fat and muscle and performing mechanical tests on skin samples using a tensile testing machine.  The common in vitro tests include simple uniaxial tests, compression tests,  and sinusoidal stretching tests. The findings, types and size of specimens, experimental conditions and strain rates of these tests are specified in Table 2.2. The size of the specimen and the strain rates used rangefrom2* 10 mm to 38.1 x38.1mm and 0.02 mm/s to 4.4 mm/s, respectively.  Uniaxial tests are often used to evaluate the  behavior of collagen fibers and elastin by loading a skin sample and measuring the resultant stress (force per unit area) and strain (change of length with respect to original length).  43  According to the findings in Table 2.2, skin exhibited a non-linear stress-strain relationship, and from this relationship; the Modulus o f Elasticity (Pa) or the stiffness o f skin could be evaluated by determining the slope o f the linear region o f the stress-strain curve.  Elastin was found to play a mechanical role in small stress and  strain, while collagen fibers contributed to the mechanical properties o f skin in large stress and strain (Silver et al., 2001).  However, the stiffness o f skin was mainly due  to the stretching o f the collagen fibers and would slowly increase during maturation as fibers' diameter increased (Figure 2.16).  Sinusoidal stretching test involves using  different sequences o f sine waves to strain the tissue and study the effect on skin fibroblast's morphology and viability.  Martel et al. (2001) demonstrated that a  normal fibroblast with intact plasma membrane, nuclei and normal rough endoplasmic reticulum lost its cytoplasmic adhesions with E C M and rounded up when skin was stretched in a sinusoidal manner (20% strain) (Figure 2.17).  The fibroblast was then  prompted to cell death and fragmented into membrane-bounded bodies, which were later phagocytized by macrophages.  44  Table 2.2  The findings, types and size of skin specimens, experimental conditions and strain rates for some in vitro tests Stretch rate or strain  Study  Saline  0.02-6  Lanir et al.  solution  mm/s  (1974)  no  1.5%/s  Belkoffet  In Vitro Techniques  Findings  Temperature Bath (pH Animal control) (number of control) animals used,skin size)  -uniaxial tensile  Nonlinear stress-strain  Rabbit (47,  test  relationship (NSSR):  35 x35mm)  yes  stiffness increases with 'increased strain NSSR:  stiffness is 14  Rat (63);  no  al. (1991)  (lmonth)-36 M P a (4  (38.1x38.1  month)  mm)  NSSR:  Human,  1) Elastic fibers play a role  dermal  buffer  in small stress & strain  graft,  solution  (spring constant = 0.4  processed  MPa)  skin;  2) Collagen fibers play a role in large stress & strain (spring constant = 4.4 GPa)  (50x10mm)  no  Phosphate  0.17%/s  Silver et al. (2001)  Table 2.2. Continue In Vitro Techniques  -sinusoidal sketching test  Findings  Fibroblasts lose cytoplasmic extension & round up -compression test Orientation of Collagen fibers: 0°-10° (through thickness of skin)  Temperature Bath (pH Animal control) control) (number of animals used, skin size)  Stretch rate or strain  Study  Rat(3);(2xl0 mm)  yes  Krebs' solution  20% in 1 Martel et Hz al. (2001)  Pig(l); (30x30 mm)  no  no  0.083m m/s  Hepworth etal. (2001)  Figure 2.16 Scanning electron micrographs of 1-month-old rat skin (a) relaxed; (b) stretched; 4-month-old skin (c) relaxed; (d) stretched (Belkoff et al., 1991).  Figure 2.17 Fibroblasts' morphology after skin was stretched in a sinusoidal manner (20% strain) (Martel et al., 2001).  In compression tests, a skin sample is compressed vertically to study the effect o f mechanical load on the orientation of the fibers through the thickness of the tissue. According to Hepworth et al. (2001), the orientation angles of the collagen fibers ranged from 0°-10°.  Although the findings o f the above studies illustrated the  mechanical behavior o f skin, the experimental conditions in which the skin samples were examined in these studies might have negative impact on their results.  With  blood supply, living skin tissues are normally exposed to temperature o f 37°C, p H o f 7.4-7.5 and have adequate moisture content, air, and nutrient supply.  According to  47  Table 2.2, most o f studies do not have temperature control and bath solution that provide p H control, moisture content and nutrient supply to the testing specimens. Without these essential living factors, the skin specimen, especially the structure of its collagen and elastic fibers and the morphology o f fibroblasts, will be altered, and this may have a great influence on its mechanical behavior.  To control p H and provide  moisture and nutrients to the skin specimens, saline solution, ringer's solution, phosphate buffer solution and krebs' solution can be used (Lanir et al., 1974; Oxlund et al., 1988; Silver et al., 2001; Martel et al., 2001).  To maintain the physiological  solution in the bath at 37°C, Lanir et al. (1974) installed a thermoregulation system, which is monitored by an electronic thermometer, to the bottom of the bath tray, and Martel et al. (2001) installed a heater cartridge in one of the walls ofthe bath.  2.3.3 Studies o f fibroblasts populated collagen lattice  Many past studies have only focused on the roles o f collagen and elastic fibers and failed to recognize that fibroblasts may be involved in tensegrity o f skin tissue. Recently, studies have been carried out to examine fibroblast's motility and its response to mechanical stress. Human fibroblast's motility has been studied on artificial floating and restrained collagen matrix (Figure 2.18).  The floating matrix has a low cell density with 10  5  48  cells per ml of collagen gel. Fibroblasts in this matrix formed dendritic networks (actin) to explore and interact with the environment (Grinnell et al., 2003).  Tamariz  et al. (2002) demonstrated that fibroblasts exerted endogenous force on the floating matrix to allow local remodeling of collagen fibers, since fibers offered little resistance to the cell in a low cell density matrix.  The restrained matrix has a high cell density  with 10 cells per ml o f collagen gel. When fibroblasts exerted endogenous force on 6  the restrained matrix, global remodeling o f collagen fibers occurred, at which fibers offered resistance to the cell and caused stress fibers (actin) and focal adhesions to form (Tamariz et al., 2002; Beningo et al., 2001).  Fibroblasts in a restrained matrix  were stellate and bipolar in form, and in the presence o f growth factors, including platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA), fibroblasts exhibited extended and retracted dendritic network, respectively.  However,  fibroblast's morphology and motility in unnatural collagen gel with no temperature, p H , moisture control or nutrient supply, might be very different from those in living dermis, a complex tensional system with both cellular and extracellular components. Thus, the findings of the fibroblasts in these studies may not be applicable to those in living animal tissue.  49  LPA  MXJF •  * V  .\ K F i g u r e 2.18  WFM  F i b r o b l a s t s ' m o r p h o l o g y i n f l o a t i n g (LD)  m a t r i x ( T a m a r i z et a l . ,  and restrained  (HD)  2002).  In addition to studying the morphology o f fibroblasts, mechanical studies o f fibroblasts populated collagen lattice (FPCLs) have been carried out to evaluate the mechanical aspect o f human dermal fibroblasts. Brown et al. (1998) and Prajapati et al. (2000) cultured fibroblasts from normal human skin and placed them on native acid soluble rat tail type I collagen gel. They used a tensional force control monitor to apply and measure the mechanical response o f fibroblasts in gel and compared the response to the natural endogenous tension generated by the cellular actin cytoskeleton.  However, the mechanical behavior o f fibroblasts in an unnatural matrix  may not accurately mimic their actual behavior in the skin dermis, since fibroblasts interact with other tensional components (ie. collagen fibers and elastin) in the dermis. Nonetheless, Brown et al. (1998) showed that FPCLs reacted oppositely to external loads to minimize the damage o f the cells.  In other words, loading caused a decrease  in endogenous tension o f a cell, while unloading caused an increase in endogenous  50  tension o f a cell.  To maintain their temperature and moisture content, FPCLs were  kept in a humidified 37°C, CO2 incubator.  On the other hand, Prajapati et al. (2000)  recognized the importance o f another cell response to loading, the release o f E C M proteases (ie. M M P - 3 , 9 , tPA & uPA) by fibroblasts in different alignment o f collagen lattices.  E C M proteases are responsible for degradation of E C M molecules to  promote E C M remodeling, a process that is essential for normal skin tissue development and wound closure.  During wound closure, fibroblasts in the skin  dermis form new collagen matrix and generate endogenous force to stimulate E C M remodeling by releasing E C M proteases. The changes in E C M in turn cause fibroblasts to form tight adhesions with the newly formed matrix to close the wound. In their study, the two types of collagen lattices used were a low aspect ratio lattice and a high aspect ratio lattice. The low aspect ratio lattice had a length to width ratio o f 0.33 to 1 and was found to have no high strain gradients under loading; the high aspect ratio lattice had a length to width ratio of 3 to 1 and was found to have high strain gradients with cells aligned parallel to the direction of the applied load.  Their study  showed that long duration o f loading of fibroblasts in high aspect ratio lattice resulted in the highest proteases release rates. However, the experimental conditions in these studies must be improved to resemble real-life conditions to obtain more reliable data.  51  Accordingly, both studies o f skin and fibroblasts provided insights into the contribution o f E C M components and fibroblasts to the natural tissue tensegrity that existed in the dermis. In vivo studies are recommended, since the experimental skin specimens are maintained in physiological condition with temperature o f 37 °C, p H o f 7.4-7.5, moisture, air and nutrient supply, and the actual mechanical behaviors of skin components in living dermis can be observed.  However, as outlined in this review,  considerable progress has been made to provide skin specimens in in vitro studies with the above living factors, thus resulting in more accurate evaluation o f the mechanical properties o f skin.  2.4 Cell Death Focal adhesion plaque allows cell anchorage to its extracellular matrix via integrins, which is extremely important to its viability.  They regulate cell adhesion  and cell shape to promote cellular responses towards external stimuli.  They relay  information regarding its continuously changing environment to the cell and therefore, affect the cell's viability.  The cell's fate is dependent upon several environmental  factors, including death-receptor ligands, growth factors and physical stimuli, such as mechanical stress and radiation (Boehringer Mannheim, 1998).  When a cell is  exposed to environmental threats, such as mechanical stress, cytotoxic drugs, and  52  ultraviolet radiation, it experiences degradation of its focal adhesion plaque.  When  the cell loses the proper integrin-interactions with its extracellular matrix, its growth  and death receptor distribution changes to trigger death-signaling cascades.  The  degradation of focal adhesion plaque then begins, and the cell can no longer receive  external stimuli and respond to them.  This will result in extensive extracellular  denaturation and destruction of its tensegrity.  The unanchored cell then becomes  prompted to a distinctive type of cell death, known as apoptosis or programmed cell  death.  Cell death can happen by either of two specific mechanisms, necrosis or  apoptosis.  Both types of cell death can involve cytotoxicity, a cell-killing property of  a chemical compound (ie. food, cosmetic, or pharmaceutical) or a mediator cell  (cytotoxic T cell).  Cell death that is caused by either cytotoxic T lymphocytes or  natural killer cells is experimentally shown to contain some aspects of both necrosis  and apoptosis (Boehringer Mannheim, 1998).  However, there are significant  morphological and biochemical differences between necrosis and apoptosis.  Figure  2.19 illustrates the major differences in the morphological features of necrosis and  apoptosis.  53  mitochondrial morphotogv  JM  *  WW ^ fragments  CP  1 • yaf cfiances normal  3pOpt0t(C tKXJl^i  conifeftsaliftii (coll Webbing)  Figure 2.19 Illustration of the morphological features of necrosis and apoptosis (Boehringer Mannheim, 1998)  Necrosis is also known as "accidental cell death" (Bwhringer Mannheim, 1998).  It occurs when cells are exposed to intensive non-physiological stimuli,  including physical or chemical damage (ie, hypothermia (extremely low temperature), hypoxia (low oxygen concentration)) that results in destruction of the plasma membrane.  At the initial stage o f necrosis, a cell will lose its ability to maintain  homeostasis, resulting in an influx o f water and ions from the external environment. The cytoplasm and the mitochondria will then swell, causing an inflammatory response.  Finally, the cell will prompt to total cell lysis.  During cell lysis, the  breakdown o f the plasma membrane causes the cytoplasmic contents, including  54  lysosomal enzymes, to be released to the extracellular fluid.  The necrotic cell will  then be phagocytized by the macrophages or digested by the released lysosomes. Apoptosis, on the other hand, is a very different mode o f cell death.  The term  "apoptosis" first appeared in the biomedical literature in 1972 . It is also known as "normal" or "programmed" cell death.  It describes a stmcturally-specific mode o f  cell death responsible for cell loss within living tissue.  