Huber Final Thesis Write-Up_21 May 2015

Influence of Hyaluranon (HA) Preparations on
Ionizing-Radiation-Treated Collagen-based Tissues
By
Aaron Huber
June 15, 2015
A thesis submitted to the
Faculty of the Graduate School of
the University at Buffalo, State University of New York
in partial fulfillment of the requirements for the
degree of
Master of Science
Biomaterials Graduate Program

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Table of Contents
Table of Contents ........................................................................................................................ ii
List of Figures ........................................................................................................................... vii
List of Tables............................................................................................................................. xii
ACKNOWLEDGMENTS........................................................................................................xiii
ABSTRACT.................................................................................................................................. xv
1.0 INTRODUCTION .................................................................................................................... 1
1.1.1 XEROSTOMIA.................................................................................................................. 1
1.1.2 Effects of Xerostomia..................................................................................................... 1
1.1.3 Causes of Xerostomia..................................................................................................... 2
1.1.4 Assessment of Xerostomic Conditions........................................................................... 4
1.1.5 Management and Treatment of Xerostomia ................................................................... 6
1.2.1 ROLE OF SALIVA............................................................................................................ 9
1.2.2 Properties of Saliva....................................................................................................... 10
1.3.1 PROPERTIES OF COLLAGEN AND ITS RELATION TO ORAL MUCOSA............ 12
1.4.1 OVERVIEW OF OM  STRUCTURE AND FUNCTION........................................... 13
1.5.1 ORAL MUCOSA AND SURROUNDING AREAS: HOW THEY ARE AFFECTED BY
GAMMA IRRADIATION........................................................................................................ 17
1.5.2 Mucositis ...................................................................................................................... 17
1.5.3 ɣ-Irradiation’s Effects on Collagen: Cross-linking and Chain Scission....................... 20
1.5.4 ɣ-Irradiation-Caused Osteoradionecrosis ..................................................................... 22
1.5.5 Radiation-induced Glossitis.......................................................................................... 24
1.6.1 RADIATION-INDUCED XEROSTOMIA SYMPTOMS.............................................. 24
1.6.2 Minimizing the Effects of Radiation-Induced Xerostomia .......................................... 25
1.6.3 Different Forms of Radiation to Treat Head/Neck Cancer and Their Effects on
Patients’ Quality of Life (QoL): 3D-CRT and IMRT ........................................................... 26
1.7.1 BOVINE PERICARDIUM (PC): STRUCTURE, COMPOSITION, AND FUNCTION28
1.8.1 HYDROXYPROLINE (HYP) ASSAY TESTING ......................................................... 29
1.9.1 HYALURONIC ACID (HA): AN OVERVIEW............................................................. 30
1.9.2 HA: How Does it Bind in the Body?............................................................................ 35
1.9.3 Hyaluronic Acid: Functionality in the Knee Joint........................................................ 35
1.9.4 HA’s Present Utilization in Xerostomia-Treating Agents for Cancer Victims [68] .... 36
1.9.5 Assessment of HA’s Inherent Antimicrobial Properties .............................................. 37

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1.9.6 Addition of Antimicrobial Properties to HA by Grafting of Antimicrobial Peptide.... 38
1.10.1 THE CLINICAL REVELANCE OF FRICTION TESTING ........................................ 39
1.10.2 Forces Involved in Friction......................................................................................... 40
1.10.3 Types of Lubrication .................................................................................................. 41
1.10.4 Static vs. Kinetic Friction Coefficients (µ)................................................................. 42
1.11.1 HA USED IN FRICTION TESTING: PREVIOUS MASTER’S THESES .................. 43
1.12.1 HA VS. SALIVA SUBSTITUTES IN FRICTION TESTING: PRIOR MASTER’S
THESES .................................................................................................................................... 43
1.13.1 HA USED IN CLINICAL STUDY [1].......................................................................... 44
2.0 PURPOSE............................................................................................................................... 46
2.1.1 MOTIVATION ................................................................................................................ 46
3.0 MATERIALS AND METHODS............................................................................................ 49
3.1.1 HUMAN ORAL MUCOSA EXTRACTION .................................................................. 49
3.1.2 Oral Mucosa Preparation for Testing ........................................................................... 54
3.2.1 FABRICATION OF 0.5% HA SOLUTION.................................................................... 56
3.3.1 MULTIPLE-ATTENUATED INTERNAL REFLECTION INFRARED (MAIR-IR)
SPECTROSCOPY METHODS [8] .......................................................................................... 57
3.4.1 IRRADIATION OF TISSUE SAMPLES........................................................................ 66
3.5.1 CHEMOMECHANICAL TENSILE TESTING.............................................................. 68
3.5.2 Tensile Testing Crossover Study.................................................................................. 75
3.6.1 STATIC FRICTION TESTING....................................................................................... 75
3.6.2 Static Friction Testing  A Focus on Oral Mucosa Pre/Post Irradiation’s Effects on
Hyaluronic Acid/Phosphate-Buffered Saline Application as a Lubricant............................. 81
3.7.1 WEIGHT TESTING ........................................................................................................ 82
3.7.2 Weight Testing  Crossover Study of Previously Soaked/Baked Oral
Mucosa/Pericardium.............................................................................................................. 82
3.8.1 VOLUME TESTING....................................................................................................... 83
3.9.1 HYP ASSAY TESTING TO ANALYZE TISSUE COLLAGEN LEVELS  PILOTS 85
3.9.2 Altered HYP Assay Testing (Removal of the HCl Hydrolysis and Subsequent Baking
Steps From Pilot Protocol) .................................................................................................... 89
3.10.1 HISTOLOGY AND LIGHT MICROSCOPY OF ORAL MUCOSA/PERICARDIUM
SAMPLES, HYALURONIC ACID/DISTILLED WATER-SOAKED ................................... 92
3.11.1 STATISTICAL EVALUATION OF DATA ................................................................. 93
4.0 RESULTS ............................................................................................................................... 95
4.1.1 CHEMOMECHANICAL TENSILE TESTING.............................................................. 95
4.1.2 Tensile Testing Crossover Study................................................................................ 102

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4.2.1 STATIC FRICTION TESTING..................................................................................... 103
4.2.2 Static Friction Testing  Oral Mucosa...................................................................... 103
4.2.3 Static Friction Testing  Bovine Pericardium .......................................................... 106
Hyaluronic Acid/Phosphate-Buffered Saline Application as a Lubricant........................... 108
4.3.1 WEIGHT TESTING ...................................................................................................... 109
4.3.2 Weight Testing Crossover Study of Previously Soaked/Baked Oral
Mucosa/Pericardium............................................................................................................ 111
4.4.1 VOLUME TESTING (Figure 63 and Figure 64)........................................................... 114
SPECTROSCOPY .................................................................................................................. 116
4.6.1 HYP ASSAY TESTING................................................................................................ 127
4.7.1 HISTOLOGY AND LIGHT MICROSCOPY OF ORAL MUCOSA/PERICARDIUM
SAMPLES, HYALURONIC ACID/DISTILLED WATER-SOAKED (Figure 78-Figure 103)
................................................................................................................................................. 129
5.0 DISCUSSION....................................................................................................................... 143
5.1.1 CHEMOMECHANICAL TENSILE TESTING............................................................ 143
5.1.2 Tensile Testing Crossover Study................................................................................ 144
5.2.1 STATIC FRICTION TESTING..................................................................................... 146
Hyaluronic Acid/Phosphate-Buffered Saline Application as a Lubricant........................... 150
5.3.1 WEIGHT MEASUREMENTS ...................................................................................... 150
5.3.2 Weight Testing Crossover Study of Previously Soaked/Baked Oral
Mucosa/Pericardium............................................................................................................ 151
5.4.1 VOLUME MEASUREMENTS..................................................................................... 151
SPECTROSCOPY .................................................................................................................. 153
5.6.1 HYP ASSAY TESTING................................................................................................ 154
5.7.1 HISTOLOGY AND LIGHT MICROSCOPY OF OM/PC SAMPLES, HA/DW
SOAKED................................................................................................................................. 155
6.0 CONCLUSIONS................................................................................................................... 157
7.0 LIMITATIONS OF THE STUDY........................................................................................ 159
7.1.1 LACK OF INVESTIGATION WITH HOW BLOOD FLOW AFFECTS LIVING OM
IN XEROSTOMIC PATIENTS.............................................................................................. 159
7.2.1 ALTERATION OF RADIATION DOSAGE................................................................ 159
7.3.1 VARIANCE OF HA/DW TISSUE SOAKING TIME .................................................. 160

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7.4.1 MINOR TISSUE SLIPPAGE (UNSENSED) MAY HAVE LED TO SKEWED
TENSILE TESTING DATA................................................................................................... 160
7.5.1 ASSUMPTION OF ISOTROPIC STRAIN OF ORAL MUCOSA TISSUE WHILE
TENSILE TESTING............................................................................................................... 161
8.0 FUTURE DIRECTIONS ...................................................................................................... 162
8.1.1 PROSPECTIVE BODILY APPLICATIONS FOR HA OUTSIDE OF THE ORAL
CAVITY.................................................................................................................................. 162
8.1.2 Vagina Lubricant for Menopausal Women ................................................................ 162
8.1.3 Stem Cell Research..................................................................................................... 162
8.1.4 HA’s Prospective Usage for Amputees Suffering from Phantom Limb Syndrome... 165
8.2.1 VOLUMETRIC ANALYSIS OF PC AND OM AS A RESULT HA/DW SOAKING 165
8.3.1 MEASUREMENT OF XEROSTOMIC PAIN RELIEF WITH HA APPLICATION.. 166
8.4.1 CONFOCAL INFRARED IMAGING OF TESTED TISSUES.................................... 166
8.5.1 TESTING OF IRRADIATED OM AND PC, HA-SOAKED VS DW-SOAKED, WITH
GRAFTED ANTIMICROBIAL AGENT............................................................................... 166
8.7.1 TEST TO COMPARE CHAIN-SCISSION/CROSS-LINKING AMOUNTS FOR 70 GY
(ONE-TIME ADMINISTRATION) VS. CLINICAL TREATMENT DOSAGES (TOTALING
TO 70 GY) .............................................................................................................................. 167
8.8.1 CHEMOMECHANICAL TESTING ALTERATIONS SEEKING MORE ACCURATE
RESULTS................................................................................................................................ 167
8.9.1 IN-DEPTH ANALYSIS OF FOOTBALL LEATHER UPON HA TREATMENT ..... 168
8.10.1 Observation of Remnant Cancer Cell Motility in the Oral Mucosa After Radiotherapy
................................................................................................................................................. 169
9.0 APPENDICES ...................................................................................................................... 170
9.1.1 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue 
Introduction............................................................................................................................. 170
9.1.2 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue  Materials
and Methods ........................................................................................................................ 171
9.1.3 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue  Results
............................................................................................................................................. 177
Discussion............................................................................................................................ 178
Conclusion........................................................................................................................... 179
9.2.1 IRB EXEMPTION NOTICE ......................................................................................... 180
9.3.1 HYP PROTOCOL USED FOR PILOT STUDY........................................................... 181
9.3.2 HYP Equipment and Reactive List/Locations (Table 6)............................................ 189
9.3.3 pH Meter Operating Instructions................................................................................ 190

