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 FUNCTION 28

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


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


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 (TopBottom) ............................. 120

Figure 70 - PC HA vs. PC DW Post-Bake (TopBottom) ....................................................... 121

Figure 71 - OM DW vs OM HA Post-Bake (TopBottom) ..................................................... 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].

32

It has been proposed that intramolecular hydrogen bonding exists between the

glucosamine ring oxygen and glucuronic acid hydroxyl, and also between the glucosamine

hydroxyl and the glucuronic acid carboxyl group. These hydrogen bonds “stiffen” portions of the

large molecule of HA and provide for two distinct versions of HA: a condensed 4-fold conformer

and a three-fold extended conformation. HA also has a secondary structure that is a two-fold

helix [17]. Cross-linking derived from this double helix structure is thought to provide HA its

viscoelastic properties [70].

HA, in the absence of neutralizing cations, is an anionic polysaccharide. Strong

intermolecular electrostatic repulsions between its carboxyl groups on glucuronic acids will grow

the total length of the HA molecule [71]. These electrostatic repulsions stiffen HA, while

increasing NaCl concentrations will weaken it [71].

Divalent cations, such as calcium, crosslink carboxylate groups of adjacent glucuronic

acid residues across two extended perpendicular HA molecules, therefore creating three-

dimensionally-cross-linked networks. This suggests why calcium/HA solutions have less

mobility and result in the synovial fluid’s preferential binding of divalent cations [68].

The properties of the linear polymer (no side chains) HA in solution have been well-

reported [62]. This is relevant because the HA solution used for testing in this research was

composed only of 0.5% HA, carried in distilled water (DW). Additionally, HA when applied to a

xerostomic patient’s mouth will be mixed with water or other liquids that the patient imbibes.

Strong electrostatic and steric repulsions (because of the large size of HA) enable HA to swell in

water, while polymer entanglements give viscoelastic traits, especially in an acidic environment

[72]. Contrasting, in a basic environment, HA’s viscosity lessens without changes in molecular

weight [66]. It should be noted that the water-binding potential of HA, contingent on MW

33

(molecular weight), can be up to six liters of water/ one gram of HA [73]! When HA is in a well-

hydrated environment, it can block other macromolecules from its area and entangle at low

concentrations (down to 0.1% HA) [74]. In dilute concentrations below 0.5%, HA molecules of

varying MWs will sediment at separate rates, and the HA remaining in solution exists as a

viscous liquid. However, at 0.6%+, all HA molecules come together because of physical

obstruction, rendering this HA solution a gel [66]. These entanglements are what protect HA

molecules from being removed from tissues, along with covalent bonding to surrounding tissue

proteoglycans to aggregate them [70]. The existence of interpenetrating networks of HA into

adjacent collagen structures explains the substantiality under low sliding velocity and high load

[70]. This key principle is the basis for HA’s proposed use as an extended-relief mouthwash for

xerostomic patients, because it lasts longer than other remedies that do not penetrate into the

OM.

As noted, HA (nature’s lubricant), is located throughout skin, lung, and intestinal tissues.

High concentrations are specifically found in the synovial fluid, embryonic tissues, skin,

umbilical cord, the pericellular coat of unfertilized eggs [62], and the vitreous humor of the eye

[1]. It enables diarthrodial joints, like the elbow and knee, to endure millions of loading cycles

over the course of an individual’s lifetime, being able to support many times a person’s body

weight [75]. In embryonic tissues, HA serves an integral role in cell movements by separating

cells in tissues, enabling motion and inhibiting receptor-mediated aggregation [72]. HA shows a

crucial role in tissue regeneration, and tumorigenesis as well [62].

Interestingly, HA (in identical form) can be found in organisms as diverse as

Pseudomonas slime, Ascaris worms and mammals such as the rat, rabbit and human [62]! HA’s

presence has been documented in human tissues as diverse as the skin, aorta, cartilage and brain

34

[64]. HA is involved in water homeostasis, and creates a viscoelastic solution in aqueous media

[1]. Present HA-containing products are used for medical purposes such as ophthalmic surgical

aids [76], to prevent fibrosis in post-surgical wound sites [77], as dermal scaffolds, to treat

cartilage defects, for glial cell culture, tissue regeneration, spermatic motility assessment, and an

equivalent to a corticosteroid as a Tempomandibular Joint (TMJ) arthritis lubricant (decreasing

CoF 50-75% in porcine TMJs [78]).

Cosmetic implants for facial tissue augmentation [1], and for interarticular injections to

treat knee osteoarthritis, are becoming commodity procedures. The latter can prove advantageous

because they restore elastic and viscous properties of synovial fluid, have anti-inflammatory

effects, and normalize hyaluronan synthesis by synoviocytes [67]. Although HA treatment of

osteoarthritis is not the first line of action, it is FDA approved, and has had reported successes.

HA injections have been shown to be most effective when injected into collagenous meniscal

cartilage itself. These injections have been shown to provide pain relief for several months [79].

HA is also available in capsule form. It can then be taken orally to remedy osteoarthritis (by

serving as a joint lubricant).

Concerning the tissue regeneration property of HA, it is interesting to note that HA-rich

fetal wound matrix has pioneered a new understanding of scarless fetal wound healing [80]. The

higher concentrations of hyaluronan orchestrate fetal wound healing via regeneration, rather than

by scarring [81]. In the human mouth, intraoral wounds heal rapidly with minimal scarring, even

without sutures [82]. Additionally, the oral mucosa does not appear to age as exterior skin does

[83]. The relationship of saliva with OM may be responsible for these regenerative properties.

Linking the latter two topics, the similarity of intraoral wound healing to scar-free fetal wound

healing is astounding. Concerning fetal wound healing, high HA levels have been seen and HA’s

35

niche in fetal wound healing has been determined [80, 81]. The healing attributes of saliva have

been recognized by many investigators [84, 85]. HA’s role in saliva, in addition to providing

lubrication, is in carrying growth factors, namely epidermal growth factor [77].

1.9.2 HA: How Does it Bind in the Body? Because HA is negatively-charged, it can bind to any positively-charged molecule, such

as several proteins. These interactions are affected by pH and ionic strength [70]. HA binds to

hyaladherin proteins, which share 30-40% sequence homology in their HA binding domains

[86]. Some of these aforementioned proteins bind to HA via a specific line of 100 Amino Acids

(AAs), deemed the “link module” [87]. Interestingly, some proteins bind HA and aggregate as

proteoglycans by utilizing two link modules. Another sect of hyaladherins binds HA with

particular cell surface receptors, like CD44, which possesses only one link module, and the

receptor for HA-spurred motility [86]. Site-focused mutagenesis (genetic mutation) of residues in

the HA binding area of CD44 has recognized arginine 41, a location preserved with a basic

residue in all HA binding proteins, as an essential mediator in HA binding [88]. Mutation at this

location eliminated ability of HA to bind to CD44 [88].

1.9.3 Hyaluronic Acid: Functionality in the Knee Joint It has been observed that HA, while existing in synovial fluid (at 1-4 mg/ml in healthy

individuals, 0.1-1.3 mg/ml in arthritic patients [89]), provides minimal friction diminishing

effects alone, but rather needs to be absorbed into the articulating cartilage of the femur, patella,

and tibia in order to cause the knee joint to become more lubricious (by aiding in boundary

lubrication) [89]. Generally, HA is thought to promote joint lubrication due to its unique water-

retention properties and squeeze-film action on fluid-film lubrication [90]. HA may function in

36

the knee joint “by being retained at or between the articular cartilage surfaces under relative

motion during testing. Such adsorbed layers of HA at the articular surface may have facilitated

sliding, due to their inherent slipperiness and ease of disentangling, and therefore reduced

friction between asperities in contact.” [89]

1.9.4 HA’s Present Utilization in Xerostomia-Treating Agents for Cancer Victims [68] Self-secreted HA is utilized by malignant cells as a mechanical liquid “wedge” to spread

as cancer. Because mucosal cancer originates in the Basement Membrane and can protrude to the

mucosal surface via HA secretion, it is possible that this same pathway may be open in the

opposite direction. By applying extraneous HA to the surface of the OM, the HA may be

absorbed into the OM and penetrate down for incorporation into the collagen-rich Basement

Membrane. This hypothesis is worth examination because, if accurate, the duration of HA’s

lubricious effects would be extended as compared to other saliva substitutes.

However, a valid topic has arisen that with HA application to the oral mucosa of dry

mouth patients is that the added HA might enable increased cancer cell movement that might

possibly exist in that area of the mouth if the radiotherapy was not entirely effective. This could

potentially become detrimental to the health of the patient, even if they are experiencing

lubrication relief in their mouths. Because this study dealt with extracted OM (non-living tissue

in vitro experiments), this proposition was not able to be investigated. It could provide to be an

intriguing future study on living oral cancer patients undergoing radiotherapy treatment.