Body tissues, especially skin,  the continuously renewing tissues, regulate their cell growth and differentiation by a subtle balance of inhibition and activation between proliferation and apoptosis through the expression o f anti-apoptotic and pro-apoptotic factors or proteins (Allombert-Blaise, 2003). Apoptosis is a physiological process that involves the elimination o f unwanted or useless cells, and it usually results from fibroblast-collagen matrix remodeling (ie. changes in tissue tensegrity) during normal development and other growth processes.  It is also induced by physiological stimuli including the lack  o f growth factors, and changes in hormonal environment and accounts for cell death after exposure to cytotoxic compounds or viral infection and from T-cell killing.  It is  commonly found in tissue homeostasis, embryogenesis, negative selection in immune system, development o f nervous system and endocrine-dependent tissue atrophy. Furthermore, apoptosis is also an important contributor to cancer, since cancer depends upon a selective, failure o f apoptosis to allow cell proliferation after mutagenic D N A  55  damage.  The current investigation is focused on the characteristic, promotion and  detection o f apoptotic fibroblasts in skin tissue.  2.4.1 The physical characteristics o f a viable and an apoptotic fibroblast  B y verifying some o f the cell's ultrastructural characteristics, a dead fibroblast can easily be distinguished from a viable fibroblast. Figure 2.20 shows an example o f a typical fibroblast observed in the dermis o f a skin sample soaked in Kreb's buffer under the transmission electron microscope.  According to the figure, a viable cell has  continuous plasma and nuclear membranes, a normal mitochondria, a non-swollen rough endoplasmic reticulum (RER) and an intact nucleus.  However, cells  undergoing apoptosis reveal both morphological and biochemical transformation (Figure 2.19).  The early morphological features o f an apoptotic cell include  membrane blebbing, shrinking o f cytoplasm, condensation o f nucleus, aggregation o f chromatin at the nuclear membrane.  Then the biochemical features become apparent  when the cell's mitochondrial function is disrupted, causing its mitochondrial permeability to be altered.  Specific apoptosis protease activators, especially A I F and  apoptosis inducing factors, are then released from mitochondria in the cytoplasm. AIF has proteolytic ability that can induce apoptosis directly.  Meanwhile, changes in  the mitochondrial membrane cause redistribution and the release o f cytochrome C, the  56  apoptotic pathway activator, into the cytoplasm o f the cell, promoting the apoptotic pathway, known as Caspase-3.  Finally, apoptosis ends with fragmentation o f  cytoplasm and nucleus into smaller membrane-bounded bodies, also known as the apoptotic bodies, which contain ribosomes, mitochondria and nuclear material.  In  pre-lytic D N A fragmentation, non-random fragmentation of the genome D N A causes the cell to die. The fragmentation has been shown to result from the activation of an Ca2+ and Mg2+ - dependent nuclear endonuclease, an enzyme that selectively cleaves D N A to produce mono- and oligonucleosomal D N A fragments (ie. low molecular weight D N A fragments). In vivo, these apoptotic bodies are quickly recognized and phagocytized by either macrophages or adjacent epithelial cells (Boehringer Mannheim, 1998). In vitro, the apoptotic bodies as well as the remaining cell fragments ultimately swell and finally lyse.  This final phase of in vitro cell death has  been termed "secondary necrosis" (Boehringer Mannheim, 1998).  Figure 2.20 An apparently normal dermal fibroblast observed in a skin sample soaked in Kreb's buffer for 90 minutes before fixation (Martel et al, 1999).  57  2.4.2 2-Deoxy-D-Glucose as an antimicrobial agent that promotes apoptosis  2-Deoxy-D-Glucose has a molecular formula o f C6-H12-05, and a molecular weight o f 164.18 g/mol. It is a white powder that is soluble in water and has a melting or freezing point o f 146 to 147 °C. As an oxidizing agent, it can generate hazardous decomposition products including carbon monoxide, irritating and toxic fumes and gases, and carbon dioxide ( M S D S ) .  2-Deoxy-D-Glucose is also known as a potent  herbicide and a glucose antimetabolite that inhibits glycolysis, the metabolic pathway that occurs in all living cells and serves as the starting point for aerobic respiration.  It,  therefore, prevents the synthesis o f Adenosine Triphosphate (ATP). Aft et al. (2002) investigated the effects of the anti-metabolite 2-deoxy-D-glucose on breast cancer cells in vitro.  This compound has been shown to  inhibit glucose metabolism, causing breast cancer cell death.  The cell death induced  by 2-deoxy-D-glucose was found to be due to apoptosis as verified by promotion o f <  caspase 3 activity. This agent can also inhibit the cellular anaerobic process.  With  the disruption and termination of both aerobic and anaerobic metabolism, the treated cell is then subjected to altered structural activity (ie. rounding of cells) with subsequent loss o f focal attachments to the extracellular matrix (Martel et al. 1999). Cells with disrupted focal adhesions are thus prompted to apoptotic cell death.  58  2-Deoxy-D-Glucose was used previously in another study by Martel (1999) to investigate its effect on the mechanical stress o f skin tissue and viability o f fibroblasts [8].  In her experiment, she subjected her tissue soaked in Kreb's buffer (Table 3.1)  with or without the addition o f 2-Deoxy-D-Glucose (30 mmol) to 4% strain for 40 minutes.  She then measured the maximum stress generated when the tissue was  stretched and determined the viability by studying the morphology o f the cells in electron microscopic prints. The results indicated that the maximum stress o f the tissue soaked in solution with the Poison (2-Deoxy-D-Glucose) was decreased in a statistically significant manner compared to that of the tissue soaked in solution without the Poison, though the cell viability with or without treatment o f the Poison was not statistically different.  2.4.3 Apoptosis assay methods  With increased understanding o f the events that happen during apoptosis, a number o f assay methods have been developed for detecting apoptosis.  These assays  can measure fragmentation o f D N A in a population o f apoptotic cells or in individual apoptotic cells, activation o f apoptotic caspases that terminate the normal cell functions, and finally, destruction o f the plasma membrane.  The current assays can  59  be divided into two types, including assays that detect apoptosis in both cell populations and in individual cells. A number o f methods have now been developed to study apoptosis in cell populations.  They mainly focus on two key apoptotic events in the cell:  fragmentation of the genomic D N A into shorter fragments prior to fragmentation o f the nucleus and cytoplasm, and caspase activation in the early stages of apoptosis. Fragmentation of the genomic D N A is an important irreversible feature that commits the cell to die.  Since D N A fragmentation by nuclear endonuclease produces mono-  and oligonucleosomal D N A fragments (low molecular weight D N A ) , many methods developed are based on the detection and or quantification o f either these low molecular weight D N A fragments, which increase in apoptotic cells or high molecular weight D N A , which is reduced in apoptotic cells.  Using Apoptotic D N A Ladder Kit,  one can easily isolate apoptotic D N A fragments for analysis.  The apoptotic cells are  first treated with apoptosis-inducing agent, such as campothecin. incubated with lysis buffer.  They are then  The lysed samples are centrifuged to separate the L M W  D N A from the very large, chromosomal length D N A ( H M W D N A ) .  The D N A  fragments are then subjected to agarose gel electrophoresis to be analyzed.  In the gel,  these D N A fragments are displayed as a distinctive ladder pattern consisting o f multiples o f an approximately 180 base pairs subunit (Figure 2.21).  This method is  60  best to detect and isolate apoptotic D N A fragments that do not replicate in vitro; in other words, it is used for cells that cannot proliferate and cannot carry out D N A synthesis in vitro.  form & chromatfrt frtfetfftr-OM&t  "DNA ladder after gi-i elect; opnojcsia  Mono- and Otlgonucleosomes (IMW-DNA)  Hi <•' i;i ome core UiStfilKfi Mtmsri Hits * maltip* 180 base pairs  gel eiectiGpnrjfesis  1  Figure 2.21 The biochemistry of DNA fragmentation and the appearance of the "DNA ladder" (Boehringer Mannheim, 1998).  E L I S A is another cell death detection test that can effectively detect and differentiate apoptosis from necrosis. inducing agent.  The cell samples are first treated with apoptosis  The cells are then centrifuged, and the supernatant which contain  necrotic D N A that leaked through the membrane during the incubation is discarded. The cells are then incubated with lysis buffer and centrifuged to separate the cleaved D N A fragments resulted from apoptotic cell death.  Finally, the supernatant is  61  incubated with anti-DNA immunoreagent, and the amount of colored product generated from the reaction between the D N A fragments and the reagent is measured spectrophotometrically, i.e. measuring the absorbance o f the colored product (Boehringer Mannheim, 1998)). Thus, the amount o f D N A fragments produced by the apoptotic cells can be determined. In order to detect caspase activation in apoptosis, Activity assay is commonly used. It can detect caspase-3 activation, an event that plays a key role in the initiation o f cellular events during the apoptotic process.  The cells are subjected to similar treatments as in other assays, except the  centrifuged supernatant is treated with anti-caspase 3 solution.  The captured caspase  3 is then isolated and treated with fluorescent caspase substrate, called A c - D E V D - A F C , to generate free fluorescent A F C .  Lastly, the amount o f activated  caspase 3 present is quantified by measuring the free fluorescent A F C that is produced. Recently, a number of methods have also been developed to study apoptosis in individual cells.  They mainly focus on D N A fragmentation and changes o f plasma  membrane in individual cells, since they are the characteristic apoptotic events in the early stages of cell apoptosis.  Cleavage of the D N A results in double-stranded, L M W  D N A fragments, including mono- and oligonucleosomes, and single strand breaks in HMW-DNA.  These D N A strand breaks can be detected by enzymatic labeling of the  free 3'-OH ends of the genomic D N A strands.  The developed methods involve  62  enzymatic labeling of the free D N A ends with modified nucleotides, such as X - d U T P , where X can be biotin, D I G or fluorescein, using the exogenous labeling enzymes, known as the optimized Terminal deoxynucleotidy transferase (TdT). The most common enzymatic labeling kits are In Situ Cell Death Detection Kit, Fluorescein and In Situ Cell Death Detection Kit, P O D . paraffin-embedded tissue sections.  They both can be used to assay frozen or  Using In Situ Cell Death Detection Kit,  Fluorescein, one can identifies cell death (apoptosis) by labeling and detection of D N A strand breaks in individual cells by fluorescence microscopy. first fixed and permeabilized.  The apoptotic cells are  They are then incubated with the reaction mixtures  containing TdT and fluorescein-dUTP. During the incubation, TdT induces the binding o f fluorescein-dUTP to the free 3 ' O H ends in the genomic D N A .  Finally, the  cells with the fluorescein are visualized with a fluorescence microscope to detect and quantify the D N A strand breaks resulted from apoptosis. On the other hand, In Situ Cell Death Detection Kit, P O D can measure cell death (apoptosis) by detecting D N A strand breaks in individual cells by light microscopy. The apoptotic cells or tissue sections are subjected to similar treatments as in the former kit.  However, the cells that contain attached fluorescein in their D N A strands  are exposed to an anti-fluorescein antibody P O D conjugate.  The D N A strand breaks  are then quantified by detecting the presence of the fluorescein. This detection step  63  can be done easily by visualizing (analyze) the resulting cell samples under light microscopy.  Finally, in order to detect damage or leakage o f the plasma membrane,  Trypan Blue exclusion assay can be used.  This is a dye exclusion method that can  distinguish viable cells with intact plasma membranes from dead cells with damaged plasma membrane.  Cells with disrupted plasma membrane permeability are stained,  while healthy (viable) cells are not stained with dyes.  The dye can only penetrate the  damaged plasma membrane and bind to the intracellular proteins of the leaky cell. The stained or damaged cells are then determined under a light microscope. However, although the method is quick and requires only a small fraction o f total cells from a cell population, it is not suitable for detection o f apoptosis, and the dye stains only necrotic cells or very late apoptotic cells that are in secondary necrosis. Out of all the apoptosis assay methods mentioned, the most suitable method for the current investigation is the In Situ Cell Death Detection Kit, POD.  The methods  for studying apoptosis in cell populations are not chosen, since the current study focuses on apoptosis in individual skin fibroblasts.  As well, D N A cleavage is the  crucial property of apoptosis; thus, the assays that measure pre-lytic D N A fragmentation are the most accurate for the determination of apoptotic cell death. The current study uses paraffin-embedded skin tissue sections, so In Situ  Cell Death  Detection Kit, POD is suitable for the identification of apoptosis o f the skin  64  fibroblasts.  The kit is proven to be a fast, specific and sensitive method for detecting  apoptosis.  In terms of the efficiency of the In Situ Cell Death Detection Kit, POD, the sensitivity of the enzymatic labeling o f D N A strand breaks is found to be so high that it can accurately detect an early apoptotic event.  In terms of timing, d U T P only  requires 30 minutes to completely attach to the 3'- O H ends of the genomic D N A . The assay has less working steps compared to other assay methods, making this test simpler.  