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9.3.4 Visual Spectrometer Protocol used............................................................................. 191
9.4.1 STATIC COEFFICIENT OF FRICTION RESULTS WITH INTERVENTION
INDICATED OVER TIME .................................................................................................... 194
9.4.2 Static Coefficient of Friction Results With Intervention Indicated Over Time  PC
(Figure 126-Figure 131) ...................................................................................................... 194
9.4.3 Static Coefficient of Friction Results With Intervention Indicated Over Time  OM
(Figure 132-Figure 138) ...................................................................................................... 197
9.4.4 Static Friction Testing: A Focus on OM Pre/Post Irradiation’s Effects on HA/PBS
Application as a Lubricant  Intervention Indicated Over Time (Figure 139-Figure 141)204
9.4.5 Static Friction Testing: Bone Resurfacing Study  Intervention Indicated Over Time
(Figure 142-Figure 145) ...................................................................................................... 205
9.5.1 0.5% HA SOLUTION’S MANUFACTURING............................................................ 207
9.5.2 Certificate of Analysis ................................................................................................ 207
9.5.3 Test Results Obtained by PureBulk............................................................................ 208
9.5.4 Storage Conditions ..................................................................................................... 208
9.6.1 ORIGINAL TENSILE TESTING CHARTS (Figure 146-Figure 154)......................... 209
9.7.1 PILOT STUDY OF TANNED COLLAGEN-BASED LEATHER (UB’S UNUSED
GAME BALLS, Figure 155)................................................................................................... 218
10.0 REFERENCES ................................................................................................................... 222

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List of Figures
Figure 1 - 3-D View of Irradiation Dosage Effects in the Head and Neck Region ........................ 3
Figure 2- Severe Radiation-Related Dental Caries Caused by Xerostomia and Inadequate Dental
Treatment [2] .................................................................................................................................. 5
Figure 3 - Basic Structure of OM [24].......................................................................................... 14
Figure 4 - A Zoomed-out View of the OM Structure [24] ........................................................... 14
Figure 5 - Diffuse, Radiation-induced Early Grade 2 Mucositis With Solitary Ulcer at Lateral
Aspect of Palatal Mucosa [2]........................................................................................................ 19
Figure 6 - Mandibular Bone (Exposed) Attribute of Osteoradionecrosis [2] ............................... 23
Figure 7 - Biochemical Structure of HA (polymerization of these 2 molecules) [68] ................. 31
Figure 8 - Scheme of Nisin Grafting [97]..................................................................................... 38
Figure 9 - Jim Kelly at His Hospital Bed During Cancer Treatment, With His Consoling
Daughter, Erin, By His Side [108]................................................................................................ 47
Figure 10 - Jim Kelly is the only quarterback in NFL history to lead his team to the Super Bowl
in four straight seasons.................................................................................................................. 48
Figure 11 - Initial Incision of Cadaver Cheek .............................................................................. 52
Figure 12 - OM Extraction, after Initial separation from Underlying Lipid Layer (Bottom Right)
....................................................................................................................................................... 53
Figure 13 - Diagram of Tissue Cutting Procedure........................................................................ 54
Figure 14 - Further Cutting/Separation of the OM from Underlying Fatty Tissue ...................... 55
Figure 15 - Laboratory Spectrophotometer used for analyses of dehydrated OM/PC during this
investigation.................................................................................................................................. 58
Figure 16 - Schematic of Infrared Ray Path ................................................................................. 61
Figure 17 - Fastening Components for Mounting KRS-5 Prism onto the Stage .......................... 62
Figure 18 - Fastened Components of KRS-5 Prism, Ready for Insertion into Spectrometer....... 63
Figure 19 - Position of KRS-5 Prism on Testing Jig .................................................................... 64
Figure 20 - Prism and Test Jig before Top Plate Application....................................................... 65
Figure 21 - Isomedix Gammator Unit........................................................................................... 67
Figure 22 - Fastening Devices Used for Tensile Testing.............................................................. 69
Figure 23 - Experimental Set-up of Measuring Initial Strain ....................................................... 70
Figure 24 - Notched OM Segments in DW Bath.......................................................................... 71
Figure 25 - Notched Pericardium Tissue Bathing in DW............................................................. 71
Figure 26 - Modulus of Elasticity Depiction [127]....................................................................... 72
Figure 27 - Stress (X-Axis) vs. Strain (Y-Axis) Curve for Oral Mucosa (As Depicted by Data
Recorder)....................................................................................................................................... 73
Figure 28 - Data Recorder Strip Chart used for ChemoMechanical Tensile and Friction Testing
....................................................................................................................................................... 77
Figure 29 - Static Friction Device................................................................................................. 77
Figure 30 - Parafilm-Sealed Cardboard Friction Testing Stage.................................................... 79
Figure 31 - OM Pinned Tissue to Parafilm-Sealed Cardboard Stage ........................................... 80
Figure 32 - Goniometer Set-up ..................................................................................................... 84
Figure 33 - Pilot Study, Post-Bake for OM/PC Samples.............................................................. 87
Figure 34 - Pilot OM/PC Samples After Digested in 6M HCl ..................................................... 88
Figure 35 - HYP Assay Supplies .................................................................................................. 89
Figure 36 - Funnel Dried with TechniCloth, Cleansed of Juice with Air Hose. Rinsed with DW91

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Figure 37 - OM HA IRR 1  Note the pink, cloudy OM pulp.................................................... 91
Figure 38 - Stress-Strain Results for Irradiated/Non-Irradiated Tissue, PC/OM, DW/HA. n= 8
HA OM Irr, 9 DW OM Irr, 9 DW PC Irr, 9 HA PC Irr, 6 OM HA NonIrr, 6 OM DW NonIrr, 2
PC HA Non Irr, 2 PC DW NonIrr, 3 PC PO NonIrr..................................................................... 97
Figure 39 - Elastic Moduli of DW and HA-Soaked Samples of OM and PC, NonIrradiated vs.
Irradiated Samples, n= 35 Irr, 19 NonIrr ...................................................................................... 99
Figure 40 - Elastic Moduli of Irrad. and Non-Irrad Samples of HA and DW-Soaked Samples,
OM vs. PC Samples, n= 22 PC, 29 OM ....................................................................................... 99
Figure 41 - Elastic Moduli of Irrad. and Non-Irrad Samples of OM and PC, HA vs. DW-Soaked
Samples, n= 26 DW, 25 HA ....................................................................................................... 100
Figure 42 - Elastic Moduli of Irrad. and Non-Irrad Samples of OM and PC, HA-Irr vs. HA-
NonIrr Samples, n= 17 Irr, 8 NonIrr........................................................................................... 100
Figure 43 - Elastic Moduli of Irrad. and Non-Irrad Samples of OM and PC, DW-Irr vs. DW-
NonIrr Samples, n= 18 Irr, 8 NonIrr........................................................................................... 101
Figure 44 - Elastic Moduli of HA and DW-Soaked Samples of OM, Irr vs. NonIrr Samples, n=
17 Irr, 12 NonIrr.......................................................................................................................... 101
Figure 45 - Elastic Moduli of HA and DW-Soaked Samples of PC, Irr vs. NonIrr Samples, n= 18
Irr, 12 NonIrr............................................................................................................................... 102
Figure 46 - Elastic Moduli of DW and HA-Soaked Samples of OM and PC, Crossover Study, n=
4 HA x DW OM, 2 DW x HA OM, 6 DW x HA PC, 6 HA x DW PC...................................... 103
Figure 47 - OM DW vs OM HA, PreIrrad, n= 4 DW, 3 HA...................................................... 104
Figure 48 - OM DW vs OM HA, PostIrrad, n= 4 DW, 3 HA .................................................... 104
Figure 49 - PreIrrad vs. PostIrrad, OM HA, n=3........................................................................ 105
Figure 50 - PreIrrad vs. PostIrrad, OM DW, n=4 ....................................................................... 105
Figure 51 - PC DW vs PC HA, PreIrrad, n= 3 DW, 3 HA ......................................................... 106
Figure 52 - PC DW vs PC HA, PostIrrad, n= 3 DW, 3 HA........................................................ 106
Figure 53 - PC DW, PreIrrad vs PostIrrad, n=3.......................................................................... 107
Figure 54 - PC HA, PreIrrad vs PostIrrad, n=3 .......................................................................... 107
Figure 55 - OM Pre/Post Irradiation, HA/PBS Application, n=3............................................... 108
Figure 56 - PC and OM Weight (g) Change After Soak in HA and DW, Bake, Storage, n= 6 OM
DW, 6 OM HA, 5 PC DW, 5 PC HA ......................................................................................... 110
Figure 57 - % PC and OM Weight (g) Change After Soak in HA and DW, Bake, Storage, n= 6
OM DW, 6 OM HA, 5 PC DW, 5 PC HA.................................................................................. 110
Figure 58 - Weighing: Post X-Soak after Original Bake, n= 3 OM DW-DW, 3 OM DW-HA, 3
OM HA-DW, 3 OM HA-HA, 3 PC DW-DW, 2 PC DW-HA, 2 PC HA-DW, 2 PC HA-HA ... 111
Figure 59 - % Weight Change During X-Soak/Bake Study After Original Trial, n= 3 OM DW-
DW, 3 OM DW-HA, 3 OM HA-DW, 3 OM HA-HA, 3 PC DW-DW, 2 PC DW-HA, 2 PC HA-
DW, 2 PC HA-HA...................................................................................................................... 112
Figure 60 - X-Study Weight Replenishment/2nd Bake, n= 6 OM DW, 6 OM HA, 5 PC DW, 4
PC HA......................................................................................................................................... 113
Figure 61 - Change Proportions For Each X-Weight Study Abscissa, n= 6 OM DW, 6 OM HA, 5
PC DW, 4 PC HA ....................................................................................................................... 113
Figure 62 – OM Baked-To-Complete-Dryness .......................................................................... 114
Figure 63 - PC and OM Volume (mm^3) Change After Soak in HA and DW, Bake, n= 6 OM
DW, 6 OM HA, 5 PC DW, 5 PC HA ......................................................................................... 114