HA is commercially available as sodium hyaluronate in combination with

polyvinylpyrrolidone (PVP) and glycyrrhetinic acid. The trade name of this concoction is

Gelclair® (Sinclair Pharma Ltd, London, UK). It has US FDA approval for management of oral

mucositis associated with chemotherapy.

37

One of the components of Gelclair®, PVP, has remarkable properties such as good

chemical and biological inertness, very low toxicity, high media compatibility, and cross-

linkable flexibility. The latter helps in providing stability to the formulation of Gelclair®. Each

of the ingredients in PVP-SH (sodium hyaluronate and polyvinylpyrrolidone) performs a specific

role, as listed in the following:

“PVP is a hydrophilic polymer with muco-adherent and film-forming properties, which

enhances tissue hydration [68].” Topical HA, in 0.2% concentration, forms a protective coating

around the oral cavity to shield exposed or sensitized nerve endings from overstimulation. It also

enhances tissue hydration, and accelerates healing [68]. Lastly, glycyrrhetinic acid has anti-

inflammatory properties that aid in ulcer healing. Together, this compound represents a

promising xerostomic treatment.

However, because of all of the ingredients that have been added to HA, it is extremely

difficult to determine the true properties of HA and how it interacts, solely, with OM. As stated

by Kapoor et. al. [68], HA’s advantageous properties seem to be more obvious than any other

ingredient in this formulation. This is another reason, therefore, that HA should be tested, alone,

to determine its true, uncompromised benefits with OM in xerostomic individuals.

1.9.5 Assessment of HA’s Inherent Antimicrobial Properties HA has been utilized in applications such as contact lenses [91-93] and wound dressings

[94, 95]. While having some microbe-limiting success, infections still arose in its presence.

Some recent studies suggest a bacteriostatic effect of HA [96]. However, HA’s overall

antimicrobial activity is not clearly apparent [97]. HA’s antibacterial attributes seem to rely on

the concentration and molecular weight of HA and on the species of bacteria. Interestingly,

Ardizzoni et al. [96], have shown that HA solutions (of high molecular weight) 0.25-4 mg/mL

38

had no effect on Gram-negative bacterial growth, such as Pseudomonas aeruginosa and

Escherichia coli, but a dose-reliant inhibition was found for Staphylococcus epidermidis.

Concerning Staphylococcus aureus, a minor inhibition of bacterial growth was observed, but for

only for high HA concentrations.

In order to avoid dependency of HA’s antimicrobial properties on HA concentration and

bacterial species, HA must be used in combination with other overt antimicrobial agents such as

silver [98], polyhexanide [99], or Nisin [97].

1.9.6 Addition of Antimicrobial Properties to HA by Grafting of Antimicrobial Peptide In a recent study [97], Nisin (an antimicrobial peptide, 34AAs-long, cationic, and

hydrophobic [100]) was attached to HA to create an antimicrobial biopolymer under solution

form (as shown in Figure 8). Nisin, in various amounts, was grafted onto the carboxylic acid

groups of HA through a monitored reaction (as depicted in Figure 8), to attain covalent grafting

by establishment of amide bonds.

Figure 8 - Scheme of Nisin Grafting [97]

The antimicrobial activity of the modified HA was examined against Staphylococcus

epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa bacteria [97].

39

The results were favorable. In solution, modified HA exhibited a high antimicrobial

effect on the aforementioned bacterial species [97]. Nisin-enriched HA may be of a great interest

to avoid bacterial contamination in applications such as wound dressings, contact lenses, and

HA-based mouth rinses.

1.10.1 THE CLINICAL REVELANCE OF FRICTION TESTING The lubrication concept (minimizing friction) has a multitude of clinical applications

such as minimizing the symptoms of dry eye, arthritis, TMJ disorders [17], and for the present

study, xerostomia. This can be attained by lessening the intimate atomic connections across

biological interfaces. Gaining a firm grasp on the requirements for lubrication is critical to

alleviating these aforementioned conditions.

A tribological (derived from the analysis of test components in motion) protocol has risen

as a promising model for friction testing in vitro [101]. This method utilizes a pin-on-disc testing

configuration to analyze biological materials. Pericardium, PC (primarily composed of collagen

[102]) and OM (less-composed of collagen) were evaluated with HA and DW, as lubricants

applied before and after 70 Gy of ɣ-Irradiation administration.

As utilized in the friction experiments, static friction was the minimal force required to

commence motion and was determined by the atomic structure at the interfaces and their

adhesive interactions [102]. Scission of the weakest trans-interface atomic bonds, as well as

beginning a plastic flow at the tissue-tissue interface, was essential to diminishing the static

coefficient of friction (CoF, μ) [102]. Contrastingly, kinetic friction is known as the force

required to keep to objects that are in contact in motion.

The Frictional Force Laws of Amonton state that frictional force is proportional to load

and independent of apparent surface area of contact and sliding velocity [102]. Although, visibly

40

to the naked eye, the surface contact of two samples interfacing each other might be great, the

actual contact plane is only 1-10 square micrometers (µm2)! With increasing load, this area can

increase as well. Plasticity and the deformation ability of a material also can impact coefficient

of friction (CoF) [102].

In the pin-on-disk friction testing set-up used in this study, stroke length and sliding

velocity controlled the time presented for tissue rehydration [103]. An arcing stroke length of 2.6

cm on the base tissue under the stationary pinned-tissue was utilized, with a sliding velocity of

32 cycles/minute (1 stroke length = 1 cycle).

1.10.2 Forces Involved in Friction In OM and PC, glycosaminoglycans (GAGs) and proteoglycans (PGs) can also play an

integral role in interstitial load support capacity of articulating (joint) tissue. The charged sugar

functional groups of the strongly-bound molecules that exist in synovial cavities resist flow of

interstitial fluid under compression by holding counterions [103]. Connective tissue

polysaccharide solutions have less than ideal osmotic behavior in that osmotic pressure rises

much faster than concentration [66]. This characteristic amplifies the osmotic restoring force

under compression-induced fluid loss. This provides a mechanism for rehydration of the

interface.

Hydration force is imperative to consider when discussing osmotic influences to load

support and lubrication. This type of force is a short ranged, ionic-strength independent,

repulsive force that is seen in molecules of neutral and charged nature [104]. As molecules

strongly adhere to solvent and approach each other, they repel. This will occur unless the

attraction of the molecules for each other outweighs that of the solvent molecules. The

interaction of water-soluble molecules (like those of HA) with water contributes to the strength

41

of biologic structures (like cell membranes), whose stability is inexplicable when only

considering electrostatic and van der Waals forces [104].

1.10.3 Types of Lubrication It has been proposed that in a dynamic physiological environment, several varying types

of lubrication can exist simultaneously. Lubrication can be categorized into boundary or

hydrodynamic lubrication. Boundary lubrication involves only a thin layer of lubricant that

incompletely splits two sliding surfaces, existing in the area encompassing points of contact

between asperities [102]. As previously described, boundary lubrication is active in synovial

fluid at low sliding velocity and high compression. This lubrication shields the cartilage surface

and preserves it physically and chemically [75]. Boundary lubricants rely on both lubricant

viscosity and on chemical make-up. When force is applied, boundary lubricants that are adsorbed

to surfaces hinder the squeezing of boundary lubricants from contact regions. This means that the

force of friction of a legitimate boundary lubricants should be the force needed to rupture the

lubricant film layer [102]. Additionally, because molecular structures of the adsorbed layer and

the unbound interfacial layers are different, shearing between these two lubricant phases is

possible [102]. The underlying layers can influence the molecular organization of an adsorbed

film extensively as compared to solution molecules. The lubricant is solidified on the substratum

surface when it is 3-5 molecules in depth. In this position, the lubricant will act as a solid under

the influence of the surface [102].

Contrasting, hydrodynamic lubrication (HL) occurs when a lubricant entirely separates 2

sliding surfaces, minimizing contact between surface asperities with a fluid film [105]. The

molecules in the shear plane of the liquid increasingly drag the intervening fluid between them to

generate hydrodynamic pressure to keep the skidding interfaces apart as sliding occurs [105].

42

The force of friction created is dependent on the molecular orientation and rheology of the fluid

film contrasting with the incidence of contacting asperities in boundary lubrication. Friction and

lubricant viscosity have a direct relationship at particular sliding velocities; nevertheless, this

relationship does not apply for non-Newtonian lubricants [105].

Squeeze film lubrication (SFL) is likened to HL in its dependency on viscosity. HL is

contingent on viscosity to pull the film around a surface while SFL depends on viscosity to resist

being displaced from the interface [106]. The viscosity in both aforementioned instances aids in

preserving film thickness. Mixed film lubrication primarily alludes to circumstances with

attributes of both boundary and hydrodynamic lubrication [106].