As well, the specificity of the test is very high, and since the amount of D N A  strand breaks in apoptotic cells is so large, the intense labeling in these assays accurately distinguishes apoptotic cells from necrotic cells.  65  3. Materials and Methods From the literature review, it is evident that information is lacking on the strength contribution of fibroblasts to skin.  To eliminate the artificiality of the implantation  experiments of fibroblasts imbedded in regenerated collagen matrices, it was decided to use whole rat skin with the fibroblasts in place. Four equal skin samples were recovered from the back o f each Wistar-Moller male rat (7 to 8 weeks old) and immediately immersed in aerated Kreb's solution held at 37°C.  Two o f the samples,  were soaked in the Kreb's solution with or without 30 mmol of 2-Deoxy-D-Glucose. The other two samples were soaked in the Kreb's solution with or without 30 mmol o f 2-Deoxy-D-Glucose and subjected to biomechanical tests. To carry out the biomechanical experiments, a load frame was used.  The principle components of this  load frame as well as the PC-Based data acquisition ( D A Q ) system are described in this chapter.  For this project, experiments were carried out on skin samples from  Wistar-Moller male rats (300-350g).  The procedure used to harvest the skin from the  animal is presented followed by a description o f the biomechanical experiments performed on the tissue samples.  As mentioned earlier, light microscopy is used to  study the changes in fibroblast morphology and determine the viability o f the fibroblasts.  Before a tissue sample can be analyzed under the microscope, it is  submitted to diverse chemical treatments and apoptosis test procedures.  After a brief  description of the principal steps involved in the processing of samples to be used in 66  the light microscope is presented, this chapter ends with a presentation o f the way apoptosis analyses were done using the light microscopy.  3.1 Load frame A custom-built load frame was used to stretch the skin samples: and unloading tests can be performed with this load frame.  Both loading  The basic features o f the  load frame are described in this chapter.  3.1.1 Basic components of the load frame Figure 3.1a presents the basic components o f the load frame.  The load frame has  two parallel stainless steel bars on the top that can be used as handles for positioning the equipment and a metal base that is used as support.  The tissue sample is held  horizontally by two stainless steel clamps. The grooves in the inner surfaces of those clamps allow a good grip on the tissue and therefore prevent it from slipping. 3.1b shows the front view of the clamping portion o f the load frame.  Figure  Figure 3.1c  shows the actual view of the clamping portion o f the load frame. The left clamps is fixed and connected to a force transducer which can support a maximal force o f 10 lbf (model:  L C H D - 1 0 , Omega Engineering, Inc., Quebec).  67  probe tip displacement transducer  left clamp right clamp  force transducer  I I I I 1I 1 T I I I I I I I  wheel  Figure 3.1a Basic components of the load frame (top view). Stainless steel clamps to hold the tissue sample, force transducer, pulley, screw, wheel, displacement transducer and extendable probe tip.  straining direction  to load cell 4  1  left clamp  tissue  right  i  *  clamp  Figure 3.1b Close up front view of the clamping portion of the load frame.  68  Figure 3.1c  Close up view of the clamping portion of the load frame.  The right clamp which is attached to on the opposite end of the sample is connected to and moved by a screw which moves it along the parallel bars and whose position is controlled by manual turning o f a wheel. ( L V D T ) (model:  A linear variable displacement transducer  LD300, Omega Engineering, Inc., Quebec) with an extendable  probe tip pressed against the side of the moving cross-head is clamped onto one of the parallel bars.  Care is taken to ensure that it has a linear response over the entire range  of the transducer.  When the wheel is turned, the moving cross-head will move  causing the probe tip of the L V D T to move as well. Two pieces of thick Styrofoam are placed under the load frame to provide support and minimize vibration.  69  As mentioned earlier in the literature review section, it is very important to mimic the conditions that a living skin tissue is exposed to.  A fluid circulation system as  shown is Figure 3.Id was used to keep the excised tissue alive. The skin tissues in the current study are exposed to temperature of37°C, p H o f 7.4-7.5 and adequate moisture content, air, and nutrient supply.  The skin tissues are soaked in Kreb's solution,  which is maintained at 37°C in the bath by circulating warm water through the base o f the polyethylene bath pumped from a circulatory water heater (Thermo Haake, model: 003-3046,Germany).  As seen in the flow diagram (Figure 3.2), the Kreb's solution  flows by gravity from 1 - L aerated storage beakers to the test bath.  Tests were run  initially to calibrate the temperatures o f all the liquid streams to hold the desired temperature conditions during a test.  The p H was adjusted in the newly made Kreb's  solution and tested daily.  70  Water Heater  Tubing system  beaker (Kreb's)  Air (Poison)  Flask  glass heat exchanger  dripper Vacuum pump Control Bath  Experimental Bath  Figure 3.1d Overview of the fluid circulation system. The principal components of the circulation system used for the maintaining the viability of the tissues are presented in this figure. The blue line represents the flow of water; the yellow line represents the flow of Kreb's solution; the red line represents the flow of Kreb's solution with Poison; the orange line represents the flow of waste solution.  To ensure the viability of the cells during biomechanical testing, the tissue, which is mounted between the clamps, is submerged in a 2.5cm by 5cm by 2.5 cm tissue bath made of polyethylene (ie. non-reactive polymer) with an open top in which the physiological solution can be circulated.  A hole was machined 1cm from the bottom  of the bath, and a tube connector was mounted onto each of the hole openings.  The  physiological solutions with and without addition of 2-Deoxy-Glucose are placed in two separate 1-L beakers and elevated on a shelf such that the solution can be fed by  71  gravity.  From each beaker, the solution is drained down to the bath through the glass  heat exchanger and then through the dripper that is equipped with a flow rate adjustor. To maintain the physiological solution at 37°C, a water heater is used, and a plastic vinyl tubing system (ID: 5/16 inch; OD: 7/16 inch) is built to circulate the warm water. Water is first poured into the water heater, and the heater is then adjusted to 68°C, so that the water will be heated up to and remained at that temperature for the entire experiment.  Prehminary tests showed that 68°C is the appropriate heating  temperature to overcome heat losses by the plastic tubes and the bath tank and heat the solution in the bath to 37°C.  The heated water first flows to each heat exchanger  through the plastic tubing to heat up the physiological solution in the exchanger and then flow through the hole at the bottom of each tissue bath to heat up the solution in the bath.  To prevent overflow ofthe solutions in each tissue bath, a thin plastic tube  is connected to a flask and then to suction inlet of a vacuum pump (model: 07530-40, Barnant Company, U.S.A).  When the vaccum pump is turned on, a suction force is  created that sucks the excess fluid from the bath to the flask. To provide aeration of the solution in each bath, another thin plastic tube connected to the outlet of the vacuum pump is placed in each elevated 1-L beaker. bath will have limited physical disturbances.  In this way, the tissue in the  The p H ofthe bathing solution is  maintained at 7.4-7.5 in the bath by the addition o f Hydrochloric acid and or Sodium  72  Hydroxide, and the pH was measured by using a p H meter (model:  HI8519, Hanna  Instruments, Singapore).  3.1.2 PC-based data acquisition system and control components of the load frame A diagram ofthe complete system for biomechanical experiments is presented in Figure 3.2. To acquire the data from the load frame, a PC-based Data Acquisition System (DAQ) (National Instruments Corporation 2003) was used.  Obtaining proper  results from a PC-based D A Q system depends on each o f the following system elements, including the PC, linear variable displacement and force transducers, signal conditioning, D A Q hardware and software.  During the experiment, a piece o f rat  tissue is held by two clamps of the load frame. The tissue is elongated when the wheel is turned in clockwise direction.  The displacement transducer measures the  change in distance and produces an electrical signal (ie. voltage) that the D A Q system (Data Acquisition System) measures.  A t the same time, the force transducer also  senses the changes in the force on the tissue and generates electrical signals (ie. voltage).  Each transducer is connected to signal conditioning box that amplifies the  low-level signals and isolates and filters them for more accurate measurements. signals are then transmitted to the D A Q hardware via a connector block.  The  The D A Q  hardware converts the analog signals to digital values.  73  force transducer  displacement transducer  Personal Computer  DAQ software  Figure 3.2  PC-Based Data Acquisition System.  The software used with the P C is LabVIEW™ (National Instruments 2003).  It  transforms the P C and D A Q hardware into a complete data acquisition, analysis, and display system.  Starting up, initiating the calibration routine, adjusting calibration  factors, recording data at different frequencies, on-line graphing and display of the time, tension and position signals and termination of the sampling process are some of the functions that can be performed using this software.  During the experiment, the  P C allows the display and recording of the clamp position and the tension (ie. voltage) of the tissue sample sensed by the transducers.  Both signals are read at the same set  intervals (sampling frequency), specified at the beginning of the experiment by the user.  In the current study, the sampling frequency is set at four samples per second.  74  3.1.3 Linearity ofthe displacement and force transducers To ensure an accurate reading o f both displacement and force, the transducers must be calibrated.  For displacement calibration, the voltages and the corresponding  distances are recorded, and their relationship is plotted.  The relationship between the  voltage and displacement was found to be linear between approximately negative 3.1 volts and positive 3.7 volts. Thus, the calibration is performed at this range for each experiment.  For the load calibration, standard weights (ie. 28.85g, 142.07g, and  283.23g) are placed on a pan made o f metallic foil and hung to the left clamp by a fine fishing line through a low friction pulley installed on the extension of the right clamp as shown in Figure 3.3.  Before the calibration, the initial voltages with and without  the pan hanging on the clamp are noted.  Each standard weight is placed on the  hanging pan four times and the corresponding voltages are recorded.  The voltage  produced by the pan is subtracted from each o f the voltage produced by the standard weights.  The results of calibration tests showed an excellent linearity for both the  displacement and load transducers; the coefficient of determination or R value (R = 2  1-SSE/(SSR+SSE), with SSE:  error sum o f squares and SSR:  2  regression sum o f  squares) corresponding to the linear regression of the calibration data for displacement and load were 0.9996±0.0001 and 1.0000±0.0000 respectively.  A n example o f  calibration result for both force and displacement transducers are shown in Appendix A.  75  straining direction  to load cell 4  left clamp  1  fish line  right clamp  1  *  -  metallic foil pan  •  Figure 3.3 Close up view of the calibration setup.  3.2 Light microscope Light microscopy was necessary to monitor the changes in fibroblast morphology in both control and experimental tissue samples and to examine the apoptotic dead cells in the excised skin tissue caused by stretching and treatment with 2-Deoxy-glucose.  The light microscope used was a Leica Galen III, operated at 120V  (Buffalo, N Y in U.S.A).  76  3.3 3.3.1  Experimental procedures Preliminary tests Preliminary tests were carried out to determine the appropriate sections o f skin  tissues to be used in tests, initial stress condition of the tissue samples required and the strain level to be used in the mechanical test. These tests were done to ensure all the tissue samples used were exposed to the same standard conditions to minimize the mechanical variations between these samples. Stress-strain experiments were performed to determine the appropriate sections o f skin tissues to be used in the mechanical tests.  The results showed that tissues from  the frontal dorsal part o f the body gave stress-strain curves with less noise and higher maximum stress values at fixed strains compared to those from the rear dorsal part o f the body. Therefore, tissues from the frontal dorsal part of the body are used for the biomechanical tests.  Figure 3.4 shows the stress-strain curves of the tissues from the  frontal dorsal and the rear dorsal part o f the body.  The tissues from the rear dorsal  part o f the body are used for the soaking tests.  77  2500  2000  • Upper Body • Lower Body  0  0.02  0.04  006  0.06  0.1  0.12  0.14  Strain (cm/cm)  Figure 3.4 The stress-strain curves of the two pieces of tissues from the frontal dorsal part (red curve) and the rear dorsal part of the body (blue curve).  Before performing the biomechanical tests, it is important to minimize the noise level of the force transducer.  