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Figure 64 - % PC and OM Volume (mm^3) Change After Soak in HA and DW, Bake, n= 6 OM
DW, 6 OM HA, 5 PC DW, 5 PC HA ......................................................................................... 115
Figure 65 - OM DW Post-Bake .................................................................................................. 116
Figure 66 - OM HA Post-Bake................................................................................................... 117
Figure 67 - PC DW Post-Bake.................................................................................................... 118
Figure 68 - PC HA Post-Bake..................................................................................................... 119
Figure 69 - PC DW, PC HA, OM DW, OM HA Post-Bake (TopBottom)............................. 120
Figure 70 - PC HA vs. PC DW Post-Bake (TopBottom) ....................................................... 121
Figure 71 - OM DW vs OM HA Post-Bake (TopBottom) ..................................................... 122
Figure 72 - OM HA - OM DW Post-Bake SUBTRACTION..................................................... 123
Figure 73 - OM HA - PC HA Post-Bake SUBTRACTION....................................................... 124
Figure 74 - PC HA - PC DW Post-Bake SUBTRACTION........................................................ 125
Figure 75 - OM DW - PC DW Post-Bake SUBTRACTION ..................................................... 126
Figure 76 - Hyaluronic Acid MAIR-IR Spectrum...................................................................... 127
Figure 77 - Pilot Study Relative Collagen Concentration Graph, n= 3 OM Irr, 3 OM NonIrr, 3
PC Irr, 3 PC NonIrr..................................................................................................................... 128
Figure 78 - OM DW 1 L 1 63 x Middle H and E........................................................................ 129
Figure 79 - OM DW 1 L 1 63 x Middle TriChrome................................................................... 130
Figure 80 - OM DW 1 L 1 250 x Middle H and E Rubbed-Off Epithelium .............................. 130
Figure 81 - OM DW 1 L 1 250 x Middle H and E Shredded Epithelium................................... 131
Figure 82 - OM DW 1 L 1 250 x Middle TriChrome................................................................. 131
Figure 83 - OM HA 1 L 1 63 x Middle TriChrome.................................................................... 132
Figure 84 - OM HA 1 L 1 250 x Middle H and E ...................................................................... 132
Figure 85 - OM HA 1 L 1 250 x Middle TriChrome.................................................................. 133
Figure 86 - OM HA 1 L 1 63 x Middle H and E ........................................................................ 133
Figure 87 - OM DW 1 L 1 400 x LEFT trichrome_basement membrane-collagen junction ..... 134
Figure 88 - OM DW 1 L 1 63 x LEFT H and E.......................................................................... 134
Figure 89 - OM DW 1 L 1 63 x LEFT trichrome ....................................................................... 135
Figure 90 - OM DW 1 L 1 400 x LEFT H and E........................................................................ 135
Figure 91 - OM DW 1 L 1 400 x LEFT trichrome ..................................................................... 136
Figure 92 - OM HA 1 L 1 63 x LEFT H and E .......................................................................... 137
Figure 93 - OM HA 1 L 1 63 x LEFT trichrome........................................................................ 137
Figure 94 - OM HA 1 L 1 400 x LEFT H and E ........................................................................ 138
Figure 95 - OM HA 1 L 1 400 x LEFT trichrome...................................................................... 138
Figure 96 - PC HA 3 L 1 63 x Middle H and E.......................................................................... 139
Figure 97 - PC DW 2 L 1 63 x Middle H and E......................................................................... 139
Figure 98 - PC DW 2 L 1 63 x Middle TriChrome .................................................................... 140
Figure 99 - PC DW 2 L 1 250 x Middle H and E....................................................................... 140
Figure 100 - PC DW 2 L 1 250 x Middle TriChrome ................................................................ 141
Figure 101 - PC HA 3 L 1 63 x Middle TriChrome ................................................................... 141
Figure 102 - PC HA 3 L 1 250 x Middle H and E...................................................................... 142
Figure 103 - PC HA 3 L 1 250 x Middle TriChrome ................................................................. 142
Figure 104 - Tensile Testing Crossover Study Elastic Moduli Chart, n= 6 OM HA, 6 OM DW, 3
OM HA x DW, 3 OM DW x HA, 2 PC HA, 2 PC DW, 2 PC PO, 2 PC DW x HA, 2 PC HA x
DW.............................................................................................................................................. 146
Figure 105 - CoF vs Time plot for unstimulated saliva from a female control [8]..................... 149

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Figure 106 - 0.5% HA in Normal Saline_Three Different PC Tissue Couples [17] .................. 149
Figure 107 - Baked to Dryness PC Samples............................................................................... 152
Figure 108 - Unstimulated saliva from P3, Black – Water leached and air dried, Blue – Water
rinsed and air dried [8]................................................................................................................ 154
Figure 109 - Images of Microtissue, Before and After HA Treatment....................................... 164
Figure 110 - Average Contractile Force (µN) of Microtissues on Day 2 (i, ii), Day 3 (iii), and
Day 4 (iv), Treated With 0.5% HA Solution for 30 Seconds (i) or 1 Minute (ii, iii, iv) ............ 164
Figure 111 - Friction Testing Apparatus at Initial Testing (Pre-Dry/Soak)................................ 171
Figure 112 – PC 1 Pinned to Cardboard for Drying (After Initial Friction Testing).................. 172
Figure 113 - PC 1 on left, PC 2 on Right.................................................................................... 172
Figure 114 - Decrease in Friction Apparent from PC 1 Test Sample (On Left) to PC 1 Test-Dry-
HA Soak-Test (On Right)........................................................................................................... 173
Figure 115 - Minimal Skidding/Tearing of Base Tissue on HA-Rehydrated Samples (PC HA 1)
..................................................................................................................................................... 174
Figure 116 - Skidding/Tearing of Base Tissue Very Apparent on DW-Rehydrated Samples (PC
DW 3).......................................................................................................................................... 174
Figure 117 - PC 4 DW-Rehydrated Post-Test ............................................................................ 175
Figure 118 - PC 3 DW-Rehydrated Post-Test ............................................................................ 175
Figure 119 - PC 2 HA- Rehydrated Post-Test ............................................................................ 176
Figure 120 - PC 1 HA- Rehydrated Post-Test ............................................................................ 176
Figure 121 - Static CoF for PC HA, Bone Resurfacing Study ................................................... 177
Figure 122 - Static CoF for PC DW, Bone Resurfacing Study .................................................. 178
Figure 123 - HYP Standard Curve Example............................................................................... 188
Figure 124 - pH Meter Depiction................................................................................................ 190
Figure 125 - Visual Spectrometer Depiction .............................................................................. 192
Figure 126 - PC DW 1_Abscissa................................................................................................ 194
Figure 129 - PC HA 1_Abscissa................................................................................................. 196
Figure 132 - OM DW 1_Abscissa .............................................................................................. 198
Figure 136 - OM HA 1_Abscissa ............................................................................................... 202
Figure 139 - OM 1 Pre/Post Irrad_HA/PBS Application ........................................................... 204
Figure 142 - Bone Replenishment PC 1 HA-Rehydration.......................................................... 205
Figure 143 - Bone Replenishment PC 2 HA-Rehydration.......................................................... 206
Figure 144 - Bone Replenishment PC 3 DW-Rehydration......................................................... 206
Figure 145 - Bone Replenishment PC 4 DW-Rehydration......................................................... 207
Figure 146 - PC Cross-over STUDY_(1) DW x HA PC_(2) HA x DW PC_NonIrrad ............. 209

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Figure 147 - OM DW vs OM HA NonIrrad ............................................................................... 210
Figure 148 - PC DW Irrad .......................................................................................................... 211
Figure 149 - OM HA Irrad.......................................................................................................... 212
Figure 150 - PC HA vs OM DW_scan ....................................................................................... 213
Figure 151 - PC HA vs OM DW_photograph ............................................................................ 214
Figure 152 - Cross-Over Study_OM........................................................................................... 215
Figure 153 - OM HA................................................................................................................... 216
Figure 154 - OM DW/HA_PC HA/DW/PO............................................................................... 217
Figure 155 - Photographs of Footballs Tested (via www.eastbay.com)..................................... 218

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List of Tables
Table 1 - OM Collagen Types [24]............................................................................................... 13
Table 2 - Tissue Turnover Times for Different Regions of Human Oral Epithelia, as Compared to
That of Skin [26]........................................................................................................................... 17
Table 3 - Cadaver Information...................................................................................................... 50
Table 4 - Sex, Age Range, and Frozen Storage Times for OM-Extracted Cadavers ................... 51
Table 5 - Pilot Study Relative Collagen Concentration Chart, n= 3 OM Irr, 3 OM NonIrr, 3 PC
Irr, 3 PC NonIrr........................................................................................................................... 128
Table 6 - HYP Equipment and Reactive List/Locations............................................................. 189
Table 7 - Concentration of HA in Human Saliva [77]................................................................ 219