Elastodynamic lubrication is contingent upon at least one of the sliding materials

deforming under hydrodynamic pressure to support loads [17]. By modeling the sliding nature of

soft tissues, such as in the lungs’ serosal cavity, it was discovered that superficial tissue

smoothing could occur in response to rotational shear. It was concluded that the force of friction

barely was contingent on load, even though a diminishing fluid thickness occurred under a load

increase. In conclusion, it was found that more-elastic materials are most appropriate to support

loads in elastohydrodynamic lubrication [17].

1.10.4 Static vs. Kinetic Friction Coefficients (µ) A friction coefficient, CoF (Force due to Friction = CoF * Normal Force Exerted), is

observed when two bodies slide against one another. The resistance to motion produced by

friction is deemed the Force due to Friction. The friction coefficient needed to begin interfacial

motion (and overcome normal force) is called the static friction [107]. Then, “the friction

coefficient decreases approaching the lowest stationary value, which is called the kinetic

friction” [107]. The kinetic friction coefficient can be described as the coefficient needed to

overcome the normal force of the “rubbing” object to continue motion. Pertaining to this study,

43

static coefficient of friction was determined by utilization of a friction-testing device employing

a stop-start “rubbing” pattern.

1.11.1 HA USED IN FRICTION TESTING: PREVIOUS MASTER’S THESES A recent Biomaterials Master’s Degree Thesis project at the University at Buffalo utilized

a tissue-on-tissue friction test to examine the lubricating effects of hydroxypropyl guar

galactomannan (HPGG) compared to a control consisting of HA. An experimental formulation

of HA showed superior lubrication and resistance to desiccation for up to eight hours [17]. HA’s

lubricity was checked (while friction testing of the PC-PC tissue interface) in the presence of

water, salt water, acid and base. HA’s lubricating properties were demonstrated as being

effective in all of these conditions. Additionally, 0.5% HA solution in DW was determined to be

the ideal concentration of HA. This was due to that at 0.6% HA, the solution became a gel and

“plowing” would occur between the rubbing pieces of PC during friction testing, therefore

increasing the coefficient of friction. Furthermore, the solution in a gel form would not be

realistic to be used clinically, as it would be difficult for a xerostomic patient to rinse their mouth

with a gel to provide lubrication relief.

1.12.1 HA VS. SALIVA SUBSTITUTES IN FRICTION TESTING: PRIOR MASTER’S THESES The prior Ganesh Biomaterials Master’s Degree Thesis utilized the same tissue-on-tissue

friction test to investigate commercially available salivary substitutes alone, mixed with, and

compared to human saliva samples from control and xerostomic subjects. The HA formulation

demonstrated superior results when compared to both natural saliva and the salivary substitutes

on the market today [8]. The most effective elements were rinse-resistant organic

polysaccharides such as linseed extracts and xanthan gum, at neutral pH, while formulations at

44

both acidic or basic pH or predicated on synthetic carboxymethylcellulose,

hydroxyethylcellulose, hydroxypropylcellulose, as well as natural esters and glycerin, were

usually not lubricious or were less substantive (with the exception of undisclosed silicone

components in some products). Intrinsic lubricities of saliva from both patient groups were

excellent, lessening CoF values from over 0.4 down to around 0.1 in all instances, and saliva

aliquots sustained these CoF reductions after additions of 4 diverse saliva substitutes designated

to examine their admixture effects on continuing lubricity and substantiality. It was proven that

small amounts of natural saliva can deliver their high lubricity to saliva substitutes that are not

very lubricious on their own. Predicated on these outcomes and comparative MAIR-IR spectra

displaying similar carbohydrate-to-protein ratios, despite lower pH data for xerostomic saliva

specimens, xerostomic saliva was revealed to be lacking mainly in amount secreted, rather than

function or composition as a superior tissue lubricant.

1.13.1 HA USED IN CLINICAL STUDY [1] Citing the aforementioned prior research at the University of Buffalo, it was discovered

that HA could preserve tissue-on-tissue (PC) lubricity in desiccating conditions (in a low-

humidity environment) for up to 8 hours, well exceeding the 30-minute lubricity times of natural

and xerostomic human subjects’ saliva and of commercial ‘dry mouth’ relief products during

laboratory testing [8]. Thus, HA was then evaluated in a preference test at Roswell Park Cancer

Institute (RPCI), using human volunteers also examined at the UB School of Dental Medicine

(UBSDM) [1]. Used for dry mouth treatment effectiveness trials, in a trial with 20 consenting

human subjects (UB HSIRB Project # SIS0891111E, 2011-2013) subjects were offered a 2-week

supply of HA, consenting to use it as much as desired. The subjects were instructed to apply the

HA solutions 3x per day, after each meal. They were instructed to lubricate their mouths with

45

water if their mouth became dry despite this thrice-daily HA application. At the onset of the

study, 70% of the subjects assessed their ‘dry mouth’ condition as “considerably or extremely

severe”, and 80% needed to carry of a bottle of fluid “very often, when leaving home”, while

80% also stated they would “wake up at night and need to drink water”. Of this group, after the

two-week trial, 60% said that the HA formulation was “much better” to “lubricate your mouth,

compared to other oral dryness relief products used”, while 50% stated that HA lasted a “longer

time” for the “oral comfort, compared to other products used.” Oral Medicine experts performed

intra-oral examinations at the commencement and termination of these trials. From these

assessments, there were no observable differences pertaining to formulation use by the examined

patients. Most subjects (65%) endorsed the HA formulation for other dry mouth patients “as-is”,

while some also recommended that a spray bottle version of HA be fabricated with flavoring

added. Coupled with the outstanding tissue-on-tissue lubricity observed during laboratory testing

in dry air conditions (on bovine pericardium tissue, PC), along with human patients’ indication

of the formulation’s effectiveness in home use, it became probable that a final HA solution could

be fabricated to treat dry mouth [1].

It should be noted that the subject group only included 2 patients that suffered from

complete lack of salivary function, the rest of participants did not have that most-serious dry

mouth suffering [1]. The current study had its goal to examine the mechanisms by which an

optimum 0.5% HA formulation might provide relief for irradiated oral mucosa and other

collagen-based tissues.

46

2.0 PURPOSE

Hyaluronic Acid, with its advantageous properties of being a natural lubricant, its role in

tissue regeneration, as well as its promising abilities demonstrated in prior Master’s students’

works, rendered it a potential ground-breaking treatment as a mouth-applicant for xerostomic

patients. The goal of this investigation was to determine if, even after irradiation treatment, Oral

Mucosa tissue can still be lubricated by HA formulation application in vitro.

2.1.1 MOTIVATION Hall of Fame Buffalo Bills quarterback, Jim Kelly (Figure 10), was stricken with oral

cancer in 2013. Kelly’s cancer was described in an interview with Peter King of Sports

Illustrated (Monday Morning Quarterback columnist) by Kelly’s oncologist Dr. Peter Costantino

as “countless microscopic (tumors)” in his head, existing dangerously close to the carotid artery

and infraorbital nerve (in the Spring of 2014) [108]. Because of this possibly-lethal proximity,

surgery to remove the tumors was not initially realistic. If doctors operated and all the cancer

was not able to be eradicated, weeks could pass before chemical or radiation therapy could

commence while Kelly recovered from surgery, and that crucial time could permit the cancer to

propagate into his brain unrestricted. Thus, several weeks of aggressive chemotherapy and

radiation were undergone by Kelly, five days a week. The goal of Kelly’s treatment was to

kill/limit the spread of the malignancies as much as possible in order to enable a “safe” surgery

to remove the remaining tumors. The toll that it took on his body and the family was terrible. It

pained people all over the world that so richly admired him, to see him so weak and frail (as seen

in Figure 9).

47

Figure 9 - Jim Kelly at His Hospital Bed During Cancer Treatment, With His Consoling Daughter, Erin, By His Side [108]

Although reportedly cancer free, Kelly now faces a long road to recover his health and

strength. His speech, eating difficulty, and xerostomia, and other side-effects will harshly affect

him for the rest of his life.

The current area of research into remedies for cancer survivors, like Jim Kelly, deals with

overcoming chemo/radiotherapies’ lasting effects.

48

Figure 10 - Jim Kelly is the only quarterback in NFL history to lead his team to the Super Bowl in four straight seasons.

(Rick Stewart/Getty Images)

The pain that cancer patients must endure should be mollified through this research.

49

3.0 MATERIALS AND METHODS

The following sub-categories depict, in-detail, the necessary steps taken to perform each

experiment performed in the study.

3.1.1 HUMAN ORAL MUCOSA EXTRACTION Human Oral Mucosa was obtained from fresh cadavers via the UB Anatomical Gifts

program. The cadavers were retrieved within 2 days of death and were subsequently stored in the

cadaver freezer at the UB School of Medicine Anatomy Lab. The average time between death

and OM extraction for the cadavers was 15 days, with the majority of that time having the tissue

“preserved” frozen. The allotment of all of so-called “ideal” specimens was as-embalmed for

dental/medical students; “rejected” specimens (due to their physical condition i.e. disease,

obesity, etc.) were provided for OM extraction for this investigation. The actual procedure was

quite simple. A mouth gag was used to pry each cadaver’s teeth apart, opening the mandible.