Due to the noise inherent in the load frame, initial tests  were made which illustrated that the tissue sample should be prestretched to obtain stress-strain curves that are similar to the ones shown in other biomechanical studies. In the current study, all tissues that were subjected to mechanical tests were preloaded to a stress level of about 50 N / m or Pa. 2  In most biomechanical projects, the skin tissue was preconditioned prior to any actual mechanical tests. This was done by repeated loading and unloading of the tissue sample to the strain to be used in the tests. Preconditioning is performed before  78  the actual biomechanical experiment in order to obtain reproducible experimental results.  In this research, preliminary stress-strain tests showed that the tissue is  preconditioned when the loading and unloading cycles are repeated three times, as shown in Figure 3.5.  Thus, the fourth curve of each test was chosen to be analyzed.  As previously mentioned (section 2.3), under normal in vivo conditions, skin is usually subjected to small extensions.  Therefore, we decided that the strain during  mechanical experiments should now exceed 4%, since 4% strain is not enough to dislodge the cells from their environment in such a way as to make obvious their loss of viability.  We have experimented and found that tissue with 10% strain showed a  lot of noise in the curve, and tissue with 16% strain had force that exceeded the load limit.  Thus, we chose 13% to be the strain level that the tissues used for the  mechanical tests were subjected to.  79  2500  2000  • 1st cycle • 2nd cycle 3rd cycle 4th cycle  0  0 02  004  0 06  008  0.1  012  0.14  0 16  Figure 3.5 Preconditioning of the tissue. The loading and unloading cycles are repeated three times for preconditioning purpose, and the fourth curve of each test is used in the biomechanical analysis.  3.3.2  Calibration o f the load frame Before each experiment, the calibrations of both the displacement and the load  transducer were performed to ensure an accurate reading of the distance and the tension during testing (Section 3.1.3).  Both displacement and force calibration curves  are shown in Appendix A .  3.3.3  Solution preparation The physiological solution used for this project was an altered version of the  classic Kreb's solution.  The altered Kreb's solution has calcium chloride eliminated,  since Ca** ion, may facilitate the denaturation o f collagen in the tissue and thus may affect the mechanical response o f the sample (Reddi, 1984). The ingredients o f the  80  altered Kreb's solution are presented in Table 3.1.  The p H of the solution was  adjusted to 7.4 - 7.5 using Hydrochloric Acid and Sodium Hydroxide.  Once the  solution was made, 500 ml of Kreb's solution was poured into one ofthe 1-L beakers. The Kreb's solution will flow through the heat exchanger to be heated and then slowly dripped into the tissue bath to be warmed.  The solution in the beaker was then  bubbled with air, and the water circulation system is used to maintain the Kreb's solution at 37°C.  Table 3.1  Ingredients of the 1 L altered Kreb's solution (g/I)  NaCl  KC1  MgS0 .7H 0  KH P0  6.9  0.35  0.29  0.16  4  2  2  4  NaHC0 2.1  3  Glucose 2.0  A n additional 500 ml of Kreb's solution was set aside in which 2.4624 g of 2-Deoxy-Glucose (2-Deoxy-Glucose, Fisher Scientific Co., Ottawa, O N , Canada) was mixed in.  It is then poured into the other 1-L beaker and slowly flowed through the  heat exchanger to be heated and then to a separate tissue bath (reserved for Poison use) to be warmed.  A concentration o f 30mmol (0.0049248 g o f 2-Deoxy-Glucose  dissolved in 1 ml o f Kreb's solution) was chosen for this project according to the previous study performed by Martel (2001).  81  3.3.4 Harvesting tissue samples Wistar-Moller male rats weighing 200-250g (6-8 weeks old) were used for this research.  The rats were euthanized by carbon dioxide asphyxiation. After verifying  that the animal is lifeless, the back of the rat was shaved.  Since skin is naturally under  tension, the length o f the sample to be used in biomechanical experiment was measured and marked, before the skin was harvested.  This was done to ensure that  the tissue sample was at its natural tension at the baseline condition for experiments. A skin marker was used to mark the places (ie. 0.5 cm from the edge o f skin) on the skin where the clamps o f the load frame were to be attached.  Each piece of tissue is  measured 5cm long by 2cm wide. Thus, the stretching area of each piece o f skin tissue is 4 cm long by 2cm wide. Also, since skin is anisotropic, a line parallel to the body axis of the animal (ie. along the vertebral column) was drawn with the skin marker to serve as a guide for consistently stretching the tissue in the same physiological orientation (Vogel, 1997).  The tissues are also marked to indicate  whether i f they are harvested from the frontal dorsal part or rear dorsal part o f the body. To facilitate access of the chemicals, mcluding those from Kreb's solution, 2-Deoxy-glucose, fixative (ie. formalin), apoptosis testing and staining, the fat layer under the dermis was gently removed with a surgical knife while the skin was being harvested.  This was done by pinning it to a wax sheet and carefully removing the  82  muscle layer from the dermis with a surgical knife.  On the other hand, the epidermis  was left intact, since it is a thick layer difficult to remove. A l l of the experiments performed in this study were approved by the Animal Care Committee of the University o f British Columbia (Vancouver, B C , Canada), Protocol Number A 0 3 - 0 1 6 1 .  3.3.5 Treatment groups From each rat, four pieces o f tissue with a dimension o f 5 cm wide and 2cm long were harvested.  Two pieces o f tissues are from the frontal dorsal part, and two pieces  of tissues are from the rear dorsal part of the back o f rat. The two pieces of tissues from the rear dorsal part ofthe body were immersed in the same solution as used for the biomechanical experiments, i.e. Kreb's buffer or Kreb's buffer containing 2-Deoxy-glucose (30mmol), for 60 minutes.  Thus, two  baths, with one containing Kreb's buffer and the other containing Kreb's buffer with 2-Deoxy-glucose, were needed.  The two pieces of tissues from the frontal dorsal part  o f the body were used for the biomechanical experiment in which the skin was stretched using the load frame described previously.  Since each piece of tissue is  stretched 13% by turning the wheel manually, it is important to determine the displacement caused by turning the wheel once.  This is determined by using the  83  change o f displacement divided by the total number of turns that is needed to cause the displacement. The number of turns required to cause 13% strain is determined for each biomechanical tests. Then one piece of tissue was then stretched while immersed in Kreb's buffer, and the other piece was stretched while immersed in Kreb's buffer containing 2-Deoxy-glucose.  The treatment was carried out for the  same total time (60 minutes) as used for the tissue subjected to soaking only.  The  unstretched tissues are placed at the bottom o f the bath to be soaked without any disturbances by the tissue that are being clamped and stretched by the load frame.  To  preserve viability o f cells, the solutions in both baths were bubbled with air and the temperature was maintained at 37°C.  These same environmental conditions were  applied to the skin sample subjected to biomechanical experiments. Once the biomechanical experiment was done, both the piece o f skin used in mechanical test and the skin only immersed in the bath, were cut and placed in labeled cassettes. They were then immediately fixed in 4% formalin. were fixed within 100 minutes o f harvesting:  A l l tissue samples  60 minutes to carry out an experiment  (either biomechanical experimental or static soaking in buffer), about 20 minutes for removing the muscle layer and 20 minutes o f manipulations. The experimental procedure described above, produced four treatment groups for tissues which were harvested from each animal (Figure 3.6):  1) skin soaked in Kreb's  84  buffer, 2) skin soaked and stretched in Kreb's buffer, 3)skin soaked in Kreb's buffer with 2-Deoxy-glucose (30 mmol) (i.e. Poison treatment) and 4) skin soaked and stretched in Kreb's buffer with 2-Deoxy-glucose (30 mmol) (i.e. stretched in Poison). Two tissue samples from each treatment group were taken to ensure that the best samples were chosen for further processing.  Figure 3.6 The four treatment groups harvested from each rat.  The experimental procedure was carried out on 30 rats, and each rat was assigned a letter (ie. group B to group AF).  A naming system was developed to name each o f  the four treatment groups of tissues from each rat.  Group B rat, for instance, has 2  skin samples soaked in Kreb's buffer, named B . K . I and B . K . 2 , 2 skin samples soaked  85  and stretched in Kreb's buffer, named B . K . S . l and B . K . S . 2 , 2 skin samples soaked in Poison, 2-Deoxy-glucose, named, B.P.I and B.P.2, and 2 skin samples soaked and stretched in Poison, 2-Deoxy-glucose, named B . P . S . l and B.P.S.2.  3.3.6 Biomechanical experiments The long side o f each tissue sample was parallel to the body axis and was tested along that axis in the load frame. One c^timeter o f the skin tissue was mounted between the clamps (ie. 0.5 cm o f skin tissue for each end of the original 5 cm tissue sample).  Thus, 4cm was taken as the original length o f the tissue.  After mounting  j  the sample, the exact dimensions ofthe tissue were measured using a micrometer. The specimen used for the biomechanical experiments was about 2 cm wide, 1mm thick and 4 cm long. Each piece of tissues subjected to mechanical tests was first preloaded to a stress level of about 50 N / m or Pa. 2  The tissue was then allowed to relax (i.e., the tissue  tension decreased with time) until the tension reading stayed constant during the first 30- 60 seconds.  The tissue was then preconditioned with the loading and unloading  cycles repeated three times, as shown in Figure 3.5 with constant strain o f 13%. There is a 30 seconds of rest period between every loading and every unloading step that illustrates the elastic recovery of each tissue sample.  However, only the data  86  from 4 curve will be used in the mechanical analysis.  Both the tension, strain  applied on the tissue sample, and the time during the stretching process were recorded on the P C and analyzed subsequently.  The tension values are manually converted to  stress values (a) (ie. force divided by tissue area), and distance values are manually converted to strain values (ie. the quotient o f final length minus initial length and initial length).  Four data points are recorded per second to ensure an accurate  stress-strain pattern tracing. Upon observation of the stress-strain curves, the relaxation during the 30 seconds o f rest period is found to illustrate the quantitative difference between tissues that are soaked in Poison and stretched and those that are soaked in Krebs' solution only and stretched.  The tissue samples from 20 rats, which show stress relaxation  differences (ie. differences between Kreb's solution treatment and Poison treatment) ranging from 0 N / m to 110.9962 N / m , are selected from the 30 groups for further 2  2  processing and morphological analysis.  3.3.7 Processing tissue for light microscopy Upon completion of the biomechanical test, the tissue was cut and immediately fixed using 4% formalin. Vlady Pavlova.  The remaining processing steps were carried out by M s  After an overnight fixation at room temperature, the tissues were  87  washed three times in Phosphate Buffer Saline (PBS).  After washing in distilled  water, the tissues were dehydrated through a graded series of alcohol solutions.  The  dehydration using alcohol involved two changes o f 70%, followed by two changes o f 95%, and then three changes o f 100% absolute alcohol.  Then, the tissues were  subjected to three changes of xylene and three changes o f melted wax at 65°C under pressure.  This was done to ensure the penetration of the wax throughout the tissue.  Infiltration was done using Miles Scientific Tissue-Tek VIP processor.  Using a  Tissue-Tek III Embedding Center, embedding was done with wax by placing properly oriented tissue samples in a metal mold and covering them with hot wax. then cooled to room temperature to produce a paraffin block.  They were  A l l pieces were  embedded in paraffin and sectioned such that a cross-section o f skin perpendicular to the dermis was observed in every section in the light microscope. Thus, cells in the entire dermal layer can be examined. Four paraffin sections, 4um thick, were obtained from each block and picked up on 25mm by 75mm by 1.0mm labeled glass slides. The sectioning was done using Leica RM2135 ultramicrotome.  Sections were then stained with Roche Scientific In  Situ Cell Death Detection Kit.  88  3.3.8 Performing apoptosis test on processed tissue using Roche Scientific In Situ Cell Death Detection Kit A n In Situ Cell Death Detection Kit procedure was used to detect the presence o f apoptotic cells in the tissues.  Professionals from the Waxit Company in U B C were  hired to perform this procedure.  To carry out an apoptosis test on paraffin-embedded  sections, the slides were first dewaxed by heating at 60°C.  Then, they were  rehydrated through a grade series o f xylene and ethanol, including absolute, 95%, 90%, 80%, 70%, and diluted in double distilled water.  To ensure that the residual  wax was eliminated from the tissues sections on the slides, the rehydrating of the sections using each concentration o f alcohol was performed for 5 minutes.  The  sections then went through the protease treatment at which they were soaked in 0.1 M Citrate buffer at a p H o f 6 and subjected to microwave irradiation.  For microwave  irradiation, slides were placed in a plastic jar containing 200ml 0.1 M Citrate buffer with p H 6 and then subjected to 350 W microwave irradiation for 5 minutes. cooled rapidly by adding 80 ml of distilled water (20°C-25°C) to the jar.  