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ACKNOWLEDGMENTS
I would like to express sincere gratitude to the following individuals for their involvement in the
completion of this research endeavor:
Dr. Robert E. Baier, for serving as my research major advisor. I am truly appreciative for the
intelligence, experience, patience, and guidance you have shown me throughout the past two
years. Your passion for research and teaching are attributes I will incessantly try to emulate in
my career.
Dr. Anne E. Meyer for serving as a member of my research committee and for always providing
needed input and advice in the direction of my research. Her aid in the friction protocol portion
of the study yielded these results being the most valuable asset to my research findings. I have
sincerely appreciated your stern expectation to always perform at my absolute best!
Dr. Ruogang Zhao for serving as a member of my research committee and providing valuable
insight into the applications of Hyaluronan beyond oral cavity treatment through his own
research endeavors.
Dr. Pilar Ortiz-Alias for providing the laboratory space as well as valued guidance and advice in
Hydroxyproline Assay Testing.
Mr. Jeffrey Slawson and Mr. Brian Frazer (of SUNY at Buffalo Environmental Health and
Safety) for their friendly and accommodating accompaniment and surveillance, enabling me to
irradiate my tissue samples.
UB Football Equipment Managers Mr. Dave Borsuk and Mr. Tom Hersey for providing
friendship throughout my time playing on the team, as well as for providing footballs that I could
begin leather treatment and testing with.
Miss Elizabeth Hatton for supplying the 0.5% HA needed to execute my experiments, as well as
needed knowledge throughout the thesis research/writing process.
Mr. Peter Bush for his aid in obtaining digital images of my microscopically-viewed tissue
samples.
Mr. Tom Wietchy and Mr. Kevin Matthew, of UB School of Medicine’s Anatomical Gifts
Program, for their aid in providing me the cadavers necessary for oral mucosa extraction.
To Cadaver #s: 14082, 14098, 14100, 14102, 14119, 14112, 14138, 14337, 14332, 14316,
14340, 14309, 14384, 15027, 15052, 15053, and 15040 for the sacrifice of their bodies for UB’s
Anatomical Gifts Program, which I am sincerely appreciative to be able to benefit from.
I would like to thank my beautiful wife, Mrs. Jennifer Huber, for your incessant love and support
of my academic aspirations over the past five years.

xiv
I would like to express sincere gratitude to my parents (Todd and Deena) and siblings (Taylor,
Jared, Jordan, Caleb, Joshua and Toriana) for their love and support of me in everything that I
have done over the past 23 years.
I would like to dedicate this work to my Lord and Savior Jesus Christ, that without Him, this life
would have no meaning!

xv
ABSTRACT
It has been previously observed that moderate-molecular-weight hyaluronic acid (HA),
also known as hyaluronan, provides reversible physical protection to collagen-rich tissues as
monitored by a unique tissue-on-tissue friction test. Considering the possible benefits of such a
formulation to irradiated head-and-neck cancer patients who have lost all natural salivary
lubrication, human oral mucosa (OM) was collected from fresh anatomical donations and tested
against a chemically cross-linked standard pericardium reference material for its ability to take
up HA reversibly and preserve desirable tissue properties after simple drying and re-wetting, as
well as after exposure to clinically-used doses of gamma irradiation (usually productive of dry
mouth symptoms). The research methods included, before-and-after 70 Gray Cs-137 irradiation,
tensile testing and tissue-on-tissue friction testing, with HA preparations applied prophylactically
or subsequently. Collateral data on each preparation was obtained by Contact Angle goniometry
for Critical Surface Tension determination and Multiple Attenuated Internal Reflection Infrared
(MAIR-IR) spectrometry for surface compositions. It was discovered that the added HA
significantly relieved the tensile strain of both normal and irradiated samples, and also provided
some modulation of the radiation-induced crosslinking and “embrittling” of oral mucosal tissues.
Re-wetted oral mucosal physical damage during tissue-on-tissue friction was significantly
reduced by HA-solution application, but not by water alone. Weight measurements illustrated
that HA was actually taken up into the native and irradiated tissues, and was completely
reversible by plain water exposure, so the effect was more than superficial lubrication which
lasts only short times. However, the weighing disparities between HA and DW (distilled water)
were not statistically significant. Radiation-induced chain scission might have also occurred,
although studies of the release of hydroxyproline showed a minimum of such effects. Therefore,

xvi
the predominant mechanisms of protection of HA formulations are friction reduction and strain
relief, which now remain to be correlated with subjective pain relief and improved oral function.

1
1.0 INTRODUCTION
1.1.1 XEROSTOMIA
Xerostomia, also known as dry mouth, can be a result of salivary gland hypofunction, or
a result of an alteration in salivary chemical composition [1]. It is the most common side-effect
related to radiation treatment in the head/neck region [2]. “Dry Mouth” can be caused by a lack
of salivary flow to the oral cavity, or a decrease in the quality of the saliva [1]. In the case of this
study, the focus is concerning xerostomic patients with total lack of salivary production as a
result of gamma-irradiation treatment stemming from cancer in the head and neck region of the
patient. Other causes of xerostomia, that might not totally eliminate saliva production, can be use
of prescription medication and systemic diseases [3]. While saliva is not essential to survive, a
lack of sufficient supply of it does diminish one’s quality of life (QOL) in a variety of ways [4].
1.1.2 Effects of Xerostomia
Multiple symptoms/consequences have been associated with xerostomia including an
overall dry feeling in the oral cavity, halitosis, soreness, oral burning, difficulty swallowing, and
an altered sensation of taste [1]. Additionally, a patient suffering from dry mouth is increasingly
susceptible to development of dental caries and periodontal disease, removable denture
discomfort, change in voice quality, difficulty chewing and swallowing, and an increased risk of
developing oral infection [5]. Nocturnal oral discomfort is another common complaint among
xerostomic individuals. In addition to the variety of dental issues that can result from xerostomia,
an individual suffering from dry mouth can suffer on an emotional and social level as a result of
decreased quality of life.

2
1.1.3 Causes of Xerostomia
Prescription drug use (individually or in combination with other drugs) is the most
frequent cause of xerostomia [3]. There are over 400 medications that have been linked to
xerostomia. This includes sedatives, antipsychotics, antidepressants, and diuretics, and
decongestants. These drugs cause an inhibition in salivary production by disrupting signaling
pathways in salivary tissue causing a reduction in fluid output [1]. As of 2008, there were over
25 million people experiencing medication-induced xerostomia in the United States alone [3].
Xerostomia is a linked complication associated with radiation therapy treating head and
neck cancer. Dry mouth is caused by the fact that the salivary glands exist in the treatment field,
being located superficially to the malignant tumor being treated. Decreased salivary flow rates
from radiation therapy can be seen within the first week of treatment, and can worsen over time
depending on the dosage and delivery (3-D conformal or intensity-modulated radiotherapy) [6].
Radiation treatment is often administered in weekly, fractionated doses of 10 Gray (Gy) for 5-7
weeks resulting in a total dose of 50-70 Gy [7]. Another study has administered radiotherapy to
patients with head and neck carcinomas, treated with a curative intent, with a dose of 2 Gy per
fraction delivered five times per week, up to a total dose of 64–70 Gy [2]. The most
radiosensitive glands are the parotid, followed by the submandibular, sublingual, and minor
salivary glands. When the parotid glands are exposed to doses exceeding 50 Gy, there is
permanent damage and the salivary function cannot be recovered [8]. There is a slight reduction
of the parotid and submandibular salivary flow rates even after the first week of radiation. A
50% decrease in parotid flow is reported within 24 hours after exposure to 2.25 Gy [8]. During
the first week of irradiation, a reduction in salivary excretion of 50–60% can be noticed [9].
Saliva production steadily decreases upon each additional radiation exposure [7]. A serious
reduction in salivary flow will subsist after a dose of 25–30 Gy and above 40 Gy salivary flow

3
remains very limited [10, 11]. At 22.5 Gy, salivary production is decreased by 50%, as observed
seven months post-radiation treatment [9]. After radiation-induced damage (Figure 1), saliva
possesses a different volume, consistency, and pH. There is little to no recovery of salivary gland
function after radiotherapy exposure to the parotid gland [5]. Radiation damage to salivary
glands is severe due to damage to the blood supply, interference with nerve transmission, and/or
destruction of the gland itself. Radiation therapy causes an increase in permeability of the
endothelial cells in the periductal capillaries followed with edema and obstruction.
Figure 1 - 3-D View of Irradiation Dosage Effects in the Head and Neck Region
The dose distribution obtained with the parotid-sparing irradiation technique in the coronal and sagittal plane. The highest dose
region is in red and the lowest dose is blue. The left parotid gland (contoured in blue) was irradiated to a high dose, the right
parotid (delineated in magenta) was spared from high-dose irradiation [9].

4
An innovation setting out to avoid this damage to the salivary glands, Intensity-
Modulated RadioTherapy (IMRT) targets the head and neck tumor more specifically, reducing
damage to the surrounding oral tissue. Salivary glands are usually spared, which leads to a partial
recovery of the glands’ function. However, this can take over two years after radiation therapy,
therefore affecting one’s quality of life in the meantime [6].
Excluding radiation, there are multiple systemic diseases associated with xerostomia.
These include Sjögren’s syndrome, rheumatoid arthritis, renal dysfunction, and systemic lupus
erythematosus [12].
1.1.4 Assessment of Xerostomic Conditions
Xerostomia can be assessed in the clinic from patient complaint, oral signs of lack of
saliva production/quality. Symptoms include decreased saliva, burning sensation of tongue, loss
or altered taste sensation, difficulty in swallowing, chewing, and speaking, erythematous fissured
or pebbled tongue, atrophy of filiform papillae of tongue, thick and foamy saliva, cracks at the
edges of the mouth and lips, bad breath, increased plaque, higher tendency to develop cervical
caries, increased oral infection susceptibility such as candidiasis, and purulent secretion from
salivary glands [8]. Patient complaints include difficulty with speech, chewing, and swallowing.
Clinical diagnostic signs of dry mouth include dryness of lips, dryness of buccal mucosa,
absence of saliva produced by gland palpation, and total count of the number of decayed,
missing, filled teeth (DMFT) (Figure 2). These are four clinical measures that, together, can
predict the presence of salivary gland hypofunction [8]. It has been reported that up to 50% of
saliva production decrease can occur without the patient being cognitive that they are suffering
from xerostomia [12].