Making the oral mucosa taught, it became easier to cut/extract the cheek tissue. Each cadaver’s

head was rotated transversely to the side of the mouth gag. A depiction of the cadaver mouth gag

inserted is depicted in Figure 11.

Table 3 is a list of the OM-extracted cadavers, as well as some of the cadaver’s health

information.

50

Cadaver #

Sex Age at Death

Cause of Death Date of Death Date of OM Extraction

Amount of Days Between Death and OM Extraction (Approx. Time Frozen)

14082 male 73 unknown December 21, 2014

February 28, 2014 69

14098 female 89 unknown March 7, 2014 March 13, 2014 6

14100 male 59 unknown March 12, 2014 March 13, 2014 1

14102 male 60 lung cancer March 12, 2014 March 13, 2014 1

14119 male 68 congestive heart failure & renal failure

March 24, 2014 April 1, 2014 8

14112 female 47 cervical cancer March 19, 2014 April 1, 2014 13

14138 male 55 breast and lung cancer April 3, 2014 April 7, 2014 4

14337 male 70 chronic respiratory failure

August 28, 2014 September 11, 2014

14

14332 female 86 failure to thrive August 24, 2014 September 11, 2014

18

14316 male 91 dementia August 15, 2014 September 11, 2014

27

14340 male unknown unknown September 2, 2014

September 11, 2014

9

14309 female 87 Chronic Obstructive Pulmonary Disease

August 9, 2014 September 11, 2014

33

14384 male 88 chronic heart failure and myelodysplastic syndrome

October 6, 2014 October 16, 2014 10

15027 male 76 bladder cancer January 4, 2015 January 20, 2015 16

15052 male 69

respiratory arrest caused by myocardial infarction and coronary artery disease

January 27, 2015 February 3, 2015 7

15053 male 83 cardiorespiratory arrest

January 29, 2015 February 3, 2015 5

15040 male 88 unknown January 20, 2015 February 3, 2015 14

Table 3 - Cadaver Information

There were thirteen males and four females that OM was extracted from, between the

ages of 47 and 91. Age range, sex, and freezer storage time (15 days, on average, per cadaver)

51

were uncontrolled for this study. Cadavers operated were unknown in every aspect, except for

their sex, when the OM was harvested. Information given in Table 4 was not provided by the

anatomy lab supervisors until March, 2015.

Totals

Male 13

Female 4

Male Age Range

55-91

Female Age Range

47-89

Avg Amount of Days Between Death and OM Extraction

15

Avg Female Age

77.3

Avg Male Age

73.3

Avg Age 74.3 Table 4 - Sex, Age Range, and Frozen Storage Times for OM-Extracted Cadavers

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Figure 11 - Initial Incision of Cadaver Cheek

It should be noted that the incision was always made away from the head tilt/mouth gag direction

With tweezers and a scalpel (scalpel blade was replaced for every cheek of mucosa removed),

the OM extraction was performed on the opposite side of the mouth gag. To start, an incision

cutting completely through the 500 micrometers (µm) of OM and its underlying lipid tissue was

performed transversely from just above teeth numbers 8 and 9 (top of the lips), straight towards

the ear, as far as the cheek obstruction would allow. Then a shallow cut was made from the end

of this cut to the bottom of the mouth (below teeth numbers 24 and 25 at the bottom of the lips)

through the mucosa, but not through the rest of the cheek. Then, a scalpel was utilized, starting at

53

the incision closest to the ear, in conjunction with the tweezers to separate the oral mucosa from

the underlying fatty tissue. This tissue separation was completed by working towards the bottom

and top of the lips anteriorly. The tweezers were used by pulling on the very edge (in order to

exert minimal damage on the OM) of the mucosa to create tension to enable cutting separation

from the underlying tissue (Figure 12). OM extracted were ensured to not have any portions of the

vermilion border [24] or lip tissue (which has different characteristics than OM). This process

was then repeated by cocking the head/mouth gag in the opposite direction and the other cheek

was then cut to extract the OM. Thus, two OM samples with minimal attached fatty tissue,

roughly resembling two boomerangs in shape, were harvested from each cadaver.

Figure 12 - OM Extraction, after Initial separation from Underlying Lipid Layer (Bottom Right)

*Note the “Boomerang” shape of the cut tissue samples (OM on Upper Left) and the fact that both the mouth gag and head are cocked “into the page,” while the extraction is on the “near” side of the page.

54

3.1.2 Oral Mucosa Preparation for Testing The OM samples were then stored in polymeric containers/bags and brought to the

research laboratory for further fatty tissue removal. The tissue was laid in a dissection tray in a

bath of distilled water to ensure the tissue remained moist during dissection. Following is a

diagram of how the forceps were used as a stopper for the tissue, while a scalpel was inserted in

between the points of the forceps. The edges of the prongs of the forceps were placed beside the

tissue (in order to not puncture and damage the mechanical structure of the tissue) to be cut, upon

the dissection tray. After scalpel insertion in between the forceps, the scalpel was pulled toward

the user and back out of the opening between the forceps edge “stoppers” while cutting the OM

(and removing fatty tissue) (as seen in Figure 13 and Figure 14). This same process was

performed to cut the OM into segments to be used for testing after the removal of the underlying

lipid masses of the OM.

Figure 13 - Diagram of Tissue Cutting Procedure

55

Figure 14 - Further Cutting/Separation of the OM from Underlying Fatty Tissue

[It is important to note that for cadaver #15040, the skin/mucosa was a deep blue color,

implying oxygen deprivation near death. It was noticed, after OM extraction, that the tissue

quality was noticeably more degraded than the other OM samples because of this hypoxia (even

though the cadaver had only been dead for a few days maximum before 2-weeks freezing).

Therefore, this cadaver’s OM was set aside and not used for future study.]

A similar approach was used for the cutting of reference fixed pericardium tissue (PC);

the forceps point-scalpel pull (in between forceps points) technique was also used.

Glutaraldehyde-tanned bovine pericardium (Peri-Guard® Lot # 5744401-899070 and Lot #

5746763-928687) was obtained from Synovis Surgical Innovations, a division of Synovis Life

Technologies Inc., St. Paul, MN, USA. The pericardium was completely submersed in a bath of

56

Propylene Oxide (PO), which was its storage solution, in a clean dissection tray. The purpose of

this bath was to prevent the PC from rapid moisture loss and tissue shriveling.

3.2.1 FABRICATION OF 0.5% HA SOLUTION Hyaluronic Acid manufacturing specifications can be referenced in Appendix 9.5. It

should be noted that the HA molecular weight utilized in this study was 1.37 * 106 Daltons.

The following protocol was accomplished for formulation preparation, as previously used

for the human preference study [1]. First, pre-cleaned scintillation vials, 20mL volume, made of

borosilicate glass with screw caps were ordered from Optics Planet Inc. (3150 Commercial

Avenue Northbrook, IL 60062). Over twenty-four hours, a 0.5% sodium hyaluronate pre bulk

solution was prepared. The 0.5% sodium hyaluronate solution was covered with aluminum foil

for twenty-four hours to allow complete dissolution. The glass scintillation vials were labeled

with their contents. 10mL of the 0.5% sodium hyaluronate solution was measured using a 10mL

graduated cylinder and subsequently poured into every labeled vial. Each vial was covered with

a small piece of aluminum foil and sterilized on the liquid setting of a steam sterilizer at 121°C,

15 pounds per square inch (PSI), for 15 minutes (Getinge Castle 233LS Vacuum Steam

Sterilizer). After removal from the sterilizer and a thirty minute cooling period, each vial was

covered with a sterile, polymeric cap over the remaining sterile aluminum foil. The vial contents

were then transferred to sterile 30 ml clear polycarbonate square bottles (VWR International,

Radnor, PA, USA) for laboratory testing and patient/football distribution for an ongoing parallel

study.

57

3.3.1 MULTIPLE-ATTENUATED INTERNAL REFLECTION INFRARED (MAIR-IR) SPECTROSCOPY METHODS [8]

MAIR-IR Spectroscopy is an analytical technique predicated on the interaction of

infrared energy with matter. An analytical infrared spectrum is a plot of infrared intensity against

wavelength of energy. Infrared energy exists between 0.7 and 1500 μm in the electromagnetic

spectrum, which is between the visible and microwave regions. For this study mid-range (400-

4000 cm-1 [109]) infrared rays were utilized. MAIR-IR Spectroscopy is performed by the sample

being sandwiched by plates to a prism in which infrared rays are passed through. The resultant

infrared spectrum represents the “fingerprint identity” of a sample with absorption peaks which

correlate to the bond motion frequencies of the covalent bonds of the atoms of the material

tested. Because each material possesses a distinctive combination of atoms, no two materials

yield exactly the same infrared spectrum. Thus, infrared spectroscopy can result in legitimate

qualitative analysis of every kind of material. Furthermore, the size of the peaks in the spectrum

is a direct indication of the amount of that molecule present existing in the compound. NaCl

(table salt) can be used to facilitate analysis of dried solutions by MAIR-IR Spectroscopy

because it does not react to IR rays because it is ionically, not covalently, bonded. In Figure 15, a

visual depiction of the instrument used is given.