They are  Afterwards,  the slides were rinsed twice with Phosphate buffered saline (PBS). Following the rinsing step, the T U N E L reaction mixture was prepared using, the Enzyme Solution (vial 1) and the Label Solution (vial 2) provided by the Kit.  One  pair o f vials was sufficient for staining 10 samples by using 50 ul T U N E L reaction mixture per sample and 2 negative controls by using 50 ul Label Solution per control.  89  lOOul o f Label Solution (vial 2) was removed for the negative controls. Then, the total volume (50ul) of Enzyme solution (vial 1) was added to the remaining 450 ul Label Solution in vial 2 to obtain 500ul T U N E L reaction mixture. The solution was mixed well to equilibrate the components. Before each experimental apoptosis test, a negative control and positive control should be included. apoptosis kit.  These controls were used for testing the effectiveness o f the  For negative control, the fixed and permeabilized cells were incubated  in 50ul/well Label Solution (without terminal transferase) instead o f T U N E L reaction mixture.  Without addition of any enzyme, the negative controls were expected to not  show any signs o f apoptosis.  For positive control, the fixed and permeabilized cells  were incubated with DNase I, grade I (3000 U/ml - 3 U/ml in 50 m M Tris-HCl, p H 7.5,10 m M M g C l 1 mg/ml B S A ) for 10 minutes at 15°C-25°C to induce D N A strand 2  breaks.  Thus, all the experimental slides could be compared to the positive controls  to evaluate the intensity o f the apoptotic signals. To perform the apoptosis testing, the area around each sample was dried first. 50ul o f T U N E L reaction mixture on sample was added on the samples.  The samples  were incubated for 60 minutes at 37°C in a humidified atmosphere in the dark. incubation, the slides are rinsed three times with PBS.  After  50-100 ul of D A B Substrate or  alternative P O D substrates is added to the slides. The slides were again incubated for  90  10 minutes at 15°C-25°C and rinsed three times with PBS.  Finally, the slides were  mounted under glass coverslip (i.e. with PBS/glycerol) and analyzed under the light microscope.  To examine the morphological changes of the tissues due to stretching  and or Poison and or mechanical damage by cutting, the slides from the selected groups (ie. 20 groups), which shows relaxation values ranging from 0 N / m to 2  110.9962 N / m , were counterstained with hematoxylin prior to analysis by light 2  microscope.  Hematoxylin stained the nuclei of the cells and intensified the apoptosis  signals.  3.4  Morphometric analysis  3.4.1 Cell counting To determine the number of apoptotic and non-apoptotic fibroblasts in each tissue samples, each slide is observed under a light microscope Leica Galen III, operated at 120V (Buffalo, N Y in U.S.A).  A 10 squares by 10 squares ocular grid (area:  0.225mm * 0.225mm) is placed in the ocular lens to enclose a fixed area for cell counting (Figure 3.7).  A random standardized way is used for picking the area that  has proper dermal connective tissue.  Areas, including blood vessels (Figure 3.8), fat  droplets (Figure 3.9) and hair follicles (Figure 3.10) are not chosen for cell counting. The space between two hairshafts is chosen as a proper area for cell counting (Figure  91  3.11a and b). The selection of area is carried out for three out of four sections for each slide.  The cell counting process is done at a magnification of 600 times (i.e.  ocular magnification (15) * objective magnification (40)).  Figure 3.7 The 10 squares by 10 squares ocular grid used for enclosing a fixed area for cell counting.  Figure 3.8 Blood vessels.  92  Figure 3.9 Fat lobules.  Figure 3.10 A hair follicle.  93  Figure 3.11 a and b A proper area for cell counting.  Three major types o f cells, including mast cells, macrophages and fibroblasts, can be seen in dermal connective tissue under the microscope.  Fibroblasts are the main  interest of the study, and under the microscope, they are usually stretched along collagen fibers or contain triangular membrane with round or oval nuclei (Figure 3.12). The other cells are classified as miscellaneous cells. Before counting cells, a positive and a negative control slides must be observed under the microscope to identify the  94  characteristics o f apoptotic and non-apoptotic fibroblasts.  A n apoptotic fibroblast, as  shown in the positive control in Figure 3.12, has its nuclei, but not its cytoplasm, stained brown.  A non-apoptotic fibroblast, as shown in the negative control in Figure  3.13 has its nuclei, stained blue.  Figure 3.12 The morphology of fibroblasts in a positive control.  Figure 3.13 The characteristics of the non-apoptotic fibroblasts in a negative control.  95  Prior to cell counting, the group letters of the slides were all blinded to avoid any bias in cell counting. For each of the three sections in each slide, a proper area o f connective tissue is chosen, and the number of non-apoptotic and apoptotic fibroblasts and non-apoptotic and apoptotic miscellaneous cells are counted.  3.4.2 Statistical analyses To study the biomechanical difference between tissue samples soaked in Kreb's solution and stretched and those soaked in Poison and stretched, the fourth loading curves for both stretched tissues samples soaked in Kreb's and Poison were carefully analyzed.  It was observed that the stress relaxation, i.e. drop in stress, during the 30  second relaxation period for tissue soaked in Kreb's solution were generally larger than for tissue soaked in Poison.  The maximum and minimum stress values of each  ofthe fourth curve for the mechanically stretched tissue samples soaked in Kreb's solution and in Poison are shown in Table 3.2.  The difference between the maximum  and minimum stress values for each group of rats or the drop in stress is computed for both the tissues soaked in Kreb's solution and stretched and soaked in Poison and stretched.  Each difference illustrates the relaxation o f each piece o f tissue under  constant strain. Percentage stress relaxation or reduction in stress (i.e. difference divided by the maximum stress value) was computed for each treatment  96  Table 3.2 Stress relaxation difference for tissues soaked and stretched in Kreb's solution and in Kreb's solution with poison  Rat  Max. Stress  Min. Stress  Reduction in  Reduction in  Max. Stress  Min. Stress  Reduction in  (K)  (K)  Stress (Pa)  Stress (%)  (P)  (P)  Stress (Pa)  Reduction in Stress  (%)  (reduction in  (reduction in  stress/max.stress)  stress/max. stress)  B  491.1  414.8  76.2  15.5  844.6  657.4  187.2  22.2  C  536.5  460.7  75.8  14.1  605.4  488.2  117.2  19.4  D  233.2  184.9  48.3  20.7  157.3  129.8  27.6  17.5  E  637.24  526.7  110.5  17.3  512.9  450.7  62.2  12.1  F  367.8  243.5  124.3  33.8  340.2  250.4  89.8  26.4  G  265,2  189.2  75.9  28.6  384.0  262.7  124.3  32.1  H  445.2  342.0  103.2  23.2  445.2  362.7  82.5  18.5  I  401.4  318.8  82.5  20.6  506.1  396.1  110.0  21.7  J  654.6  501.7  153.0  23.4  288.4  239.7  48.7  16.9  K  619.9  467.0  153.0  24.7  710.3  557.4  153.0  21.5  L  605.9  425.5  180.4  29.8  305.3  235.9  69.4  22.7  M  286.7  217.4  69.4  24.2  229.4  201.7  27.7  12.1  N  381.2  263.3  117.9  30.9  444.1  333.1  111.0  25.0  0  201.0  159.4  41.6  20.7  228.0  172.5  55.5  24.3  Table 3.2  Rat  Continue.  Max. Stress  Min. Stress  Reduction in  Reduction in  Max. Stress  Min. Stress  Reduction in  (K)  (K)  Stress (Pa)  Stress (%)  (P)  (P)  Stress (Pa)  Reduction in Stress (%)  P  334.3  230.6  103.7  31.1  313.5  244.4  69.1  22.1  Q  301.9  246.6  55.3  18.3  444.7  341.0  103.7  23.3  R  245.4  190.2  55.2  22.5  243.1  187.9  55.2  22.7  U  322.8  240.0  82.8  25.7  499.4  388.9  110.5  22.1  s  318.4  214.4  104.0  32.7  291.6  229.2  62.4  21.4  T  402.9  312.7  90.1  22.4  474.3  377.2  97.1  20.5  V  572.5  434.0  138.4  24.2  421.0  324.1  96.9  23.0  W  389.7  272.0  117.7  30.2  369.0  279.0  90.0  24.4  X  274.8  219.5  55.4  20.2  307.6  245.3  62.3  20.3  Y  354.9  258.1  96.8  27.3  364.1  295.0  69.2  19.0  Z  511.2  379.7  131.4  25.7  469.0  392.9  76.1  16.2  AA  501.2  376.9  124.3  24.8  282.7  234.3  48.3  17.1  AB  355.3  306.9  48.3  13.6  291.9  202.2  89.8  30.8  AC  222.8  167.6  55.2  24.8  279.5  224.2  55.2  19.8  AD  232.5  184.2  48.3  20.8  197.6  163.0  34.5  17.5  AE  218.9  170.4  48.4  22.1  279.3  203.2  76.1  27.2  AF  361.1  202.0  159.1  44.1  237.4  182.1  55.3  23.3  group in each rat group.  A paired t-test, as shown in Table 3.3, was used to compare  the stress relaxation values of the stretched tissues soaked in Kreb's solution and those o f stretched tissues soaked in Poison for all the groups.  The sample size for the t-test  corresponded to the total number of rats that are used in the experiment (i.e. n = 30). The null hypothesis (Ho) set for the t- test was that there was no difference between the relaxation values for the two treatment groups.  The alternate hypothesis (H ) set for a  the t-test was that there is a difference between the relaxation values for the treatment groups that are soaked and stretched in Kreb's solution and for those that are soaked and stretched in Poison.  The computed t value was 2.141, which is larger than the  critical t (2-tailed) value, 2.042 (Zar 1999). Therefore, there was a significant difference (p < 0.05) between the relaxation values for the treatment groups that are soaked and stretched in Kreb's solution and for those that are soaked and stretched in Poison. To investigate the rationale for the difference in the relaxation values between the tissues that are soaked and stretched in Kreb's solution and for those that are soaked and stretched in Poison, a morphological analysis was carried out on the tissue samples of the four treatment groups for the 20 rats with difference in the relaxation values that are higher than or equal to zero.  This procedure is performed in order to determine  whether there is a relationship between the difference in the relaxation values of the  99  stretched tissues soaked in Kreb's solution and in Poison and the ratio o f apoptotic and non-apoptotic fibroblasts.  100  Table 3.3 Paired t-test for evaluating the significance of the relaxation differences between tissues soaked and stretched in Kreb's solution and in Kreb's solution with poison. (D: difference in stress relaxation between 2 treatments, n: number of groups, v: degree freedom, 10.05 (2-tailed): critical values of the t distribution, Ho: Ho: Ha:  "  null hypothesis, Ha:  alternate hypothesis)  nd = 0 (id not = 0  Paired Sample Inference (Normal data [n>=30]) Rat  D  Square of D  C  -41.7  1711.0  D  20.7  427.7  E  48.4  2338.4  F  34.5  1193.1  G  -48.3  2335.1  H  20.6  425.8  I  -27.5  756.9  J  104.3  10875.8  K  0  0  L  111.0  12320.2  Sum of D  N 519.9  Table B.3 [Critical Values ofthe t Distribution] 30 t0.05(2-tailed),30  Sum of Squares  2.042  V  66012.7  Average Difference  29  t  2.141  teal > t crit  17.3 Therefore, we can reject Ho  Variance The difference between the Differences (Krebs vs. Poison) is  M  41.6  1732.5  N  6.9  48.1  44.3  significantly different.  Table 3.3 Continue. Rat  Square ofD  D  0  -13.9  192,5  P  34.6  1195.1  Q  -48.4  2342.3  R  0  0  U  -27.6  762.5  s  41.6  1731.3  T  -6.9  48.1  V  41.5  1724.3  w  27.7  766.7  X  -6.9  48.0  Y  27.7  765.9  Z  55.3  3061.2  AA  76.0  5769.8  AB  -41.4  1716.6  AC  0  0  AD  13.8  190.6  AE  -27.7  765.7  AF  103.8  10767.5  Standard Error 8.1  t 2.141  4.  Results The main purpose o f this project involved the assessment of the effects of Kreb's  buffer, stretching and exposure to 2-Deoxy-D-Glucose on fibroblast's viability in the dermis of the skin.  Once these effects were determined, the contribution of the  fibroblasts to the mechanical properties ofthe connective tissue in rat skin was analyzed. The graphical data obtained from the biomechanical tests, including the stress and strain values, for tissues stretched in Kreb's solution and in metabolic Poison, 2-Deoxy-D-glucose, for the experimental rats are presented in this chapter.  The  stress of the tissue samples during the biomechanical tests is shown as a function ofthe strain o f the stretched skin.  The results o f the morphological analysis o f the  fibroblasts, mainly the viability study, are also presented.  The relationship between  the viability of the fibroblasts and the rat skin's mechanical properties is also discussed.  4.1  Biomechanical results  The biomechanical tests were performed on 30 rats. The experimental procedures described in chapter three were followed for two o f the four pieces of tissues from each rat.  Mechanical testing was performed on one piece o f tissue  103  soaked in Kreb's solution and another piece of tissue soaked in Poison.  The other  pieces were soaked in Kreb's solution with and without Poison, but were not subjected to mechanical load.  4.1.1 Stretching in Kreb's buffer In this part of the study, skin harvested from the frontal dorsal part o f the rat body was immediately soaked in Kreb's solution and then preloaded to a stress level o f 50 Pa for 60 seconds.  To precondition the tissue, it was loaded and unloaded three times.  Thus, the fourth loading and unloading curve of each test was analyzed.  The tissue  soaked in Kreb's buffer was stretched to a strain o f 13% at a rate of0.0042 cm/s. At 13% strain, there was a 30 second rest period before unloading to 0% strain. strain, and time were recorded on the P C and analyzed subsequently.  Force,  The force  values were manually converted to stress values (a) (ie. force divided by tissue area), and distance values were manually converted to strain values (ie. displacement divided by initial length).  A stress-strain curve for tissue from rat group E soaked in Kreb's  solution is shown in Figure 4.1 as a blue-colored curve. level which is caused by the load frame.  The curve shows some noise  Upon loading, the stress values increased to  637 Pa, and after 30 seconds o f constant strain, it relaxed to 527 Pa.  104  700  I • E.K.  0  0.02  0.04  0.06  0.08  0.1  0.12  0.14  Strain (cm/cm)  Figure 4.1 Typical stress-strain curves for samples soaked in Kreb's solution and Poison, respectively.  