5
To treat dry mouth, water and other oral-moisturizing fluids are usually needed
throughout the day, and especially at night. Pain can be caused by spicy and hot (temperature)
foods, depending on the individual severity of xerostomia. This irritation could be explained on
the basis of epithelial removal (physical weakness of attachment, as a result of the dry-to-wet
friction increase) and exposure of mucosal sub-epithelial nerve endings. Additionally, patients
with removable dentures have difficulty with retention. Also, their denture adhesives may not
function properly [3]. The whole saliva flow rate (total unstimulated salivary output) can be
gauged by having an individual accumulate saliva in the mouth and expectorate every 60 seconds
for five to fifteen minutes. An unstimulated whole saliva flow rate < 0.12 – 0.16 mL/min is
generally accepted as being abnormally low and illustrating a marked salivary hypofunction [3].
Conversely, healthy individuals typically produce saliva (unstimulated) at 1-2 mL/min, while
xerostomic individuals produce saliva at a rate below 0.7 mL/min [3].
Figure 2- Severe Radiation-Related Dental Caries Caused by Xerostomia and Inadequate Dental Treatment [2]

6
1.1.5 Management and Treatment of Xerostomia
The primary goal of managing xerostomia is to relieve the symptoms associated with it.
Frequently hydrating with water is the easiest way to ease xerostomic symptoms. Caffeine and
alcohol should be avoided because both cause dehydration. The use of citrus sweet drinks or
candies accelerates the caries process and must be discouraged. Patients are advised to sip cool
water throughout the day and drink milk with their meals [8]. Water is the easiest (and cheapest)
way to temporarily remedy dry mouth. It will cleanse and hydrate the oral tissues. However,
water is a poor mucosal wetting agent that lacks buffering capacity, lubricating mucins, and
protective proteins. Whole or 2 % milk may serve as a better substitute because both exhibit
moisturizing properties that can help patients swallow a food bolus [8]. At night, a humidifier
may be used to help keep the bedroom air moistened, easing the effect of dry mouth on the
patient while asleep. Besides water, other treatments include oral rinses, mouthwashes, gels,
sprays, and artificial salivary substitutes that aim to relieve dry mouth symptoms. Products have
a mild flavor component, and have a neutral or alkaline pH [3]. Application of these products
needs to numerous times per day, by the individual. Minimizing oral tissue damage and dental
caries is incredibly important for xerostomic individuals. Other than lubrication of their mouth,
xerostomic patients should maintain immaculate dental hygiene and see a dentist at least tri-
annually [3]. Mouth rinsing after eating, coupled with brushing with a mild, high fluoride-
containing toothpaste, should be part of the daily ritual. A diet low in sugar is essential for the
prevention of dental caries. Parasympathomimetic drugs such as pilocarpine, bethanechol, and
cevimeline stimulate what is left of salivary gland function to increase their saliva production
and can relieve oral discomfort and speech. However, an increase in sweating is a negative side
effect commonly associated with the use of such drugs [1].

7
Water is commonly used as a salivary substitute. However, it lacks the appropriate
buffering capacity, lubricating mucins, and protective proteins found in natural saliva [1]. Saliva
is composed of water, proteins, and electrolytes. Salivary substitutes set out to provide moisture,
protect the tissues in the oral cavity, and inhibit any microbial colonization. Salivary substitutes
usually contain fillers such as carboxymethylcellulose, along with electrolytes, fluorides,
preservatives, and sweeteners [1]. However, most salivary substitutes do not possess the
digestive or antimicrobial enzymes that natural saliva has [8]. Thus, the essentiality of
impeccable oral hygiene is needed for the xerostomic patient. Although there is enough
knowledge about the individual molecules of saliva, there is no product that truly provides
sustained relief to xerostomic individuals. A major liability of present salivary substitutes is that
they need to be applied recurrently multiple times per day. The purpose of this study is to
investigate HA (0.5% Hyaluronan Solution within distilled water, pH of 6-7 range), as it has
been previously shown to sustain lubrication in the mouth, to provide xerostomic relief not
observed in any other dry mouth-treating product today [8].
The following is a brief description of the developmental history of salivary substitutes:
Various solutions have been created to moisten the oral cavity. Solutions with varying amounts
of glycerol, saline, sodium bicarbonate and magnesium hydroxide[8] have been shown to be
beneficial compared to water as a saliva substitute. Antacids were then incorporated to these
liquid formulations to maintain pH balance. Solutions containing betaine, olive oil, honey, xylitol
were also evaluated as salivary substitutes. Xylitol is an antimicrobial and encourages
remineralization of teeth via the replenishment of phosphate and calcium ions, causing teeth pH
to increase, therefore discouraging the acidic breakdown of the teeth that would otherwise lead to
dental cavity formation. Xylitol acts as a sweetener. It is a 5-carbon alcohol, while most artificial

8
sweeteners are 6-member, and therefore are too large to be processed by oral bacteria. Because
of its size, Xylitol can be ingested by bacteria, and due to its antimicrobial properties, kills the
bacteria of the mouth [13]! Saline has also been included in salivary substitutes. Oral rinses
containing hyetellose, hyprolose, or carmellose have also been attempted as salivary substitutes,
but are purely palliative substances that relieve the discomfort of xerostomia by temporarily
wetting the oral mucosa [2].
Some substitutes do have antimicrobial agents, such as proteins, (specifically enzymes).
Enzymes, like lysozyme, cause bacterial lysis and stop their acid creation. Lactoferrin acts by
chelating (removing metal elements) the available iron that is essential for oral bacteria to
proliferate.
In order to compensate saliva insufficiency for an extended period of time, it is
imperative that saliva substitutes attach and embed themselves into the OM (Oral Mucosa)
surface to extend their advantageous effects on the OM. Prior to this present work, no
consideration has been found in literature that lubricants might be imbibed directly into the
tissues themselves. This previous lack of investigation could serve as an explanation for the lack
of extended lubrication relief for the vast majority of salivary substitutes on the market today [8].
Some substitutes are composed of polysaccharides, proteins and glycoproteins (for
example: salivary mucin). Mucin has a water binding capacity and high resistance to shear forces
[8]. It thus enables lubrication and moistening of OM similar to saliva. In order to have a
remineralizing outcome for the salivary substitutes, extra calcium, phosphate, and fluoride ions
can be included. Many Substitutes possess enzymes such as glucose oxidase, lactoferrin and
lactoperoxidase. These enzymes produce hypothiocyanate when coming in contact with
thiocyanate found in saliva [14]. This hypothiocyanate hinders growth and acid production of

9
microorganisms, therefore helping to prevent the acidic break-down of adjacent teeth (which
causes cavities). These artificial salivas do enhance lubricity and provide antimicrobial benefits
for short times [15].
To increase solution viscosity, sodium carboxymethyl cellulose (CMC) in a phosphate-
buffered saline solution with calcium and phosphate (to limit enamel demineralization) has been
included [8]. Other polymers such as polyethylene oxide have been tried as an improvement over
CMC. The addition of a remineralizing potential to a saliva substitute has also been proposed
[16]. Furthermore, there have been comparisons of the effects of mucin-containing substitutes
with those containing carboxymethyl cellulose. While the lubricating properties of mucin-
containing formulations have been better, the re-hardening properties of CMC-containing
substitutes on softened human enamel have also been beneficial [8]. Recent use of linseed
extracts composed of water-soluble polysaccharides has revealed sufficient viscosity and
resistance to mechanical shear forces, and linseed extract has significantly reduced dry mouth
symptoms, therefore being a legitimate saliva substitute [8].
Relating to how saliva is affected in xerostomic patients as compared to healthy
individuals, the lubricities, surface energy analysis (via contact angle analysis) and IR spectral
results (analysis of covalent bonding in the sample) were not significantly different [8]. This
revealed, importantly, that the make-up of saliva does not vary between healthy and xerostomic
conditions in any important way except for secreted volume. Thus, there is no apparent
significant disparity in the quality of saliva between healthy and dry mouth individuals [8].
1.2.1 ROLE OF SALIVA
Saliva consists of two primary, and several secondary, components that are secreted by
independent mechanisms: a fluid constituent that includes ions, produced mainly by

10
parasympathetic stimulation, and a protein component generated by secretory vesicles in acini
and released with sympathetic stimulation [2]. Saliva’s presence in the oral cavity does aid in
microbial attachment, teeth mineralization, taste, lubrication, buffering, maintaining the mucosal
immune system [2], preparing the food bolus during mastication, and permselective tissue
coating [1]. Saliva also shows important antimicrobial properties which help prevent infection.
Identified chemical components of saliva include histatins, statherins, lysozymes, cystatins,
proline-rich proteins, carbonic anhydrases, amylases, peroxidases, lactoferrins, mucins, and
secretory IgA [1].
1.2.2 Properties of Saliva
Saliva provides lubrication by reducing friction between oral surfaces (by acting as a
boundary lubricant). The film of fluid between the two moving surfaces becomes very thin due
to increased load or high speed. Thus, the two surfaces that are separated by the fluid film may
come in close contact with each other and cause wear. The lubrication of saliva is provided by
the mucins and proline-rich glycoproteins of the saliva [8]. Saliva generally provides a lasting
lubricating effect for up to half an hour, and only longer if it is replenished regularly. In past
studies [17], HA has been shown to extend lubrication properties for pericardium reference tissue
(PC) for up to 8 hours (even beyond the lubricating time capability of saliva). One new aspect of
this current study analyzes how this lubrication is affected by freshly-extracted human oral
mucosa (OM) testing, as well as irradiation’s effects on friction changes.
Saliva is primarily produced by the parotid and submandibular glands. The parotids,
submandibular and sublingual glands account for 90% of saliva production [18], while the
parotid glands alone account for 60% of saliva production [9]. In addition, the oral cavity and
pharynx contain minor salivary glands which contribute less than 10% of secreted saliva [5].