58

Figure 15 - Laboratory Spectrophotometer used for analyses of dehydrated OM/PC during this investigation.

Two-millimeter thick KRS-5 prisms were used for analysis in this investigation. KRS-5

prisms are water-“etchable” and therefore cannot be cleaned with water. Instead, KRS-5 prisms

have to be rinsed with acetone and scrubbed with the wooden end of a cotton swab. The

scrubbing also helps to physically remove contaminants from the prism surface. An advantage of

a more slender prism would have been that there are 25 impacts of the ricocheting infrared

beams on each face of the prism, as compared to much less for the thicker prism. The more

impacts the IR rays have on the face of the prism, the more readings it will be able to obtain from

the adjacent sample being tested and more accurate and significant peaks will result. However,

because of the fact that MAIR-IR Spectroscopy readings for dehydrated tissue samples were

desired (KRS-5 prisms would therefore suffice because they are water-soluble), these prisms

were still very useful.

The MAIR-IR Spectrometer utilized for this study was a Perkin Elmer Spectrum 100 FT-IR

Spectrometer (Waltham, MA, USA). The prism used was a thalium halogenide material (KRS-5

H104, dimensions: 2 mm x 20 mm x 50 mm, manufacturer: Crystran Ltd, Dorset, UK)

59

For each MAIR-IR analysis of the dried PC or OM, the following protocol was used:

Background Spectrum: The “background” spectrum was measured before the sample

spectrum. The purpose of this was to measure the contribution of the spectrometer and

the air environment to any spectrum obtained. Neither prism nor sample was positioned

within the spectrometer during this initial test.

A Baseline spectrum was then completed with the MAIR prism (KRS-5 Combination

of Thallium Bromide and Thallium Iodide) that would be used, but no sample was placed

upon the prism. The purpose of this spectrum was to provide confirmation that the prism

was clean and uncontaminated. A single KRS-5 prism was used throughout the entire

investigation and the baseline recorded was found to be reproducible each time.

Each sample was placed with clamps on the prism and spectra of these samples were

obtained and analyzed. The range of values of covalent bonds being recorded was from

4000-600 cm-1. Samples used were taken from prior weight/volume studies that had

terminated with dried PC and OM specimens, soaked in HA or DW. With little to no

water involved with the spectra readings, a direct tissue covalent bond analysis was able

to be completed without hydroxide molecule obscuration that would have otherwise have

been present by the existence of water in the samples.

To “zero” the instrument in between testing samples, a Baseline with residue was

collected from the previous sample. If this Baseline was significantly different (“new”

peaks were depicted when compared to original Baseline) from the original Baseline at

the beginning of the work day, then the prism was cleaned and washed with acetone in

order to eliminate possible residue artifacts from previous samples tested.

60

Acetone rinsing of the previous sample remnant from the prism was accomplished by

spraying a stream of acetone from a distance at an estimated stress of 1 Pa. As before, the

material remaining on the prism was air-dried before being analyzed. Therefore, a

“residue” spectrum was run after each sample, before a new one was placed on that same

prism. This spectrum depicted the retentive qualities of any components of the sample

able to resist such shear stresses of the spraying of acetone.

Figure 16 is a schematic of the path of Infrared (IR) rays through the stage apparatus system

of the MAIR-IR Spectroscopy instrument. This is a “top” view of the path. The ovals on the right

and left of the diagram represent the input and output of the system. In this case, they represent

the IR ray source (right) and the sensor of the rays (left). The latter corresponds to the read-out

on the computer software used (Spectra). The red trapezoid in the center represents the top edge

view of the KRS-5 prism’s position in the apparatus and has 45° beveled edges (both of these

edges are visible) to receive and transmit reflected IR rays. It should be noted that the penetration

into the sample sitting on the shorter parallel side of the prism only is a micrometer in depth. The

curved condensers adjust the infrared rays so they can enter the prism, and then the analytical

slit, efficiently.

61

Figure 16 - Schematic of Infrared Ray Path

In Figure 17, are the components used to fasten the KRS-5 Prism into the Stage. The plate on

the left is called the “top plate”, the middle is deemed the “middle plate” and the right is termed

the prism holder. The threaded rods of metal are named “screws.”

62

Figure 17 - Fastening Components for Mounting KRS-5 Prism onto the Stage

Depicted in Figure 18, the plates, prism holder, and screws are fastened together and ready to

be installed into the Stage for analysis. It should be noted that the more sample in contact above

and below the prism, the stronger the peaks in the spectra that will result. It was imperative that

when handling this instrument, “artifact peaks” produced from the oils of the researcher’s hands

were avoided.

63

Figure 18 - Fastened Components of KRS-5 Prism, Ready for Insertion into Spectrometer

Figure 19 is a photograph of the prism held within the prism holder without the screws, nor

the top and bottom plates attached. It should be noted that the prism needed to be positioned

parallel and slightly elevated from the surface of the prism holder. It should be noted that the

three “disk holders” seen above, solidifying the prism’s position, actually have two “ridges” of

their corresponding knobs, protruding outwards. The prism was nestled in between these

“ridges”, on all three sets of disk holder knobs, to maintain the ideal position for passing infrared

rays through the prism. At the bottom-left of the image are two circular holes on the “bottom” of

the prism holder. These holes fit into pegs in the stage apparatus that guarantee the prism holder

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to be in an upright position, perpendicular to the stage’s surface beneath it. Figure 20 is a

depiction of the jig just before insertion of the fastening screws.

Figure 19 - Position of KRS-5 Prism on Testing Jig

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Figure 20 - Prism and Test Jig before Top Plate Application

The samples used for this study were the post-baked HA and DW-soaked tissues left

from the bulk weighing analysis study. These baked tissues were issued in order to not

compromise the integrity of the prism with otherwise water-moistened tissue. Because each

sample only covered a small fraction of the prism surface, all samples from each group (OM HA,

OM DW, PC HA, and PC DW) were all placed on the prism for the same “total” reading. Thus,

despite having 19 total samples tested (5 PC DW, 3 PC HA, 6 OM DW, and 5 OM HA), only 4

readings were performed. The purpose of this sample “combination” was to maximize the

sensitivity of the spectrometer by having the most possible tissue covering the prism’s surface.

Therefore, the covalent bond “peaks” could more easily be displayed by the spectrometer (and

conclusions about these recordings could more easily be generated).

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Finally, subtraction spectra were conducted by subtracting the peak magnitudes of 2

spectra, yielding the difference in peak magnitude between them.

3.4.1 IRRADIATION OF TISSUE SAMPLES A Cesium-137 Lead-Lined Gammator Irradiation Unit was used, manufactured by

Isomedix (Chester, NY), located in the SUNY at Buffalo South Campus’ Biomedical Education

Building (BEB) (Figure 21). The Cs-137 has a half-life of thirty years. According to SUNY at

Buffalo’s calibration records:

In 2004, the reactor emitted 627 Rads/minute

On May 24, 2007, it emitted 797 Rads/minute

With the present rate being 3.5 Gray (Gy)/minute (350 Rads/minute), an irradiation time

of twenty minutes was suggested to provide 70 Gy of irradiation. This estimated radiation

exposure was derived from an inspection by Best Theratronics on September 5, 2013

which calculated that the central dose rate measured with Fricke dosimeters of the

irradiator was 3.78 Gy/min.

The 70 Gy of irradiation exposure is equal to the maximum common total dosage usually

applied to a typical head/neck cancer patient undergoing radiation treatments [2, 9]. Capped

containers of the samples were placed into a large low-density polyethylene (LDPE) container.

This enabled the samples to be stacked vertically, in order to minimize the dosage cycles needed

to irradiate all of the samples. The sample-filled container was then placed on a stainless steel

circular disc that acted as a turn table that could be activated. The individual containers of the

samples had their caps loosened because of vapor pressure (caused from sample irradiation). The

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rotating table was then turned on and a few rotations confirmed if the present orientation of the

samples would enable safe gamma irradiation. After this verification, a large lever was moved

180 degrees (confirmed by a “click”, signifying the seal of the device from the outside

environment), sealing the container and rotating disc inside of the gammator. A stop watch was

then utilized to monitor the irradiation time of 20 minutes. Figure 21 is a depiction of the

Gammator with tissue samples contained within a LDPE container.

Figure 21 - Isomedix Gammator Unit

Some notable aspects of gamma-irradiation are as follows: It provides superior and

uniform (via “turn table”) material penetration. Gamma irradiation also sterilizes its contents.