The strain was then returned to its initial value. stress and relaxation values for each rat group.  Table 3.2 shows all the maximum The difference between the maximum  and the minimum stress values during the relaxation period (i.e. stress relaxation value or reduction in stress (Pa)) for rat E is 111 Pa which is a reduction in stress o f 17.35%. Table 3.2 shows that the maximum stress on tissues soaked in Kreb's solution ranged from 201 Pa to 655 Pa; the minimum stress ranged from 159 Pa to 527 Pa; the relaxation values ranged from 42 Pa to 180 Pa which is a reduction in stress ranging from 14 to 44%.  105  The average relaxation value for all 30 rat groups is 94 Pa with a standard deviation of 39 Pa and coefficient of variation of 0.47 (i.e. standard deviation divided by mean relaxation values).  4.1.2 Stretching in Kreb's solution with 2-Deoxy-D-Glucose In this part of the study, skin harvested from the frontal dorsal part of the rat body was immediately soaked in 2-Deoxy-D-Glucose, Poison, and then subjected to the same preloading, preconditioning and stretching procedure as the tissues mentioned in Section 4.1.1. The data were analyzed in the same way as well.  A stress-strain  curve for specimen E soaked in Poison is shown in Figure 4.1 as a red-colored curve. The curve showed a very low noise level that is caused by the load frame. Upon loading, the stress increased to 513 Pa, and then it relaxed to 451 Pa after 30 seconds of constant straining. The tissue was then unloaded. For rat E, the stress relaxation was 62 Pa which is a reduction in stress of 12%.  Table 3.2 shows that the maximum stress  values of the tissues soaked in Kreb's solution ranged from 157 Pa to 845 Pa; the minimum stress values ranged from 130 Pa to 657 Pa; the relaxation values ranged from 28 Pa to 187 Pa with reduction in stress ranging from 12 to 32%.  106  j  /  The average relaxation values for all 30 rat groups are computed to be 81 Pa with a standard deviation of 35 Pa and coefficient of variation of 0.44 (i.e. standard deviation divided by mean relaxation values).  4.1.3 Comparison of the tissues soaked and stretched in Kreb's solution with and without Poison The average stress relaxation for specimens treated with Poison for all 30 rat groups is 81 Pa with a standard deviation of 35.41 Pa and a coefficient of variation of 0.26.  The average stress relaxation and average reduction of stress fortissuessoaked  in Poison were lower than those oftissuessoaked in Kreb's solution by 13.19 Pa ± 3.10 Pa and 3.05% ± 1.85%respectively. Figure 4.2a shows the average stress relaxation values, and Figure 4.2b shows the average reduction in stress for specimens of the 30 rats soaked in Kreb's solution with and without Poison.  107  140  Kreb's + Stretch  Poison + Stretch  Figure 4.2a Average stress relaxation for tissues soaked in Kreb's solution with and without the presence of poison. J3  30  24.4435  m  Kreb's + Stretch  Poison + Stretch  Figure 4.2b Average reduction in stress for tissues soaked in Kreb's solution with and without poison.  108  According to the paired t-test in Table 3.3, there is a significant difference in stress-relaxation for tissues soaked in Kreb's solution and in Poison.  Tissue samples  from 20 rats which showed relaxation differences ranging from 0 Pa to 111 Pa, were selected from the group o f 30 for further processing and morphological analysis. Figure 4.3 shows the average relaxation values for tissues soaked in Kreb's solution with and without Poison for the selected 20 rats.  The average stress relaxation for  tissues soaked in Kreb's solution is 109.01 Pa with a standard deviation of38.90 Pa. The average stress relaxation for tissues soaked in Poison is 70.94 Pa with a standard deviation of29.35 Pa. The average stress relaxation for tissues soaked in Kreb's solution was higher than that for the tissues soaked in Poison by 38.07 Pa ± 9.55 Pa. The higher relaxation values indicate that the tissue is less stiff and can relax to a greater degree.  Therefore, tissues soaked in Kreb's solution and strained to 13%  become less stiff, allowing the tissue to relax more, compared to those soaked in Poison, and similarly strained.  109  160  140  109.01  I  1  70  94  111  Lv  Y Kreb's + Stretch  Poison + Stretch  Figure 4.3 Average stress relaxation for the 20 selected rats with tissues stretched and soaked in Kreb's solution with and without poison.  4.2 Morphological analysis  Upon completion of the biomechanical experiments, the tissue samples from the 20 selected rats with relaxation differences (ie. tissues stretched in Kreb's solution versus tissues stretched in Poison) greater than zero were subjected to an apoptosis test to evaluate the viability of the fibroblasts.  110  4.2.1 Fibroblasts count for the four treatment groups After the tissues samples were processed and subjected to apoptosis testing, non-apoptotic fibroblasts and other miscellaneous cells were blue, while apoptotic fibroblasts and other miscellaneous cells were brown. The number o f non-apoptotic and apoptotic fibroblasts and miscellaneous cells were counted.  Since there were  three tissue sections in each slide (ie. each rat), cell counting was carried out for each tissue section. The average number o f cells for all three sections was computed for each rat group as shown in Table B. 1 in Appendix B . To determine the effect o f each treatment (i.e. soaked in Kreb's solution, soaked and stretched in Kreb's solution, soaked in Poison, soaked and stretched in Poison) on the viability o f the fibroblasts, the sums o f the non-apoptotic and apoptotic fibroblasts were computed for each treatment group (Table B.2 in Appendix B).  For  non-apoptotic fibroblasts, the tissues soaked in Kreb's solution had 2.35 times more non-apoptotic fibroblasts compared to those soaked and stretched in Kreb's solution; the tissues soaked in Kreb's solution had 1.33 times more non-apoptotic fibroblasts compared to those soaked in Poison; tissues soaked and stretched in Poison had 1.71 times more non-apoptotic fibroblasts compared to those soaked and stretched in Kreb's solution; tissues soaked in Poison had 1.03 times more non-apoptotic fibroblasts compared to those soaked and stretched in Poison.  For apoptotic fibroblasts, the  tissues soaked and stretched in Kreb's solution had 5.56 times more apoptotic  111  fibroblasts compared to those soaked in Kreb's solution; the tissues soaked in Poison had 2.66 times more apoptotic fibroblasts compared to those soaked in Kreb's solution; tissues soaked and stretched in Kreb's solution had 1.66 times more apoptotic fibroblasts compared to those soaked and stretched in Poison; tissues soaked and stretched in Poison had 1.26 times more apoptotic fibroblasts compared to those soaked in Kreb's solution.  The results show that Kreb's solution caused more  non-apoptotic fibroblasts, while sketching caused more apoptotic fibroblasts in the tissues.  Although the Poison caused slightly more apoptotic fibroblasts compared to  the Kreb's solution, it caused less apoptotic fibroblasts in tissues compared to stretching.  The average number of non-apoptotic and apoptotic fibroblasts (i.e. total  number o f fibroblasts divided by the 20 groups) and the percentages o f non-apoptotic fibroblasts (i.e total number of non-apoptotic fibroblasts divided by total number o f fibroblasts) and apoptotic fibroblasts (i.e. total number o f apoptotic fibroblasts divided by total number o f fibroblasts) were also computed for each treatment group as shown in Table B.2 in Appendix B.  Figure 4.4a shows the number of non-apoptotic and  apoptotic fibroblasts in 20 samples for each treatment group; Figure 4.4b and Figure 4.4c shows the average number and the relative amounts o f both non-apoptotic and apoptotic fibroblasts, respectively. Tissues soaked in Kreb's solution has the highest number of non-apoptotic fibroblasts, while tissues soaked and stretched in Kreb's  112  solution has the lowest number of non-apoptotic fibroblasts. Tissues soaked and stretched in Kreb's solution have the highest number o f apoptotic fibroblasts, while tissue soaked in Kreb's solution has the lowest number o f apoptotic fibroblasts. These results illustrate that Kreb's solution prevents fibroblasts from apoptosis, and stretching effectively kills fibroblasts.  The high ratio o f non-apoptotic fibroblasts  observed in tissue soaked in Kreb's buffer suggest that the Kreb's solution provides all the nutrients necessary to keep the cells from apoptosis.  In terms ofthe poison  treatment, there is no significance difference between the number and the percentages of fibroblasts in tissues soaked in Poison and those soaked and stretched in Poison. This result illustrates that stretching in Poison does not effectively kills more fibroblasts.  However, the figure shows that tissues soaked in Poison have more  apoptotic cells and less non-apoptotic cells compared to tissues soaked in Kreb's solution.  113  600  9 non-apoptotic fibroblasts I apoptotic fibroblasts  Kreb's + stretch  Poison + stretch  Figure 4.4a Total number of non-apoptotic and apoptotic fibroblasts in 20 samples for four treatment groups.  9 non-apoptotic fibroblasts I apoptotic fibroblasts  Kreb's + stretch  Poison + stretch  Figure 4.4b The average number of non-apoptotic and apoptotic fibroblasts in 20 samples for four treatment groups.  114  115  5.  Discussion To investigate the role o f fibroblasts on the mechanical properties o f skin, rat skin  tissues were soaked and/or stretched in Kreb's solution with or without the Poison, 2-Deoxy-D-Glucose.  The effects of the 2 treatments, soaking and stretching in  Kreb's solution and soaking and stretching in Poison, on the number o f apoptotic and non-apoptotic fibroblasts will be discussed.  This chapter will also be discussed the  correlation between the relaxation values obtained from the biomechanical experiments and the number o f apoptotic and non-apoptotic fibroblasts obtained from the morphological analysis.  Finally, the sources of errors and the equipment  constraints that have an effect on the accuracy of the results are outlined.  5.1  Viability of fibroblasts The fibroblasts in the tissues of the 30 rat were subjected to four treatments, as  mentioned in section 3.3.4.  However, to evaluate relationship between the fibroblasts  count and the mechanical properties of rat tissues, only the treatments involving stretching (i.e. soaked and stretched in Kreb's solution and in Poison) will be used in the analysis.  For viability analysis, only the 20 rats with relaxation values that are  greater and equal to zero were used.  The rats, each assigned with a letter, were first  put in descending order according to their relaxation values obtained from the  116  biomechanical experiments.  They were then compared to the number o f apoptotic  and non-apoptotic fibroblasts o f all four treatments. Figure 5.1a shows the number of non-apoptotic and apoptotic fibroblasts; Figure 5.1b shows ratio o f apoptotic to non-apoptotic fibroblasts, and Figure 5.1c shows the relative amounts o f apoptotic and non-apoptotic fibroblasts for the 20 samples that were soaked and stretched in Kreb's solution. The rat groups have been arranged in order of descending stress relaxation differences.  The figures show that stretching in  Kreb's solution causes a lot more apoptotic cells than non-apoptotic cells.  The  number and relative amounts of apoptotic cells decrease as the relaxation values decrease; thus, the tissues with more apoptotic cells relax more, while the tissues with fewer apoptotic cells relax less.  117  1  • •  1, i  n  non-apoptotic ibroblasts apoptotic fibro DlSStS  j 11 I I II MII pprr IF Fr ii mr m ii n II r r m i m i I II IKS 1  JK S1  AFKS1  AAK.St  ZK S 1  EK S 1  11 i i  MKS1 TKS1  VKS 1  PKSl  FKS 1  WKSI  1 •  Y K S 1 D K S 1 H K S 1 ADKS1  NK-St  KKS1  fi.KSI  AC K S 1  Rat groups  Figure 5.1a Number of apoptotic and non-apoptotic fibroblasts for samples soaked and stretched in Kreb's solution.  LXS.1  J.K.S.1  AF.K.S.1 AA.K.S 1 Z.K S 1  EX.S l  M.K.B.1  T.K.S.1  V.K.S.1  P.K.S.1  F.K.S.1  W.KS.1  YX.S.1  DKS1  H.K.S.1  AD.K.S.1  N.KS.1  KK.S.1  MSI  ACX.S.1  Rat groups  Figure 5.1b Ratio of non-apoptotic to apoptotic fibroblasts for samples soaked and stretched in Kreb's solution.  118  B non-apoptotic fibroblasts • apoptotic fibroblasts  I i1  i i i II 1 II i i i i i i i i r i IPI P 1 II 1 II r p r r ii 1 II 1 ri i i ii1 pII i i i i i r 1 1 I I 1 II i i i « i •  •  •  r  f  #  #  i  <f #  4f #  ^  <*-  s  <t>- J *  1  J*"  ?  III .I*  -  #~ &  f fr~  Rat groups  Figure 5.1c Relative amounts of non-apoptotic and apoptotic fibroblasts for samples soaked and stretched in Kreb's solution.  Figure 5.2a shows the number of non-apoptotic and apoptotic fibroblasts, Figure 5.2b shows the ratio o f apoptotic to non-apoptotic fibroblasts, and Figure 5.2c shows the relative amounts of apoptotic and non-apoptotic fibroblasts for the 20 samples that were soaked and stretched in Poison. The rat groups have been arranged in the order o f descending stress relaxation differences.  The figures show that  stretching in Poison in general causes more non-apoptotic cells than apoptotic cells. The pattern is the reverse of the case where tissues were soaked and stretched in Kreb's solution.  The number and the relative amounts of non-apoptotic cells are a lot higher  119  than for tissues stretched in Kreb's solution. On the other hand, the number and the relative amounts of apoptotic cells are lower. Thus, the Poison, 2-Deoxy-D-Glucose, is shown to cause more non-apoptotic fibroblasts than apoptotic fibroblasts.  I non-apoptotic f i b r o b l a s t s I apoptotic fibroblasts  | •5  1 E  L.P.S.1  J.P.S.t  AAP5.1  ZP.S.l  EP.S.1  Mfi.B 1  T.P.S.1  VPX.1 PJ»J1  F.PJM  W.PS.1  Y.PS.1  DP5.1  HJ»£.1  AD.P.S.1  N.P.S 1  K.P.S.1  RPS.1 ACP.S.1  Rat groups  Figure 5.2a Number of non-apoptotic and apoptotic fibroblasts for samples soaked and stretched in Poison.  120  l  l  H [f  f j  b  F ;  !  