11
Salivary glands are composed of secretory units which contain acinar and myoepithelial cells,
and intercalated, striated, and excretory ducts [5]. Saliva is composed of both serous (protein)
and mucous (mucin) components which are secreted by the acinar cells [5]. The submandibular
gland primarily produces unstimulated saliva, while the parotid gland secretes the majority of
stimulated saliva. Stimulation of the parasympathetic nervous system produces a great quantity
of watery saliva that is low in amylase. Arousal of the sympathetic nervous system produces a
minute quantity of viscous saliva composed of a large amount of amylase along with organic and
inorganic solutes [5]. Although the minor salivary glands have only limited contribution to the
basal or the stimulated saliva flow rates, preservation of their function is also of importance
(when undergoing head/neck cancer treatment), because the minor salivary glands produce up to
70% of the total mucin secreted by salivary glands [19]. Healthy individuals can produce 1.5
liters of saliva daily [5]. Another feature of saliva worth noting is that it can lubricate both hard
and soft oral tissue. Therefore, it has a vital role in the protection of these tissues and their
functionality [20]. Prior laboratory measurements show this lubricating effect to last only about
30 minutes in a drying environment [8].
Due to lack of sufficient salivary production in the xerostomic oral cavity, increased
susceptibility to acid attacks causing demineralization of the tooth surfaces is a major concern
[8]. Therefore, to prevent demineralization of teeth, it would be most desirable to have saliva
substitutes at a neutral pH or slightly alkaline in order to mirror that of saliva (average pH of 7
[8]). This pH generally hovers in the 6.5 to 7.5 range, depending on hormonal changes
(especially the monthly hormonal swings of women) [8]. In a recent study, it was observed that
most substitutes that provided good lubricity tended to have pH’s between 5.5 and 6.7, such as
Biotène liquid, Numoisyn, Salinum, Xylimelts and Xylimelts Mint [8]. In contrast, the pH of a

12
solution of HA is naturally around 7. This fact is promising in that, coupled with HA’s extended
lubrication effects, it could potentially serve as the most effective salivary substitute.
1.3.1 PROPERTIES OF COLLAGEN AND ITS RELATION TO ORAL MUCOSA
Collagen, especially type-I, is the most distinguishing fibrous protein derived from
connective tissues such as dermis and bone. It makes up 1/3 of the body’s proteins [21]. It should
be noted that excess or lack of proper collagen amounts in the body can lead to several disorders.
Excessive collagen has been revealed in conditions such as lung fibrosis, liver cirrhosis,
scleroderma, and tumor growth. Diminishment of tissue collagen has been witnessed in
particular disorders of connective tissue, such as rheumatoid arthritis and wound/ulcer-damaged
tissues. Clinically, tissue repair and wound healing overproduction and collagen deposition are
essential to heal tissue damage [21].
Twenty-eight types of collagen that comprised forty-six unique polypeptide chains were
identified by 2009 [22]. Collagen is usually composed of three parallel alpha (single-bond)
polypeptide strands (2 α1 subunits and 1 α2 subunit [22]) in a helical coil, wound together with
non-covalent (hydrogen [23])bonds [22]. The collagen triple helices assemble in a complex,
hierarchal manner that creates macroscopic fibers and networks in bone, tissue, and basement
membranes. This molecular orientation is nearly crystalline [23], therefore possessing
exceptional tensile strength but low elasticity. The collagen macromolecule itself consists of
regions of order where the triple helix contains mainly apolar AAs (Amino Acids), and
disordered amorphous regions containing AAs with long polar side chains projecting radially
from the axis of the macromolecule. Density variations in the molecule are due to these ordered
and disordered regions. The disordered regions of adjacent nearby macromolecules have been
suggested as crosslinking sites via the reaction of radicals formed on the long flexible side chains

13
of the polar AAs. Collagen structures combine with extracellular matrix (ECM) proteins,
proteoglycans, and Glycosaminoglycans (GAGs) [22].
Alterations in the helical assembly and the proportion of non-helical sequences yields
various kinds of collagen, each possessing a unique function and structure. The location and
function of the types of collagen that exist in human oral mucosa (OM) are listed in Table 1:
Table 1 - OM Collagen Types [24]
1.4.1 OVERVIEW OF OM  STRUCTURE AND FUNCTION
Human Oral Mucosa (as shown in Figure 3 and Figure 4), isotropic in nature [25] (no
definitive fibrous directionality), has three main categories (Masticatory, Lining, and
Specialized) that each are either keratinized or non-keratinized. The keratinized mucosa covers
the dry areas of the skin and non-saliva-secreting sections of the mouth. The non-keratinized
mucosa encompasses the moist areas of the oral cavity.

14
Figure 3 - Basic Structure of OM [24]
(1) is the Stratum Basale, (2) is the Stratum Spinosum, (3) is the Stratum Granulosum, and (4) is the Stratum Corneum.
Figure 4 - A Zoomed-out View of the OM Structure [24]

15
The Masticatory Mucosa is constituted of keratinized squamous epithelium. It is located on
the dorsum of the tongue, the hard palette, and its attached gingivae [24]. Secondly, Specialized
Mucosa is found in regions of taste buds on lingual papillae on the dorsal surface of the tongue.
Its defining feature is that it contains nerve endings for sensory reception and taste perception
[24]. Lastly, the Lining Mucosa consists of non-keratinized stratified squamous epithelium. It is
found everywhere else in the oral cavity, including the soft palate, floor of the mouth, and the
ventral surface of the tongue. It is additionally located in the buccal mucosa (inside lining of the
cheeks) and the Labial Mucosa (inside lining of the lips) [24]. This study dealt with the Lining
Mucosa of the Buccal and Labial regions of the cheek interiors as its most representative tissue
type.
The oral mucosa consists of two primary layers: the Squamous Epithelium and the more
“deep” Lamina Propria [24]. The Squamous Epithelium contains the Stratum: Corneum,
Granulosum, Spinosum, and Basale in a four-layer array. Also existing is a three-layer array that
replaces the Stratum Corneum and Granulosum with a non-specific Superficial Layer.
Keratinization, referenced previously, is expounded in the following [24]: The differentiation
of keratinocytes in the Stratum Granulosum into surface cells forms the Stratum Corneum. The
cells terminally differentiate as they move to the surface from the Stratum Basale. Here,
progenitor cells are transformed into specialized cells. Progenitor cells are more specified
versions of stem cells. They differentiate into particular target cells and can only divide a finite
amount of times (Whereas stem cells can theoretically differentiate an infinite number of times).
Concerning the non-keratinized epithelium, it has no superficial layers showing
keratinization. It may transform into keratinized cells, a process deemed hyper-keratinization, via

16
functional or chemical disturbances (such as grinding or clenching of the jaw/teeth). A common
occurrence of hyper-keratinization exists when non-keratinized buccal mucosa becomes
keratinized as a Linea Alba forms, which is a white ridge of callous tissue that extends
horizontally where the maxillary and mandibular (top and bottom) teeth meet [24].
The Lamina Propria is a layer of fibrous connective tissue that is comprised of a network of
Type I and III collagen and elastin fibers. The main cells of the Lamina Propria are fibroblasts,
which generate fibers and the Extracellular Matrix (ECM). The Lamina Propria has two layers:
Papillary and Dense. The Papillary layer is more superficial and consists of loose connective
tissues within connective tissue papillae, along with blood vessels and nerve tissue. The Dense
Layer is the deeper layer. It consists of dense connective tissue with a large amount of fibers.
Between the papillary layers and the deeper layers of the Lamina Propria is the capillary plexus.
It provides nutrition for all layers of the OM and sends capillaries into the connective tissue
papillae. Submucosa may or may not exist “deep” to the Dense Layer of the Lamina Propria,
depending on the area of the oral cavity. If present, the submucosa can contain loose connective
tissue or salivary glands, as well as overlying bone or muscle inside the oral cavity.
The Basal Lamina (Basement Membrane) exists at the interface between the oral epithelium
and the Lamina Propria and consists of Type IV Collagen. Its functions include protection,
sensation, secretion, and thermal regulation. From this location, cells grow up to the surface of
the mucosa. Therefore, the Basement Membrane is where cancerous cells originate and become
malignant. The latter two paragraphs, dealing with the Lamina Propria and Basal Lamina, were
written citing information from Squier, et al. [24].
It should be noted that many reference articles in this study have dealt with the attributes of
skin, and we have correlated these findings to be relevant to OM. This is because OM and skin

17
are alike, with OM having living cells on its most superficial layer, while skin has a Stratum
Corneum that consists of dead cells that act as a protective sheath over the body. Therefore, the
turn-over rate of cells in OM versus skin is much more rapid because there is no superficial
“dead cell” layer (as seen in skin) [26]. Table 2 (below) details this.
Tissue Type
Days
Median Range
Skin 27 12 to 75
Buccal Mucosa (non-
keratinized)
14 5 to 16
Hard Palate (keratinized) 24 -
Floor of Mouth (non-
keratinized)
20 -
Gingiva-oral aspect of free
and attached gingiva
(keratinized)
11* 8 to 40
Oral sulcular epithelium 6* 4 to 10
* Indicates data from
primate
Table 2 - Tissue Turnover Times for Different Regions of Human Oral Epithelia, as Compared to That of Skin [26]
1.5.1 ORAL MUCOSA AND SURROUNDING AREAS: HOW THEY ARE AFFECTED BY
GAMMA IRRADIATION
1.5.2 Mucositis
Oral Mucositis is the inflammation of OM and is caused by the adverse effects of
radiation treatments to cancer head/neck cancer patients. Annually, there are at least 400,000
diagnosed cases of mucositis worldwide [27]. Mucositis negatively affects the patient’s
ability to tolerate the treatment, as well as having the potential of altering the cancer
treatment itself [28]. For example, a radiation treatment schedule might have to be delayed to
allow for the proper healing of oral lesions. Patients receiving radiation in the head/neck

18
region have a 30-60% chance of developing mucositis. The radiation itself interferes with the
turnover of normal epithelial cell regeneration, which leads to damaged mucosa [29].
Normally, epithelial cells undergo a turnover every 1-2 weeks (as shown in Table 2).
Radiation therapy is thought to induce a sterile inflammation that results in increased
permeability of the endothelial cells of the periductal capillaries, which produces periductal
edema. This edema causes compression of the small salivary ducts and destruction of the
duct epithelium. The end result is fibrosis, degeneration, and atrophy of the salivary acinar
cells (which are the most sensitive oral cells to radiation) [30]. Cell proliferation rates never
recover [2]. In addition to direct tissue injury, the oral microbial flora are thought to
contribute to mucositis. Although the exact mechanism is unknown, one hypothesis states
that endotoxins produced by Gram-negative bacilli are potent mediators of the inflammatory
process [28]. Resident bacteria on ulcerated surfaces enhance local injury. Mucosal-barrier
injury associated with mucositis allows attachment and invasion by oral commensal
organisms and, in conjunction with floral changes, leads to the presence of, or an increase in,
pathogens such as hemolytic streptococci. Figure 5 [2] is a visual depiction of an oral
mucositis ulcer.