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The gamma irradiation process involves gamma photons penetrating through the entire

sample, destroying any cell life, and providing uniform sterilization and treatment, even if they

are in sealed containers [9].

3.5.1 CHEMOMECHANICAL TENSILE TESTING ChemoMechanical Testing (CMT) was conducted to observe mechanical strength of the

substance when being stress and strained (via pulling to sample failure). The chemomechanical

tester was custom-built by Columbia Laboratories, Inc. (now Juniper Pharmaceuticals, Inc.

Boston, MA, USA). The electrical system operating the connection between the loading cell and

the paper data recorder was produced by Minarik Automation and Control (Minarik Cleveland,

Macedonia, OH, USA). The data recorder utilized (producing paper-outputted data), which was

the same for friction analysis in this study, was a Linseis L250E model. This tensile testing was

performed to observe the mechanical effects of irradiation on PC/OM, as well as soaking in

DW/HA. The CMT had 3 major components: the controlling unit, amplifier, and an analog data

recorder. Rotations per Minute (RPM) of a helical-threaded rod being retracted (and therefore

pulling the attached specimen upwards, elongating it from its fixed bottom end) was calculated

by measuring the mm/min it was moving upwards, and then dividing by a predetermined

calibration factor of 0.0436. On the power amplifier, an electronic dial represented the rotations

per minute, and another dial provided fine adjustment. It was important to note that, whenever

loading a sample into the brace (Figure 22), the extension/retraction of the pre-set helical rod be

stopped so that the sample could be “loaded” into the clamps while giving the material some

slack. This way, the sample was not strained/stressed before the testing even began (which would

otherwise flaw the determined mechanical properties of the material). Initial strain was logged at

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the instant when stress was first recorded to continue stretching the sample (Figure 23). This

corresponded to when the fastened sample became taut, but before stretching occurred.

Calibration occurred by placing weights in the upper clamp of the CMT and marking on

the recorder with the pen where a corresponding line occurred as a result of that specific weight

pulling the spring-loaded non-fixed upper clamp of the CMT downwards. Calibration began by

using a 1 kilogram weight, then working down to 500, 300, 200, and 100 grams. When a very

low ultimate strength was expected, calibration below 100 grams was necessary to guarantee an

accurate scale for when the material failed. Re-calibration occurred after each 5th sample tested.

Figure 22 - Fastening Devices Used for Tensile Testing.

60-Grit sandpaper was used on all sides of clamping devices, of which included a steel

clamp and a large paper clip. Sand paper was changed every-other sample because of

imminent water damage (but before the water damage became apparent)

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Figure 23 - Experimental Set-up of Measuring Initial Strain

OM and PC samples were further cut in a DW-filled dissection tray after first being

divided into rectangular strips (Figure 24 and Figure 25). The rectangles were “notched”

transversely from each lateral side of the samples, leaving a strip in the center remaining

connected, and measuring 1-2 mm in width. Although there was access to lab steel cutting jigs

that enabled a contoured approach to a 1 mm-wide dog bone section (instead of 2 notches being

made transversely on the tissue), the “notched” tissue strip technique was utilized to

accommodate the slippery, tough, and pliable tissues such as PC and OM. It was difficult, even

with a fresh scalpel blade, to cut the tissue strips with two slots. It is hoped that by keeping the

cutting technique consistent, that the results have validity in comparison other tissues tested,

while not necessarily possessing “true” ultimate strengths (traditional dog bone shapes could

have produced higher data values, possibly). Dog bone segmented tensile testing is advantageous

because the gradual contours of the shape (lack of sharp edges) diminish stress/strain

condensation that would otherwise occur at a sharp edge (and therefore cause premature failure).

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Notch tensile testing has been used in past studies, though not for biological tissue, for analysis

of crack susceptibility and fracture in metals [110].

Figure 24 - Notched OM Segments in DW Bath

Figure 25 - Notched Pericardium Tissue Bathing in DW

**All samples were cut with notches and made ready for tensile testing. The notches most visible are denoted by the s above

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It should be noted that the bottom clamp remained stationary, while the top clamp was

retracted (pulled upwards) at a constant rate to stress and strain the sample being tested (as

shown in Figure 23).

All PC samples, although quasi-isotropic, were shown to be slightly stronger when pulled

in the axial direction [57]. Therefore, this orientation was chosen. For OM, strength has been

claimed to be isotropic [25], and therefore strips were cut in any orientation possible from the

harvested “boomerang” shaped OM. The samples included in the final data inclusion criteria

were all “clean breaking” samples, which broke at the slim “dog bone” section each tissue

sample and that exhibited little to no visible slippage in the fasteners (or the corresponding

indications of slippage on the data recorder).

The speed at which the helical rod (and therefore the upper clamp) was pulled upwards

and away from the lower clamp was important to be kept constant for all samples tested.

Variability of samples occurs with varying speeds of elongation. American Standard Test

Methods (ASTM) on the Standard Test Method for Tensile Properties of Plastics [111] was

attempted to be followed at all points possible. Figure 26 is a Stress (σ)/Strain (ε) curve (Young’s

modulus being stress/strain in the elastic region) used for tensile testing of the OM and PC.

Figure 26 - Modulus of Elasticity Depiction [127]

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The primary aim of these curves was to visually assess plastic and elastic deformation as

stress exerted on the material increased. Stress was almost strictly elastic in nature. Ultimate

strength and failure points were important to note for analyzing each material’s ultimate strength.

The failure of a sample was always a tearing or shredding process, but in spite of this

visual observation - the stress-strain curves showed sudden, precipitous drops in stress at the

instant of fracture [57]. The location of the fractures occurred at the slender dog bone segment,

showing little-to-no tendency to fracture at the grips. This sudden, near-perfect elastic

deformation (with no apparent plasticity) was observed for both OM and PC, and is depicted in

Figure 27 and Appendix 9.6.

Figure 27 - Stress (X-Axis) vs. Strain (Y-Axis) Curve for Oral Mucosa (As Depicted by Data Recorder)

ε

σ

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Figure 27 is not directly indicative of stress level (MPa). Rather, the horizontal distance of

the pen marking is used (with no correlation to the numerical values shown in the figure),

through a calibration conversion (with known-weight pen displacement), to determine the

corresponding stress level at each particular failure point. Strain was measured and then

“converted” in a similar fashion (but in the vertical axis). The labels of “HA 4” and “HA 3” are

names given to OM samples soaked in HA. This conversion process took into account that the

recorder paper moved at a constant rate of 0.2 mm/sec, while the loading cell moved the upper

fixed portion of the tissue sample upwards at a constant rate of 0.4166 mm/sec. Therefore, the

strain recorded by the pen on the data recorder paper was multiplied by 2.083 to determine the

corresponding displacement of the tissue itself. Strain was then calculated using both original

length (at the instant of registered stress on the sample) and displacement during the pulling

process.

The scraps left over from cutting the samples of pericardium (PC) and OM were stored in

either 10% formalin-solution containers (for OM tissue) or in propylene oxide (for PC tissue) at

37°F. They were stored until returned for a ceremonial cremation by the UB Anatomical Gifts

Program.

Regarding the initial testing’s sample numbers, 19 notched non-irradiated specimens of

human oral mucosa (OM, 12 samples) and bovine pericardium (PC, 7 samples) and 35 notched

irradiated specimens (17 OM, 18 PC) were each soaked in distilled water (DW) or HA and

thenceforth also friction tested.

It was imperative, especially for PC, to make cross-sectional areas as small as possible to

ensure a clean breakage, ensuring an accurate ultimate tensile strength. PC samples had

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approximately a 0.5 mm thickness. Because of PC’s strength, it was essential that the width of

the sample (after 2 transversely-cut notches made) be less than 1mm width (at the break region)

to prevent fastener slippage and to ensure clean breaks.

3.5.2 Tensile Testing Crossover Study A crossover study was conducted with both PC and OM, using the aforementioned

ChemoMechanical tensile testing protocol, to observe any additional penetration effects of DW

or OM with the other liquid already saturating the tissue. This was accomplished by allowing the

OM/PC samples to soak in either DW or HA for 24-48 hours. Then, the samples were transferred

to soaking in the other solution. Tensile testing was then performed to observe the effects of

crossover soaking. It was expected, at the onset of this study, that the elastic moduli (E) would be

somewhere in the middle of the moduli of tissue soaked in one respective solution, instead of

both.

After non-clean-breaking samples were excluded from the final data collection, 4 HA x

DW OM were used (meaning samples were first soaked in HA for 24-48 hours and then

transferred to be soaked in DW for the same time duration thereafter), 2 DW x HA OM, 6 DW x

HA PC, and 6 HA x DW PC. The resultant Young’s Moduli were then evaluated as compared to

the previous tensile testing of pure HA or DW-soaked PC/OM tissue.