rl i 1  i  1 •  I1  r  i.1  1  LP .S .1. i5 P 1 S1.AFA .A P.SI 2P .S1. £PS1. S .1 . T ». V S1 . S1. SJ1 P.. RatS1 groups ;  MJ  PP  FJ>  w »A1  YP . S1 . DP . S1 .  --  •si  _n_n ,  H  HP .S .1 . A D  Figure 5.2b Ratio of non-apoptotic to apoptotic fibroblasts for samples soaked and stretched in Poison.  • non-apoptotic fibroblasts • apoptotic fibroblasts  Rat groups  Figure 5.2c Relative amounts of non-apoptotic and apoptotic fibroblasts for samples soaked and stretched in Poison.  121  To further compare the effects of the two treatments, soaking and stretching in Kreb's solution and in Poison, on the viability o f fibroblasts, the ratios of the number of fibroblasts in tissues stretched in Kreb's solution versus those stretched in Poison, ratios for both apoptotic and non-apoptotic fibroblasts are computed as shown in Table 4.1 and plotted in Figure 5.3.  The ratios for non-apoptotic fibroblasts are all below  one, indicating that tissues soaked and stretched in Kreb's solution cause fewer non-apoptotic fibroblasts compared to those soaked and stretched in Poison; however, the ratios for apoptotic fibroblasts are mostly above one (i.e. except for rats H , N and K), indicating that soaking and stretching in Kreb's solution cause more apoptotic fibroblasts compared to those soaked and stretched in Poison. again been arranged in descending stress relaxation differences.  The rat groups have As shown in the  figure, the ratios of non-apoptotic fibroblasts increases from 0.19 to 0.77 as relaxation decreases, illustrating that the tissues with more non-apoptotic fibroblasts relax less. The ratios of apoptotic fibroblast decrease from 2.96 to 1.84 as relaxation decreases, illustrating that the tissues with more apoptotic fibroblasts relax more.  122  Table 4.1 The ratios of the number of fibroblasts in tissues stretched in Kreb's solution versus those stretched in Kreb's solution with poison ratios for both non-apoptotic and apoptotic fibroblasts  non-apoptotic  Rat  apoptotic fibroblasts  fibroblasts  L  0.188  2.963  J  0.162  3.789  AF  0.226  2.613  AA  0.449  1.938  Z  0.533  1.944  E  0.565  1.778  M  0.556  1.657  T  0.528  1.771  V  0.755  1.667  P  0.630  1.871  F  0.563  2.074  W  0.623  2.000  Y  0.563  2.762  D  0.904  1.227  H  0.522  0.985  AD  0.800  1.325  N  0.892  0.964  K  1.143  0.962  R  0.95  AC  0.772  "  1.018 1.839  123  B Non-apoptotic fibroblasts • Apoptotic fibroblasts  111 if  1.5  i  I  IPirirnTr  0.5  L  J  AF AA  Z  E  M  T  V  I I I II11! I! I! III P  F  W  Y  D  H  AD  N  K  R  AC  Figure 5.3. Ratios of the number of fibroblasts for tissues stretched in Kreb's solution versus tissue stretched in Poison, for both non-apoptotic and apoptotic cells.  5.2 Correlation between stress relaxation and viability of fibroblasts in tissues To examine the role o f fibroblasts on the mechanical properties of skin, the difference in stress relaxation between the stress in tissues and the viability of fibroblast in tissues are analyzed in two ways.  Firstly, the differences in relaxation is  studied for the two treatments (i.e. stretched in Kreb's solution and in Poison) separately, and then it is studied for the two treatments simultaneously.  Formerly, the  relationship between the reduction in stress (Pa) in tissue samples for the two individual treatments (i.e. stretching in Kreb's solution and in Poison) and the viability of fibroblasts are analyzed.  Latterly, the stress relaxation differences between the two  124  treatments are compared to the viability o f fibroblasts.  For the latter relation, since  the relaxation differences reveal the mechanical difference between the two treatments, it is reasonable to compare them to the viability of fibroblasts caused by the two treatments.  The stress relaxation differences are compared to the relative amounts o f  fibroblasts in tissues soaked and stretched in Kreb's solution divided by fibroblasts in tissues soaked and stretched in Poison.  The results of both relationships are then  examined and compared to determine i f they are consistent with each other.  5.2.1 Relation between reduction in stress and relative amounts of non-apoptotic and apoptotic fibroblasts Figure 5.4 shows a plot o f reduction o f stress versus the relative amounts o f non-apoptotic fibroblasts for tissue samples stretched in Kreb's solution and in Poison against the percentage o f non-apoptotic fibroblasts. The reduction o f stress values are recorded in Table 3.2 and the relative amounts of non-apoptotic fibroblasts are recorded in Table 4.2.  125  Table 5.1 The relative amounts of non-apoptotic and non-apoptotic fibroblasts in tissues stretched in Kreb's solution and in Kreb's solution with poison Poison  Kreb's solution  % apoptotic % non-apoptotic fibroblasts fibroblasts  % non-apoptotic fibroblasts  % apoptotic fibroblasts  14.0  86.0  71.9  28.1  13.3  86.7  78.2  21.8  12.9  87.1  63.1  36.9  26.2  73.8  60.5  39.5  31.4  68.6  62.5  37.5  35.4  64.6  63.3  36.7  34.1  65.9  60.7  39.3  31.1  68.9  60.2  39.8  38.1  61.9  57.6  42.4  37.0  63.0  63.5  36.5  39.1  60.9  70.3  29.7  40.4  59.6  68.5  31.5  40.8  59.2  77.2  22.8  46.5  53.5  54.2  45.8  15.8  84.2  26.1  73.9  39.8  60.2  52.4  47.6  38.4  61.6  40.2  59.8  44.4  55.6  40.2  59.8  40.4  59.6  42.1  57.9  43.6  56.4  64.8  35.2  126  200  180  4>  *  160  •  •  140  _ S  1 2  •  0  55  *  • •  •  £ 100  4  • • • •  •  •  •  • Kreb's Solution * Poison  • 1  •  80  •  •  • • • •  « 60  •  *  •  •  40  •  • •  •  20  10  20  30  40  SO  60  70  80  90  % Non-apoptotic fibroblasts  Figure 5.4 Reduction in stress for tissue samples stretched in Kreb's solution and in Poison versus the relative amounts of non-apoptotic fibroblasts.  According to the figure, samples stretched in Kreb's solution show higher reduction in stress compared to samples stretched in Poison, and samples stretched in Kreb's solution also have lower relative amounts of non-apoptotic fibroblasts compared to samples stretched in Poison.  Thus, stretching in Kreb's solution causes less  non-apoptotic fibroblasts, allowing the samples to relax at a greater degree (i.e. decreased stiffness).  Figure 5.5 is a plot of the reduction in stress versus the relative  amounts of apoptotic fibroblasts for tissues stretched in Kreb's solution and in Poison.  127  180  160  140 — 120  •  c |  « * Kreb's Solution  100  a Poison  80  40  20  10  20  30  40  50  60  70  80  90  100  % Apoptotic fibroblasts  F i g u r e 5.5  R e d u c t i o n i n s t r e s s f o r tissue s a m p l e s s t r e t c h e d i n K r e b ' s s o l u t i o n  a n d i n Poison versus the relative amounts o f apoptotic fibroblasts.  According to the figure, samples stretched in Kreb's solution show higher reduction in stress compared to samples stretched in Poison, and samples stretched in Kreb's solution have higher percentage of apoptotic fibroblasts compared to samples stretched in Poison.  Thus, stretching in Kreb's solution kills fibroblasts apoptotically, causing  more apoptotic cells and allowing samples to relax at a greater degree (decreased stiffness).  128  5.2.2 Comparison between relaxation o f tissues and ratios o f relative amounts o f fibroblasts in tissues for the two treatments To study the two treatments simultaneously, the relaxation differences (i.e. difference in relaxation between tissues soaked and stretched in Kreb's solution and in Poison are compared to the relative amounts o f fibroblasts in tissues soaked and stretched in Kreb's solution divided by fibroblasts in tissues soaked and stretched in Poison.  The ratios of the relative amounts for non-apoptotic fibroblasts of two  treatments were computed as shown in Table 4.2.  The relative amounts o f fibroblasts  are used because the total number o f fibroblasts for the four treatment groups for each rat is different due to the different tissue areas chosen for cell counting; thus, it is reasonable to use the relative amounts of fibroblasts in the cell counting analysis. Figure 5.6 shows a plot of relaxation differences versus the ratios of the relative amounts of non-apoptotic fibroblasts in tissues soaked and stretched in Kreb's solution and the relative amounts of non-apoptotic fibroblasts in tissues soaked and stretched in Poison.  The results o f 17 o f the 20 groups are plotted; the 3 groups with zero stress  relaxation difference are eliminated. Within the range studied, i f the data points are linearly fitted, a correlation with an R o f 0.8516 is observed between the stress 2  relaxation differences and the ratios o f non-apoptotic cells.  The stress relaxation  differences decrease when the ratios of the relative amounts o f non-apoptotic fibroblasts for tissues stretched in Kreb's solution versus in Poison increase.  The  129  ratios are all below one, since tissues stretched in Kreb's solution have less non-apoptotic fibroblasts compared to tissues stretched in Poison.  When the ratios o f  the relative amounts of non-apoptotic fibroblasts o f the two treatments become closer in value (i.e. relative amounts of non-apoptotic fibroblasts in samples stretched in Kreb's solution increase), the stress relaxation differences approach zero.  120  • • • \  y = -13952X + 123.66 R = 0.8516 J  \^ *  •  60 • Percent of non-apoptoticfibroblaststorKraft" s treatment/ Percent of norvepoptotJc fibroblasts for Poison treatment  • «\ • • • \^ • • N. m  >.  •  N. I  0.2  0.4  0.6  0.8  • \ .  1  12  -20 Ratio of non-apoptotic fibroblasts .  Figure 5.6. Difference in stress relaxation for tissues soaked and stretched in Kreb's solution and in Kreb's solution with poison versus the ratio of the relative amounts of non-apoptotic fibroblasts in tissues soaked and stretched in Kreb's solution and in Poison.  130  To investigate the relationship between the relaxation differences and the presence of apoptotic fibroblasts, stress relaxation differences (i.e. difference i n relaxation values between the tissues soaked and stretched in Kreb's solution and in Poison) are also compared to the ratios of the relative amounts of apoptotic fibroblasts in tissues soaked and stretched in Kreb's solution to the relative amounts o f apoptotic fibroblasts in tissues soaked and stretched in Poison.  The ratios of the relative  amounts of apoptotic fibroblasts of two treatments are computed as shown in Table 5.2.  Figure 5.7 shows a plot o f stress relaxation differences versus the ratio o f the  relative amounts of apoptotic fibroblasts in tissues soaked and stretched in Kreb's solution to the relative amounts of apoptotic fibroblasts in tissues soaked and stretched in Poison.  The results o f 17 of the 20 groups are plotted; the 3 groups with zero  relaxation differences are eliminated.  Within the range studied, i f the data points are  linearly fitted, a correlation is observed between the stress relaxation differences and the ratios of non-apoptotic cells. The relaxation differences increase when the ratios o f the relative amounts of non-apoptotic fibroblasts for tissues stretched in Kreb's solution to the relative amounts of non-apoptotic fibroblasts for tissues stretched in Poison increase.  The ratios are all above one, since tissues stretched in Kreb's  solution have more apoptotic fibroblasts compared to tissues stretched in Poison. When the ratios of the relative amounts of apoptotic fibroblasts ofthe two treatments  131  become closer in value (i.e. relative amounts o f apoptotic cells in samples stretched i n Kreb's solution decrease).  Table 5.2 The ratios of the relative amounts of non-apoptotic and apoptotic fibroblasts of two treatments (K.S.: Kreb's solution plus stretching, P.S.: Poison plus stretching)  Rat  Stress Relaxation Differences  % ratio of non-apoptotic  % ratio of apoptotic  fibroblasts (K.S.) / % ratio blue  fibroblasts (K.S.) / % ratio  fibroblasts (P.S.)  brown fibroblasts (P.S.)  L  111.0  0.2  3.1  J  104.3  0.2  4.0  AF  103.8  0.2  2.4  AA  76.0  0.4  1.9  Z  55.3  0.5  1.8  E  48.4  0.6  1.8  M  41.6  0.6  1.7  T  41.6  0.5  1.7  V.  41.5  0.7  1.5  P  34.6  0.6  1.7  F  34.5  0.6  2.1  W  27.7  0.6  1.9  Y  27.7  0.5  2.6  D  20.7  0.9  1.2  H  20.6  0.6  1.1  AD  13.8  0.8  1.3  N  6.9  1.0  1.0  K  0.0  1.1  0.9  R  0.0  1.0  1.0  AC  0.0  0.7  1.6  132  120  • Percent of apoptoticfibroblastsfor Krct/B Percent of apoptoticfibroblastsfor Poison  0.S  1.5  2  2.5  3  3.5  4.5  Ratio of apoptotic fibroblasts  Figure 5.7 Difference in stress relaxation for tissues soaked and stretched in Kreb's solution and in Kreb's solution with poison versus the ratio of the relative amounts of apoptotic fibroblasts in tissues soaked and stretched in Kreb's solution and in Poison.  The results obtained from both sections 5.2.1 and 5.2.2 are in agreement with each other.  The plots shown in section 5.2.1 and 5.2.2 both illustrated that the  stiffness of the tissue increases when there are more non-apoptotic and less apoptotic fibroblasts, while the stiffness ofthe tissue decreases when there are more apoptotic and less non-apoptotic fibroblasts. The experimental results suggested that stretching ofthe tissue leads to apoptosis of fibroblasts.  This result further explained and verified the results obtained by  133  Martel et al. (2001).  In her experiment, she observed that fibroblasts that are  stimulated mechanically will lose the appropriate E C M and cytoplasmic contacts and round up.  Due to time constraints, she did not carry out any cell death testing.  The  apoptotic tests performed in this current study demonstrated that fibroblasts that are stimulated mechanically undergo programmed cell death (apoptosis), due to the high ratios o f apoptotic fibroblasts count.  According to the stress relaxation differences, it  is reasonable that tissue that is stretched is less stiff (i.e.  high stress relaxation), since  the tissue is in a more relaxed state when its E C M and cytoplasmic contacts are disconnected. 2-Deoxy-D-Glucose, on the other hand, did not cause a lot o f fibroblasts to undergo apoptosis.  It is possible that it killed the fibroblasts in a necrotic manner,  causing the fibroblasts to swell and undergo lysis.  However, the E C M and  cytoplasmic contacts of the fibroblasts are still connected; thus, the fibroblasts are still anchored. relax.  