19
Figure 5 - Diffuse, Radiation-induced Early Grade 2 Mucositis With Solitary Ulcer at Lateral Aspect of Palatal Mucosa [2]
Unfortunately, although there are treatments available for mucositis, such as pilocarpine,
cytokines, Amifostine, dinoprostone, antimicrobial agents, chlorhexidine, benzydamine,
sucralfate, and chamomile, none is completely effective [8]. However, Amifostine could be
beneficial for patients receiving IMRT (Intensity-Modulated Radiotherapy) when the dose to
the contralateral parotid exceeds 26 Gy. It also could be considered to treat young HNC
(Head/Neck Carcinoma) patients undergoing IMRT, regardless of dosage applied to the
contralateral parotid [31]. Amifostine functions by reducing the biological damage to the
salivary glands, regardless of the radiation dose administered [31]. Amifostine is activated to
its selective tissue-protective metabolite in healthy tissue but not in neoplastic tissue. In
1994, Amifostine, given simultaneously with each partition of radiotherapy for 6–7 weeks,

20
was tolerated and improved overall salivary gland function [2]. It has been suggested that
Amifostine is a protector against xerostomia during radiotherapy [32]. Wasserman and
associates have stated that Amifostine treatment during head and neck radiotherapy
diminishes the severity and extent of xerostomia 2 years after treatment [33]. Adverse effects
of Amifostine (sometimes serious), [34] the need for daily injections, and monetary penalties
have restricted popularity; subcutaneous treatment can be just as effective and causes fewer
toxic effects [35].
Relating to the efficacy of chlorhexidine to treat oral mucositis, it was seen that the
drug’s value was no greater than that of sterile saline. In patients who received radiotherapy,
some data suggest that chlorhexidine worsened the condition. Benzydamine, an anti-
inflammatory drug, reduced concentrations of tumor-necrosis factor and was also effective in
reducing the intensity and duration of mucosal damage [2]. Sucralfate, a non-absorbable
aluminum salt of sucrose and octasulfate [2], clings to ulcer bases and creates a surface
barrier in the gastrointestinal tract. The drug possesses antibacterial activity and binds to
epidermal growth factor, which accelerates healing [2]. Sucralfate is a direct cytoprotectant,
which was originally thought to prevent or limit radiation-induced mucositis, but studies
have not confirmed this. However, even though sucralfate does not prevent mucositis,
reduced overall oropharyngeal pain was seen in one study [2]. Additionally, systemic drugs
for pain relief, including opioid analgesics, have been used in patients receiving radiotherapy
[2].
1.5.3 ɣ-Irradiation’s Effects on Collagen: Cross-linking and Chain Scission
Amino acid (AA) analysis has revealed that tyrosine, phenylalanine, and histidine
decreased in collagen due to ɣ-irradiation. This implies that these AAs were cross-linking points

21
between the collagen subunits during irradiation [22]. It has been shown that collagen in a dry
state, post-irradiation, has gone through much more chain scission than cross-linking. This has
been evidenced by an increase in solubility and a simultaneous decrease in tensile strength [23].
However, the opposite has been observed for wet specimens of collagen [23]. This can be
explained with the following: the crosslinking reaction is inhibited by dehydration. These results
suggest that the crosslinking was caused by an indirect mechanism (irradiation-induced cross-
linking can only be completed with existence of water in the tissue).
Two competing reactions are involved in the alteration of collagenous structure, the
formation of crosslinks by an indirect mechanism dependent on the presence of fluid, and protein
chain scission leading to increased solubility by the direct action of radiation on the collagen
fibers [23]. It has been proposed that the hydroxyl radical is the sole effective cross-linking agent
produced in the midst of the radiolysis of water (therefore enabling cross-linking to occur in
hydrated tissue species) [23].
The efficiency in cross-link production from radicals in the protein structure is dependent on
the mobility of the macromolecule and the long flexible amino acid side chains [23]. All the
methods used for removing water from the fiber decrease the indirect effect of cross-linking, and
simultaneously result in decreasing the mobility of the macromolecule within the fiber structure.
Thus, the prospective contact between adjacent molecules and the probability of the formation of
intermolecular crosslinks becomes vastly reduced [23]. Both swelling (via water imbibition) and
irradiation of fibers produce some disorganization of the structure, thus increasing the flexibility
of the molecules, therefore encouraging cross-linking.
It has been observed that both collagen chain scission and cross-linking can occur
simultaneously [23]. However, it also has been shown that the degree of cross-linking of

22
collagen due to ɣ-irradiation outweighs concomitant chain-scission [22]. Radiation-induced
cross-links have been revealed by increased molecular weights of the protein strands of collagen
via re-aggregation of collagen fragments by hydrogen bonding, the formation of disulfide bonds,
and the formation of covalent links between the aromatic residues tyrosine and phenylalanine
[22]. As chain scissions occur simultaneously and a substantial proportion of these amino acids
is still intact even after 50 Mrads (500,000 Gy) [36], it is unlikely that the new bonds are entirely
of disulfide and biphenyl types. Interchain crosslinks involving hydrogen bonds are also ruled
out as a means of irradiation-induced cross-linking.
The crosslinks may occur, however, through a carbon-carbon bond. The formation of such
bonds occurs during the dimerization of hydroxy acids and is involved in the irradiation-induced
polymerization of carbohydrates [37]. The most probable site of cross-linking is between the side
chains of polar amino acids. These residues make up 22% of the total residues and therefore are
abundant sites to accommodate all the new cross-linkages spurred by gamma irradiation [38].
1.5.4 ɣ-Irradiation-Caused Osteoradionecrosis
Osteoradionecrosis is an uncommon resultant of radiotherapy, occurring in 8.2% of patients
participating in a 30-year retrospective study [39], and has been a declining side-effect over the
past 20 years [40].
Regarding pathogenesis, this tissue alteration occurs with free radical generation from
radiation and their corresponding damage to the treatment fields’ endothelial cells. Eventually,
hypovascularity (decreased amount of viable blood vessels), tissue hypoxia, destruction of bone-
forming cells, and marrow fibrosis can result [39].
Clinically, osteoradionecrosis observation can range from small asymptomatic regions of
exposed bone that remain stable to full-scale osteonecrosis that is characterized by intense pain

23
and a malodorous necrotic jaw bone (green-grey color) with pus discharge (suppuration) present
[39]. Figure 6 is a visual representation of oral osteonecrosis.
Figure 6 - Mandibular Bone (Exposed) Attribute of Osteoradionecrosis [2]
If osteoradionecrosis is diagnosed early, local debridement, antibiotic treatment, and
ultrasonography can be effective [41]. As the condition progresses, potential aid to the patient
decreases.
Pertaining to the avoidance of osteoradionecrosis, delayed radiation damages could spur
cellular reduction, lessening of vascular density, shrinking of small vessels and subsequent
fibrosis, and hypocellularity of bone-marrow components. All of these influences cause hypoxia,
a key facet of late-onset wound restoration inferior to lessened fibroblast activity and decreased

24
rate of collagen production. Finally, secondary infection, injury, and surgery lead to deteriorating
eventual morbidity [42]. Pressurized oxygen treatment leads to angiogenesis (new blood vessel
formation), higher cellular oxygen concentrations, fibroblast/osteoblast propagation, and
collagen development in irradiated tissues, and increases cellular oxygen concentrations [43, 44].
However, hyperbaric (pressurized) oxygen treatment has not been successful in treating dental
extraction sites in the irradiated mandible [45].
1.5.5 Radiation-induced Glossitis
Glossitis is a result of permanent dry mouth and therefore is relevant to this study,
addressing “patients” that have complete ceased salivary production. Glossitis is characterized by
difficult swallowing, chewing, and speaking, as well as a sore, tender, or swollen tongue [46].
Relevant treatments used to ease xerostomia symptoms include specific toothpastes which do not
contain the foaming agent sodium lauryl sulphate that dries the mouth are included in some
select sprays and moisturizing gels. Gentle tongue cleansing is also advised [46].
1.6.1 RADIATION-INDUCED XEROSTOMIA SYMPTOMS
Radiotherapy-induced damage in the OM is the result of the deleterious damage of
radiation, not solely on the OM, but also on the adjacent salivary glands, masticatory
musculature, bone, and dentition [47]. Recent findings [2] have suggested that cell-membrane
damage by radiation impairs receptor-cell signaling, which leads to compromised and incomplete
function. Damage also occurs in the parenchyma of the salivary gland, and radiation-associated
inflammation, vascular changes, and edema contribute to the overall damage severity. This
radiation-induced damage to the salivary glands (normally 60-65 Gy) leads to diminishing
salivary flow, alterations in electrolyte and immunoglobulin make-up of saliva, reduction in
salivary pH, and repopulation of cariogenic bacteria in the mouth [2].

25
In addition to direct cellular damage, an absence of wetting medium reduces the ability of
chemoreceptors on the tongue and palate to accept stimuli in foods or liquids, resulting in a
failure of the salivary gustatory response. This thickened mucinous saliva forms a barrier to
dietary, thermal, and mechanical stimulation of the taste buds, which in turn affects the salivary-
center feedback pathway of salivary gland stimulation and ultimate secretion [2].
Both resting and stimulated salivary flow are inhibited due to radiation-induced damage
to the salivary glands. However, a compensatory hypertrophy of the un-irradiated salivary-gland
tissue occurs after a few months and up to 1 year, which lessens the sensation of xerostomia;
however, little further improvement can be expected after this period [2].
Despite numerous recent advancements in cancer-related research, all anti-neoplastic
agents, including radiation, are associated with tissue toxicity. Concerning cancer patients treated
with radiation, fibrosis, necrosis, and severe organ dysfunction may appear months to years after
treatment. Radiation treatment is encouraged for head and neck squamous cell cancer because
cure rates are over 80% in early stages, and 30% in more advanced stages [48]. Except for
laryngeal cancer in early stages, most head and neck carcinomas (HNC) are treated with
radiation portals that include large portions of the parotid and/or salivary gland. Acute
xerostomia, found early-on in radiotherapy, can become complicated with the adverse
contributions of fungal infections and mucositis [48].
1.6.2 Minimizing the Effects of Radiation-Induced Xerostomia
Possible treatments to diminish the liabilities of radiation include cytoprotection.
However, cytoprotectors may counteract the efficacy of radiotherapy if protection is also exerted
indiscriminately on cancer cells. It has been suggested that cytoprotection can help in limiting
malignant tumor proliferation [31].