3.6.1 STATIC FRICTION TESTING Glutaraldehyde-tanned bovine pericardium (Peri-Guard® Lot # 5744401-899070 and

Lot # 5746763-928687) was obtained from Synovis Surgical Innovations, a division of Synovis

Life Technologies Inc., St. Paul, MN, USA. These samples were delivered in large high-density

polyethylene (HDPE) bottles, with the tissue being completely submersed in a bath of sterile

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propylene oxide. Segments exposing the smoother side of each tissue sample were used as the

flat, lower tissue on the stage of the friction device, while a smaller tissue segment was glued

(Loctite Super Glue, Henkel Corporation, Rocky Hill, CT, USA) to a brass furniture tack as the

upper, rounded tissue on the device “pin.” Unisol® 4 saline solution, supplied by Alcon

Research Inc., was used as a control solution and a diluent in the friction experiments. Freshly-

extracted Oral Mucosa was also used in this study. PC/OM tissues were cut in 4 x 2.5 cm

rectangles in order to have ample space to accommodate the arcing motion of the tissue-tissue

friction test. Thirteen (13) specimens (6 PC & 7 OM) were tested both before and after γ-

irradiation, 7 soaked in DW, 6 soaked in HA.

The reciprocating pin-on-disc device, donated by Spire Corporation (Bedford, MA,

USA), was used to articulate the bovine pericardium specimens. The smaller, rounded test

surface was attached to a vertically-loaded ‘pin’, and the opposing flat surface fixed horizontally

to a ‘disk’ that oscillated through an arc length of 26mm at 30 cycles/min. The load (metal post +

screw + cork + brass tack with tissue) on the pin was a constant normal force of 32 grams.

Friction between the two smooth, serosal surfaces of glutaraldehyde-tanned bovine pericardium

was monitored with a strain gauge and strip chart recorder system (Linseis 250 E Model, Alcon

Laboratories, Fort Worth, TX, USA). Calibration with known weights allowed for the calculation

of friction forces as a function of pen displacement on the strip chart data recorder. Figure 28 and

Figure 29 illustrate the experimental set-up:

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Figure 28 - Data Recorder Strip Chart used for ChemoMechanical Tensile and Friction Testing

Figure 29 - Static Friction Device

The glutaraldehyde-tanned bovine pericardium tissues (glutaraldehyde tanning had been

performed by the manufacturer to preserve and cross-link the PC tissue to increase and maintain

its mechanical strength [22]) and fresh human OM were used to study reduction of friction

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between the tissues when lubricated by PBS (phosphate-buffered saline, mimicking water’s

lubricious effects, and 0.5% Hyaluronic Acid Solution, HA). The PBS used was Batch # 13-

67968 (FID: 121431), made 6/25/13 for a prior laboratory experiment, and was stored, sterile, at

room temperature. The tissues were stored at room temperature in their packaged storage

solutions and rinsed in a DW (distilled water) bath to remove excess storage solution. Samples of

sizes 1 x 2.5 cm for the flat segments and 1 x 1cm for the rounded segments for the

experimentation were cut from a main sheet of tissue. The remaining bovine pericardium was

stored again in its original storage solution at room temperature. The remnant “scraps” of OM

were stored in glass jars filled with 2.5% glutaraldehyde. Concerning the PC tested, the fibrosal

side of the smaller piece of tissue was glued onto a 7/16” diameter hemispherical brass tack with

a drop of Loctite super glue (Henkel Corporation, CT, USA). For the OM samples tested, the fat-

attached side of the OM was used as the bound side for the flat segment, and also for the glued-

to-tack side for the rounded segment. Therefore, a pure OM-OM mucosal interface was created.

This tissue-on-tack set-up, for both tissue types, was then fitted to a cork on the metal post of the

friction device. Three stacked layers of lint-free Texwipe TechniCloth® TX® 609 (Illinois Tool

Works, Mahwah, NJ, USA), composed of hydroentangled 45% polyester, 55% cellulose, were

cut from 9” x 9” sheets and wetted with distilled water and placed on the horizontal reciprocating

disk. The purpose of the wet TechniCloth pieces was to maintain tissue hydration and provide

some mechanical “cushioning” to mimic oral mucosa. The TechniCloths are free from chemical

binders, have high sorbency and strength, possess low solvent extractable levels, exhibit both wet

and dry strength, and are “clean room” packaged. These sheets were placed on top of a Parafilm-

lined (Bemis NA, Neenah, WI, USA), painter’s tape-sealed (3M, St. Paul, MN, USA) piece of

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“water-proofed” cardboard that acted as a stability platform (tissue was pinned to the cardboard

Ball Point Pins, Singer, La Vergne, TN, USA) for the larger “base” tissue sample (Figure 30).

Figure 30 - Parafilm-Sealed Cardboard Friction Testing Stage

The larger piece of tissue was then placed on the lint-free cloth with the serosal side exposed (for

PC samples). Stainless steel pins were inserted around the perimeter of the tissue, puncturing

through the TechniCloth® layers and into the underlying Parafilm-sealed cardboard to ensure

tissue stability while testing (Figure 31). The vertical tissue-coated pin was then lowered to allow

contact along the path of movement of the serosal sides of the mating tissues.

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Figure 31 - OM Pinned Tissue to Parafilm-Sealed Cardboard Stage

Each experiment began with the tissues being friction tested in their original states. This

was achieved by having both the pinned and base tissue samples being bathed in DW just up

until transfer to the friction device. Before placement on the friction tester, the tissues were

dabbed lightly with a TechniCloth® to remove excess moisture obtained from tissue bathing.

Subsequent additions of 50 μl Unisol4 were made when the tissue became apparently

dehydrated, causing a swift increase in friction). If lubricating fluid began to “pool” on the tissue,

the tissue was lightly blotted with TechniCloth® to remove the excess moisture. Each trial

period lasted over 25 minutes; some trials lasted for hours. For the pin-on-disk friction device

set-up used in those experiments it was found by calibration with the standard weights that

Before September 18, 2014: Static Coefficient of Friction (CoF) = ((Pen displacement

(mm)/2) x (0.7824 gm/mm))/32 gm

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After September 18, 2014: Static Coefficient of Friction (CoF) = ((Pen displacement

(mm)/2) x (0.7471 gm/mm))/32 gm

Following completion, the elapsed time to each Coefficient of Friction (CoF) reading was

calculated by measuring the length of the output on the known-speed chart paper (0.2 mm/sec)

which was set on the chart recorder. Based on this, for every 1 min, the output length on the

paper was 12mm. CoF was calculated at regular time intervals and the CoF vs. elapsed Time

graphs were plotted on Microsoft Excel 2013.


Hyaluronic Acid/Phosphate-Buffered Saline Application as a Lubricant

Three (3) samples of freshly obtained OM were cut into segments as required for static

friction testing, as per the modified Meyer et al. protocol [101] as described previously. The

samples were then tested as-is with intermittent 50 μl PBS application. Before being tested, each

sample was lightly dabbed with a TechniCloth®. HA (50 μl) was only applied to one sample, as

HA’s effects were already well-documented in previous friction studies. The total testing time

before irradiation, per sample, was approximately 45 minutes. This auxiliary study aimed to

observe the magnitude of HA’s effects, as compared to PBS application, by being intermittently

topically applied to post-irradiated OM (rather than fully-submerging OM in DW or HA, as

performed in previous experiments). The tissues were fully submerged in DW after initial testing

until they were again friction tested post-irradiation. This post-irradiation testing’s duration was

approximately 75 minutes per sample.

After 70 Gy of irradiation, new CoFs were determined for each sample, with application

of both PBS and HA in 50 μl increments.

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This experiment set out to determine if HA addition after irradiation still had a good

lubricating effect, while also confirming that irradiation itself modified CoF for OM.

3.7.1 WEIGHT TESTING The purpose of these experiments was to determine any HA retention in each sample and

how it compared to DW uptake. If more HA would be absorbed by the tissue, then a possible

implication would be that HA better embedded itself into the tissue than DW (upon soaking).

Baking of the tissue was performed to determine how much of the remnant of the embedded

liquid was permanently left behind within the tissue. Twenty-two (22) pieces of freshly-

harvested OM and reference glutaraldehyde-tanned propylene oxide-stored PC (12 OM and 10

PC) were cut into strips in a DW-filled dissection tray and subsequently weighed after light

dabbing with a clean TechniCloth®. This recording was deemed the “wet” weight after

measurement on a Mettler AE 100 weight balance (Mettler-Toledo, Greifensee, Switzerland).

The samples were then soaked in DW/HA for a 24-48 hours and weighed again (after gentle

dabbing of TechniCloth®. After baking to complete dryness in 105°C (at least 72 hours), the

samples were then weighed again. After zeroing the balance between each sample, each sample

was either measured on weighing paper or on square polypropylene anti-static weighing dishes.