Due to the anchorage o f the fibroblasts, the tissues have reduced ability to  The tissues will therefore have higher stiffness (i.e. low stress relaxation).  134  5.3 Sources of errors There are several sources of errors that may affect the accuracy o f the results obtained from the mechanical test and the apoptotic cell count.  The sources o f errors  are mainly due to the preparation o f the skin tissues, the sensitivity o f the load frame, staining, cell counting and stress and strain measurements.  Thus, to minimize errors,  the procedural steps that were defined at the beginning of the experiments were followed carefully and rigidly.  5.3.1 Preparation o f skin tissues 31 male rats with a weight of200 - 250 grams were used. error is due to natural differences in the rat population.  The primary source of  Although they all have similar  weights, each rat has a slightly different age, health condition, and genetics, causing natural differences in skin tissues, such as stiffness, thickness and dryness, which affect cell composition and mechanical behavior. Prior to soaking and mechanical tests, the rat must be killed and the skin tissues are harvested.  During the preparation process, cells may have been destroyed due to  the handling procedures. already.  When the rat is killed, the cells in the tissues may start to die  There are artifacts in the tissue samples when they are cut from the animal  and pinned onto the wax sheet.  When the muscle layer is carefully removed from  each tissue, some cells and structures of the tissues might still be damaged due to  135  pulling by the pins and peeling by the scalpel.  During the peeling process, the tissue  was not soaked in any liquid; thus, it might become too dry, leading to further damage of the tissue cells and structures.  This process becomes more problematic with rats  that are slightly younger, since it has a thinner muscle layer, which is harder to remove as it is more tightly attached to the dermis, thus prolonging the time required to complete the procedure.  Though the procedure was completed in about 20 minutes,  the tissue was not supplied with any nutrients and oxygen during this procedural step; thus, some cells may have died.  This will directly affect the cell composition,  influencing the total number of non-apoptotic and apoptotic cells in the tissue.  5.3.2 Load frame The load frame used in this project is capable of performing a diversity o f stress-strain experiments for biological materials. Though it was custom-built for the current project, it still has a few constraints. The force transducer used for the biomechanical experiment was a lot less sensitive than that used by Martel et al. (1999).  Significant noise was noted in the  stress-strain graphs when the strain was below 10%. Therefore, 13% strain was chosen for the study.  136  Although the noise level was relatively low at the chosen strain level, there are fluctuations in the measured stress values, which affect the accuracy of the measurements.  .  Another source o f error is handling short pieces o f rat skin tissues. tissue sample in the load frame is a delicate task.  Mounting o f  While mounting, special care must  be taken to avoid stretching the tissue sample and damaging it which could influence the mechanical and viability results.  During mounting, 0.5 cm o f tissue from each  side is clamped. The tissue is not soaked in any solution during the mounting and slowly becomes dry; thus, cells may die due to the lack of water, oxygen and nutrient supply. For the mechanical test, a long sample o f tissue (i.e. 2 cm by 5 cm) is used to reduce the risk o f damaging the tissue.  The tissue samples held by the clamps o f the  load frame had a width to length ratio o f 1:2, which is high compared to the standard 1:5 to 1:8 ratio used in many studies for quantitative analysis and determination properties (Colin, 1982; Vogel, 1975; Vogel et al., 1979; Wan Abas, 1995).  5.3.3 Staining, cell counting and stress relaxation measurement After the mechanical tests, the tissue samples underwent apoptotic staining tests. The errors that may be involved in the test performed by the professionals at the Waxit  137  Company were uncontrollable; however, since the test was professionally done, the errors should be small. To compute the stressrelaxation values for the each stress-strain test, the maximum and minimum stress during the 30 second relaxation period are needed. There are errors in choosing these maximum and minimum stress values due to fluctuations in the readings During cell counting, a proper area (i.e. area between two hair follicles) was carefully chosen that includes most o f the cell types in skin tissues. strain may have an effect on the choice of the representative area.  However, eye There may also be  cells (i.e. non-apoptotic, apoptotic and miscellaneous cells) being miscounted or omitted due to the eye strain caused by the constraints of the monocular microscope. It is quite difficult to focus and count the cells using a monocular microscope. Therefore, there are errors involved in counting the cells directly influencing the recorded number o f fibroblasts and miscellaneous cells.  138  Conclusions >  The In Situ Cell Death Detection Kit, POD effectively and specifically detects the presence o f apoptotic cells (i.e. blue cells) in skin tissue and distinguishes them from non-apoptotic cells (i.e. brown cells)  ^  Kreb's solution prevents fibroblasts in skin tissues to undergo apoptosis  >  Stretching in both Kreb's solution and Kreb's solution with 2-Deoxy-D-Glucose significantly increases the number of apoptotic cells  ^  Stretching in Poison cause some apoptotic cells, but  much less than stretching  ^  Stretching o f skin tissue causes the extracellular matrix and cytoplasmic contacts o f the fibroblasts to become disconnected (i.e. fibroblasts round up [Martel et al. (2001)]) and allows the tissues to relax more easily.  ^  2-Deoxy-D-Glucose does not kill all o f the fibroblasts by apoptosis, but possibly by necrosis.  The extracellular matrix and cytoplasmic contacts o f the  fibroblasts are still connected, allowing the fibroblasts to stay anchored. Therefore, the tissues cannot easily relax y  Stiffness o f the tissue increases when there are more non-apoptotic and less apoptotic fibroblasts, and it decreases when there are more apoptotic and less non-apoptotic fibroblasts  y  Tissues stretched in Kreb's solution have higher reduction in stress values (i.e. stress relaxation) compared to tissues stretched in 2-Deoxy-D-Glucose 139  ^  There is a direct correlation observed between the stress relaxation differences (i.e. between stretching in Kreb's solution and in Kreb's solution with 2-Deoxy-D-Glucose) and the ratios o f percentages of apoptotic cells (Kreb's solution) to percentages o f apoptotic cells (Kreb's solution with 2-Deoxy-D-Glucose).  y  There is an inverse correlation observed between the stress relaxation differences (i.e. between stretching in Kreb's solution and in Kreb's solution with 2-Deoxy-D-Glucose) and the ratios of percentages of non-apoptotic cells (Kreb's solution) to percentages of non-apoptotic cells (Kreb's solution with 2-Deoxy-D-Glucose).  ^  According to the relationships observed, fibroblasts do play a role in the mechanical properties of rat skin tisssues.  140  Future Works y  Some modifications should be made to the force transducer o f the load frame to further minimize the noise level for future experiments so even smaller strain (i.e. below 10%) can be applied to the skin tissues.  It would be interesting to  test the minimum elongation that is needed to cause fibroblasts to undergo apoptosis. >  Different strain rates should be used to examine the importance o f time in straining the skin tissues.  >  Due to the constraints and unknown effects o f anesthesia in skin tissues, the tissues were not stretched in vivo. However, since skin tissues in living animals are continuously subjected to mechanical load, it is crucial to investigate the mechanical role o f fibroblasts in skin stretched in vivo. To perform stretching in vivo requires a modification o f the load frame so that the clamps, that hold the tissue sample, could be placed on the back o f the animal.  >  The apoptosis test performed in the study only detects apoptotic and non-apoptotic cells.  It would be interesting to perform other test, including  necrotic tests, to examine whether the non-apoptotic cells undergo necrosis. 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Vogel, H . G . (1977).  Arch. Derm. Res.. 264: 225-241.  Analysis of the Low Part o f Stress-Strain Curves in Rat Skin.  Influence of Age and Desmotropic Drugs. Vogel, H . G . (1975).  Arch. Derm. Res., 258:  141-150.  Strain of rat skin at constant load (creep experiments).  Influence of age and desmotropic agents.  Wan Abas, W. A . B . (1995).  Gerontology, 23:  77-86.  Stress Stabilisation Behaviours in Skin under Small  Tensile Loads In Vitro. Bio-Medical Materials and Engineering, 5(2): Zar, Jerrold H . , Biolstatisical Analysis. 4 edition, (New Jersey: th  59-63.  Simon & Schuster/A  Viacom Company, Inc., 1999), 20-39,161-169. Campbell, Neil A . , Biology, 4  th  edition, (California:  The Benjamin/Cummings  Publishing Company, Inc., 1996), 7-8.  147  Appendix A: transducers  Linearity and calibration of the force and displacement  Example of records on the calibration and linearity of the force transducer.  Figure A.1 Calibration of the force transducer with standard weights 300  0-1 0  ,  1  ,  2  ,  3  •  •  r—  4  •  ,  5  •  ,  1  6  7  Voltage (V)  148  Figure A.2 Calibration of the displacement transducer with standard weights  o-  7  «—•  y = 0.3071 x* 5.9306 R = 0.9995  5  2  4  3  2  ,  1  -  4  -  3  -  , 2  e-  1  0  1  2  3  4  Voltage (V)  149  Appendix B:  Records of cell counting  The average number of cells (i.e. fibroblasts and miscellaneous cells) o f all three tissue sections o f each treatment group was computed for each rat group as shown in this appendix.  To determine the general effect o f each treatment group (i.e. soaked in  Kreb's solution, soaked and stretched in Kreb's solution, soaked in Kreb's solution with Poison, soaked and stretched in Kreb's solution with Poison) on the viability o f the fibroblasts, the sums o f the blue- and brown- colored fibroblasts are also computed for each treatment group and displayed in this section.  150  Table B.1 The average number of cells of all three tissue sections in each treatment groups for each rat (20 rats) Fibroblasts (Blue)  Fibroblasts (Brown)  Miscellaneous (Blue)  Miscellaneous (Brown)  D.K.1  30.3  3.3  9.0  3.7  D.K.S.1  15.7  18.0  3.3  10.3  D.P.I  12.3  15.7  5.7  5.0  D.P.S.l  17.3  14.7  4.7  9.0  E.K.1  19.0  4.3  8.0  2.7  E.K.S.1  11.7  21.3  2.3  8.3  E.P.I  16.3  12.3  8.3  5.7  E.P.S.l  20.7  12.0  5.0  9.3  F.K.1  24.0  2.3  13.3  2.3  F.K.S.1  12.0  18.7  1.7  10.0  9.7  16.3  9.0  8.3  F.P.S.l  21.3  9.0  2.7  8.7  H.K.1  24.7  4.3  7.7  1.0  4.0  21.3  1.3  10.7  21.3  9.0  4.3  7.7  7.7  21.7  5.0  6.7  29.0  2.3  14.7  2.0  3.7  24.0  1.3  9.7  J.P.I  22.3  7.3  12.0  4.3  J.P.S.l  22.7  6.3  3.3  7.7  K.K.1  25.7  6.0  12.3  2.7  K.K.S.1  13.3  16.7  8.0  5.7  K.P.1  18.7  3.3  8.0  2.3  K.P.S.1  11.7  17.3  7.7  4.7  L.K.1  22.3  2.3  10.7  1.3  4.3  26.7  1.0  11.0  L.P.I  13.0  12.0  5.0  7.3  L.P.S.l  23.0  9.0  5.7  8,3  F.P.I  H.K.S.1 HP.l H.P.S.1  J.K.1 J.K.S.1  L.K.S.1  151  Table B.1  Continue Miscellaneous (Blue)  Miscellaneous (Brown)  Fibroblasts (Blue)  Fibroblasts (Brown)  M.K.1  25.3  1.3  12.3  1.7  M.K.S.1  10.0  19.3  2.3  8.0  M.P.I  18.7  6.3  12.0  5.7  M.P.S.l  18.0  11.7  5.3  9.7  N.K.1  23.7  4.3  14.3  3.3  N.K.S.1  11.0  17.7  3.3  12.3  N.P.I  11.0  17.3  9.7  2.7  N.P.S.l  12.3  18.3  4.0  10.3  P.K.1  25.0  3.0  15.3  3.7  P.K.S.1  11.3  19.3  9.0  5.7  P.P.I  18.7  4.3  8.3  2.3  P.P.S.l  18.0  10.3  4.7  6.0  R.K.1  15.0  6.3  11.3  4.0  R.K.S.1  12.7  18.7  6.0  9.0  R.P.1  15.3  13.0  4.0  7.7  R.P.S.1  13.3  18.3  7.7  7.0  T.K.1  26.7  1.0  14.3  1.0  9.3  20.7  1.3  12.0  T.P.I  26.7  7.0  8.3  4.3  T.P.S.l  17.7  11.7  5.3  8.3  V.K.1  24.0  2.0  9.3  1.3  V.K.S.1  12.3  20.0  3.7  10.3  V.P.I  18.3  6.7  10.0  5.3  V.P.S.l  16.3  12.0  6.3  7.0  W.K.1  25.7  1.0  12.0  1.7  W.K.S.1  12.7  18.7  6.0  11.3  W.P.I  24.0  4.3  6.7  5.3  W.P.S.l  20.3  9.3  4.0  8.7  T.K.S.1  152  Table B . l  Continue Fibroblasts (Blue)  Fibroblasts (Brown)  Miscellaneous (Blue)  Miscellaneous (Brown)  Y.K.1  23.0  9.0  5.7  8.3  Y.K.S.1  13.3  19.3  1.0  12.3  Y.P.I  18.0  12.7  7.0  5.7  Y.P.S.l  23.7  7.0  3.3  9.7  Z.K.1  25.0  3.3  9.7  1.7  Z.K.S.1  10.7  23.3  1.0  10.3  Z.P.I  20.0  8.3  5.3  6.7  Z.P.S.l  20.0  12.0  2.7  7.7  AA.K.1  23.7  2.0  7.0  0.7  7.3  20.7  2.7  10.0  AA.P.1  20.3  9.7  10.0  5.0  AA.P.S.1  16.3  10.7  2.7  8.3  AC.K.1  18.3  8.3  4.7  5.3  AC.K.S.1  14.7  19.0  5.3  10.0  AC.P.l  13.7  14.0  6.0  8.3  AC.P.S.l  19.0  10.3  4.0  7.0  AD.K.1  26.7  4.0  13.0  1.3  AD.K.S.1  11.7  17.7  3.0  7.0  AD.P.l  23.0  7.3  2.7  7.3  AD.P.S.l  14.7  13.3  7.3  4.3  AF.K.1  25.7  2.3  10.7  1.0  4.0  27.0  2.7  11.0  AF.P.l  20.3  7.0  3.3  9.0  AF.P.S.l  17.7  10.3  3.3  8.3  AA.K.S.1  AF.K.S.1  153  Table B.2  The sum and average number of apoptotic and non-apoptotic fibroblasts for each treatment group. apoptotic fibroblasts non-apoptotic fibroblasts -  Overall Trend for Fibroblasts only (sum)  sum (Blue)  average (Blue)  sum (Brown)  average (Brown)  % ratio blue  % ratio brown  Kreb's  482.7  24.1  73.0  3.7  86.9  13.1  Kreb's + stretch  205.7  10.3  408.0  20.4  33.5  66.5  Poison  361.7  18.1  194.0  9.7  65.1  34.9  Poison + stretch  351.7  17.6  245.3  12.3  58.9  41.1  Kreb's + stretch vs. Kreb's vs. Kreb's + stretch  2.3  Kreb's  5.6  2.6  0.2  Kreb's vs. Poison  1.3  Poison vs. Kreb's  2.7  1.3.  0.4  1.7  0.6  1.6  1.3  1.1  0.8  Kreb's + stretch vs. Poison + stretch vs. Kreb's + stretch  1.7  Poison + stretch Poison + stretch vs.  Poison vs. Poison + stretch  1.0  Poison  154  

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