26
Surgical transfer of submandibular glands prior to head/neck radiation treatment, as well
as acupuncture, have also been used as means to preserve saliva production post-irradiation, but
with limited success [6]. The surgical transfer of the submandibular glands can be a method to
preserve salivary production during radiation therapy because this region is often shielded from
the main radiation dosages and only confronts around 5% of the total dose (3–3.25 Gy). The
surgery is called the Seikaly-Jha procedure; a method that involves the transfer of one
submandibular salivary gland into the submental space (to protect it from irradiation), while
pedicled on the facial artery, facial vein, and submandibular ganglion [49]. This procedure is
administered solely to patients with clinically negative cervical lymph nodes, using the gland on
the contralateral side of the primary tumor. It is not suitable for all patients. For individuals that
underwent this treatment, post-radiotherapy data indicated fewer complaints of xerostomia as
well as garnering few surgical complications [50].
1.6.3 Different Forms of Radiation to Treat Head/Neck Cancer and Their Effects on
Patients’ Quality of Life (QoL): 3D-CRT and IMRT
Recent innovations have been made to spare the salivary glands, particularly the two
parotid glands of each head/neck cancer patient. 3-Dimensional Conformal Radiotherapy (3D
CRT) and Intensity-Modulated Radiotherapy (IMRT) are radiotherapy techniques predicated
on 3D reconstruction of the tumor and adjacent anatomical structures (based on CT scans)
and on computer technology that creates a “beam eye-view,” which is the virtual shaping of
radiation portals to precisely envelope the tumor, minimizing influence on adjacent
tissue/organs [31].
In a study conducted by Golen, et. al. [18], even after six weeks of radiotherapy (average
dose being 33.8 Gy/patient), saliva excretion fractions (SEFs) decreased by 34%. 3D CRT

27
and IMRT are means which allow application of high doses of radiation through minimizing
toxicity [51]. 3D CRT has been described to exclude 1 parotid during irradiation (laryngeal
cancer patients only with a high percentage early-stage laryngeal cancer irradiated) [52].
However, The IMRT technique confers better dose homogeneity as compared to 3D-CRT in
patients with early glottic (laryngeal) cancer [53]. Additionally, salivary gland production
can be maintained with IMRT [54]. It has been supported by several clinical studies that
IMRT can reduce the radiation dose to the contralateral (or both) parotids [52].
Relating to the risk of radiation-induced carcinomas, there is likely to be an increased
incidence for IMRT compared with 3D-CRT due to the dose distribution (larger volume
irradiated at lower doses). In 3D-CRT, 0.5% of surviving patients will develop a second
malignancy as a result of this factor. In IMRT, an additional 0.25% of surviving patients will
develop a radiation-induced malignancy because of this. Thus, a total of about 0.25% of
surviving patients would be expected to develop a second malignancy as a consequence of
the change to IMRT from 3D CRT, which is approximately a doubling of incidence observed
for more conventional radiation therapy [55].
Concerning unilateral vs bilateral 3D CRT radiation treatment in the head/neck region
(having radiation exposure on corresponding sides of malignancy or solely at the cancerous
site), radiation-induced patient-rated xerostomia and sticky saliva was significantly worse
after bilateral compared to unilateral radiation [56]. After unilateral radiation, patient-rated
xerostomia and sticky saliva recovered to the baseline level, while there was barely a
recovery for bilateral radiation patients [56]. It is important to note that with unilateral
radiation-treated patients, recovery post-treatment could be accompanied with the
compensatory overproduction of saliva in the contralateral parotid and submandibular

28
glands. Therefore, it suggests that the spared salivary gland compensates for the loss of
function, thereby limiting xerostomia. This compensation is observed up to one year after
radiation treatment. After this period, any “recovery” or hoped-for compensating with saliva
production likely will not occur [2].
1.7.1 BOVINE PERICARDIUM (PC): STRUCTURE, COMPOSITION, AND FUNCTION
Pericardium, which is almost isotropic because it possesses 3 layers of collagen aligned
in varying directions, is a composite material made up of collagen and elastin fibers in a viscous
ground substance matrix. The tissue acts as a viscoelastic material under stress because of its free
rearrangement of fibers, with the matrix penetrating around and through the bundles [57].
The strong mechanical properties of the pericardium are due primarily to collagen. The
initial extensible portion of the stress-strain curve is due to initial rearrangement and aligning of
collagen fiber weave under stress in the plane of applied force [57]. Mechanically speaking,
actual plasticity of the pericardium, long accepted as fact, is not present [57]. The strong collagen
and weak elastic fibers, as well as the viscous ground substance matrix, play interrelated roles in
determining the dependence of mechanical function on the tissue structure. Each of collagen’s 3
layers is aligned at approximately a 60° angle relative to the adjacent layers [57]. Assuming that
bovine PC (glutaraldehyde-tanned bovine pericardium tissue) structure is histologically similar
to that of the canine [57], previous authors indicated that fiber direction was relatively constant
over a test dimension. The pericardium triple-layer structure results in a quasi-isotropic material.
Directional discrepancies of tissue modulus and ultimate tensile strength (UTS) are within 20-
30% of each other (axial to transverse fiber directions) [57]. The material can be considered a
surrogate for other collagen-dominated tissues during laboratory testing.

29
It has been found that PC has a Hydroxyproline (HYP) concentration of 7.98% [58]. This
equates to roughly 67% collagen (assuming collagen content is 8.44x the HYP concentration
[59]). This high collagen content, as related to OM, is correlated with PC’s strength.
1.8.1 HYDROXYPROLINE (HYP) ASSAY TESTING
HYP is an amino acid that composes about 1/3 of collagen (the third most abundant
amino acid in collagen, with glycine and proline) in human tissue. Collagen is one of the few
proteins that contains HYP (the other protein is elastin [60]), and collagen contains the most
amounts of HYP of any protein in the body [61]. Hydroxyproline is produced as the co-
translational hydroxylation of proline by the proline hydroxylase enzyme, which transpires even
before the conclusion of the polypeptide chain synthesis. The carbon atom in the “4” location of
proline residues, which come before glycine residues in the sequence Pro–Gly–Xaa–Yaa,
experiences this hydroxylation [60]. According to a previous protocol (private communication,
Mark Lauren), HYP content is 11.9% of collagen’s composition [59]. This quantity was used in
our study as a correlation value between the amounts of collagen and hydroxyproline. This
calculation falls closely in-line with another study, which cited HYP concentration in collagen in
the range of 12.8-14.7% [60], and another analysis: 12.5% collagen is HYP [21]. This serves as
confirmation that HYP’s concentration in collagen falls in the 12-13% range.
In summary of the HYP Assay protocol utilized in this study, it required the degradation
of biological tissue into its Amino Acid components. The amino acid, HYP, was then visually
“highlighted” by select chemical agents and reactions. These “highlighted” intensities of color
then corresponded to the varying amounts of HYP in each tissue sample examined. There is a
high utilization of this assay in order to monitor collagen quantities in many pathological

30
conditions, such as tumor invasion and metastasis, rheumatoid arthritis, diabetes mellitus,
chronic ulcers, and muscular dystrophy [21].
The relevance to our study was to analyze the effects of Hyaluronic Acid and Distilled
Water (HA/DW) on OM/PC before/after irradiation. Irradiation, by causing free radical
formation and the breaking of bonds, produces free AAs. As chain scission occurs, more HYP
should be made available to be able to be sensed via a coloring agent and a visual spectrometer.
However, collagen cross-linking occurs simultaneously and has been cited to outweigh chain
scission [22]. Therefore, noting the prior research of Inoue et. al., it is expected that irradiated
tissue would cause more crosslinking in the tissue, and less HYP would be detected by the HYP
Assay Testing.
1.9.1 HYALURONIC ACID (HA): AN OVERVIEW
HA was first discovered in the vitreous humor of the eye in 1934 [62] by Meyer and
Palmer [63] and subsequently synthesized in-vitro in 1964 [64]. Hyaluronan (as depicted in
Figure 7), or hyaluronic acid (HA), is a non-sulphated anionic glycosaminoglycan (GAG)[65] that
is found throughout the extracellular matrix of connective tissue, and serves as the major non-
protein component of joint synovial fluid [66]. Synovial fluid acts as a lubricant, a shock
absorber, and helps control the movement of cells and larger molecules within joints [1].
Hyaluronic acid, which is readily water-soluble [64], as the major component of synovial fluid,
apparently aids in joint lubrication across articular surfaces [67]. Alternate repeating units of β
(1-3) linked D-glucuronic acid and β (1-4) linked N-acetyl-D-glucosamine (these two molecules
are sugars) make up the HA polymer chain [63], which can extend over 30,000 repeating units in
length [64]. The molecular weight of HA utilized in this study was 1.37 * 106
Daltons (as noted
in Appendix 9.5). The pKa of the carboxylic acid group (on the glucuronic acid molecule) is 3.2

31
(the larger the pKa, the more dissociation of the molecules in solution, thus more acidic),
rendering HA charged in the majority of physiologic conditions [17].
Figure 7 - Biochemical Structure of HA (polymerization of these 2 molecules) [68]
Hydrogen bonding, additionally, is an integral aspect of HA molecules in solution. An intriguing
aspect of HA is the aptitude for inter-residue hydrogen bonding between the N-acetamido group
on the glucosamine and the carboxyl group on the glucuronic acid that is able to affect mobility
and local structure [69]. HA-water interaction can include hydrogen bonding and polar bonds to
the hydroxyl groups and the charged carboxylic acid [66]. It has been proposed that HA can
influence water structure going out into solution predicated on its dielectric characteristics,
rendering it similar to ice [66]. HA has a profound effect on water activity and flow resistance,
giving HA-containing tissue compartments elastic traits [70].

Huber Final Thesis Write-Up_21 May 2015

Huber Final Thesis Write-Up_21 May 2015

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