3.7.2 Weight Testing Crossover Study of Previously Soaked/Baked Oral Mucosa/Pericardium OM/PC soaked and baked samples, which were used in the aforementioned weight

testing, were left to dry (after they were baked) for three and a half months. These samples were

then measured again for post-storage weights. Then, the samples were left to rehydrate in DW or

HA solution. There were 21 samples used (9 PC and 12 OM). The groups used were: 3 OM HA-

DW (where the HA, in this case, was the initial soaking solution before initial baking and

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subsequent storage. DW served as the rehydrating solution in this instance), 3 OM HA-HA, 3

OM DW-DW, and 3 OM DW-HA. These groups for the PC samples were 2 PC HA-DW, 2 PC

HA-HA, 3 PC DW-DW, and 2 PC DW-HA. These samples were left to rehydrate for three days.

Their post-rehydrated weight was then recorded after light dabbing with a TechniCloth®. The

samples were then baked, for a second time at 105°C for three days. At the conclusion of this,

the samples were then weighed again.

3.8.1 VOLUME TESTING Volumetric research changes were also performed simultaneously to weight

measurements (and therefore the sample size for each respective group was the same).

Dimensional analysis comparing HA to DW-uptake was done to correlate between the levels of

liquid uptake that PC and OM had done upon soaking. After baking, the samples were again

analyzed for permanent dimensional changes (even after soaking liquid evaporation).

Concerning PC, the average published thickness was 0.36 (𝜎 = 0.03)mm for adult bovine

pericardium [112]. What was measured for the samples used in our study generally aligned with

this data, but 0.5 mm was the average PC thickness. Manufacturer specifications could have

contributed to this 0.14 mm thickness difference. The samples, after weighing for each step (as-

is, soak, bake) (after light cloth dabbing), were then dimensionally analyzed with a goniometer

(Figure 32).

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Figure 32 - Goniometer Set-up

The left side of the instrument is the eyepiece and magnification instrument used to

visually observe the sample under magnification. The center apparatus is the stage. The knobs

beneath it can adjust the stage up, down, left, right, and forward, and backward (forward and

backward movement occurs via the track in which the stage apparatus sits), all in reference to the

eyepiece. The instrument on the right is the light, which helps to create a silhouette of the sample

on the material, aiding in moving the crosshairs (which are adjusted adjacent to the eyepiece) to

measure the sample. The knobs operating the sample’s movement left to right were shown to

have 1 complete knob rotation = one millimeter (mm). The knobs were notched and therefore

were accurate to 0.01 mm. The other knob (beneath the stage) was utilized to measure vertical

thickness. The tissues were placed on a piece of DW-moistened TechniCloth® on the stage. The

tissues were positioned for dimensional analysis by using DW-moistened stainless steel forceps

(in order to prevent the tissue from sticking to the forceps and therefore get displaced out of

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proper position). When measuring OM, some minor pieces of fatty tissue were still connected to

the mucosa. A diagram of the tissue position was recorded so that sample orientation could be

consistent for dimensional analysis after soaking and after baking in a 105°C oven. When

undergoing baking, the OM/PC samples were placed between 2 glass cover slips (VWR

Scientific micro cover glasses, 1 oz., 24 x 50 mm, Univar USA, Tonawanda, NY) and taped

tightly together (polyacrylic Scotch® Tape, 3M, Minneapolis, MN, USA) to ensure uniform

drying and to prohibit curling of the tissue. This technique was proven relatively successful,

although some samples of PC tended to become wavy (like a fried piece of bacon) if the sample

was not “clamped” tightly enough.

The tissues were not perfect rectangles and therefore had heterogeneities in length and

width when moving across the sample. This was especially evident after baking. Additionally,

slight errors in tissue positioning relative to the original measurements taken (as-is) were evident

for post-soak and post-bake data analysis.

3.9.1 HYP ASSAY TESTING TO ANALYZE TISSUE COLLAGEN LEVELS PILOTS HYP analysis was performed in order to determine the level of chain scission and cross

linking in the OM and PC samples as a result of irradiation treatment. With literature [22] and

this study’s (through tensile testing) showing that collagen chain cross-linking is more significant

than chain scission, HYP assay testing set-out to confirm this. Additionally, oral mucosa HYP

concentration was also measured (which, through literature research had never been done

before).

The HYP Assay testing can be summed-up in three steps: hydrolysis, oxidation, and

development of a chromophore (coloring agent, in this case: pink). At the conclusion of these

steps, two curves were developed relating the intensity of the pink color of the samples with the

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amount of hydroxyproline in the sample. One curve consisted of known standard concentrations

of HYP, 0-65 ppm (parts per million). Using this known-value curve, corresponding

%Transmittance (%T) values of the tissue samples, with previously-unknown HYP

concentrations could therefore be determined by utilizing this standard curve.

It should be noted that Bovine Achilles tendon was used as a “control” in our experiment

because it is around 86% collagen (and therefore 10.8% HYP) [61], and represents the highest

concentration of HYP of any other protein (another protein in humans that contains much HYP is

elastin).

The particular procedure to determine HYP levels was based on the reaction of Ehrlich’s

reagent with the HYP oxidation process. The chemical mechanism of this reaction can be

elaborated as follows [60]: The hydroxyproline structure possesses a pyrrolidine

ring, which can, via oxidative dehydrogenation, transform into to a pyrrole ring, which can be

subsequently determined using a reaction with Ehrlich’s reagent, The resulting quinoid

compound is deeply colored (the color, ranging from orange to lilac, depends on

substituents).

Chloramine-T (N-chloro-4-toluenesulfonamide, sodium salt) was used as an oxidizing

agent. The advantages of this oxidizing agent are that it is inexpensive, has an ease of

decomposing Chloramine T’s excess, and the absence of colored reduction products, which

could otherwise skew the results.

The oxidation reaction was executed in a buffer solution with pH 6. Due to Ehrlich’s

reagent (Solution C) being water-insoluble at that given pH, n-propanol, which is miscible with

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water, was added. Because p-dimethylaminobenzaldehyde (p-daba) can be oxidized by

chloramine, an excess of chloramine was dissolved by the addition of n-propanol. The separation

of the decomposition of an excess of the oxidizing agent (Chloramine T) as an individual stage

of the process before the addition of Ehrlich’s reagent possessed advantages over the

simultaneous addition of Ehrlich’s reagent and the acid. This was because Ehrlich’s reagent

could partially oxidize in the latter instance [60].

Proteoglycans, which are often associated with collagen in tissues, do not interfere with

the analysis because they begin a reaction with Ehrlich’s reagent only after pretreatment with

acetylacetone, which was not used in this particular protocol (Appendix 9.3). An overview of the

HYP Assay Pilot Study Protocol involved these steps: (1) Digestion of samples in HCl while

heated (Figure 34) after complete sample baking (Figure 33),

Figure 33 - Pilot Study, Post-Bake for OM/PC Samples

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Figure 34 - Pilot OM/PC Samples After Digested in 6M HCl

(2) Addition of a color reagent that reacts with HYP in the samples/standards, (3) Dilution of the

samples and addition of a reagent, (4) Insertion and Reading %T/Absorbance in a Visible

Spectrometer (5), Comparing Results to a Curve created from Standard Samples (set amounts of

ppm of pure collagen).

A few quick notes for the explanation of some of the reactives used in the protocol are

these: Solution A is required to be mixed with Chloramine T (acts as an oxidizing agent [60]).

The purpose of Solution A is to neutralize any remnant of the HCl that was involved in the

hydrolysis step at the beginning of the test [61]. Solution C involves P-DABA, which is the

chromophore (coloring agent). P-DABA is light-sensitive, and requires dark storage in order to

remain stable for up to two months [61]. Concerning the acid-hydrolysis step, biological tissues

(such as OM and PC) contain high quantities of GAGs (primarily, chondroitin sulfate), which are

un-degradable even under severe acid hydrolysis environments. This causes them to be “left

89

behind” after acid hydrolysis; therefore the remnant is able to be examined in the HYP Assay

Test. This HYP Assay Methodology involves preliminary chromatographic separation of AAs,

which enables the subsequent spectrophotometric detection of HYP, and gives the most accurate

results of any HYP detection technique [60]. The success of spectrophotometric determination of

HYP in tissues containing GAGs depends on the completeness of GAG hydrolysis.

A list of the required reactives and equipment needed, along with their locations in the

UB SDM in order to carry out this protocol for HYP Analysis, is located in Appendix 9.3. The

majority of the supplies used are shown in Figure 35.

Figure 35 - HYP Assay Supplies

3.9.2 Altered HYP Assay Testing (Removal of the HCl Hydrolysis and Subsequent Baking Steps From Pilot Protocol)

The purpose of this alteration in HYP protocol was to determine if HA could alter

the amount of HYP able to be “sensed” at the conclusion of the test. When taking into account

that both irradiated and non-irradiated samples were tested, it could be gleaned that HA could

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possibly protect a patient from irradiation’s effects if it is existent in the mouth at the time of

head/neck radiotherapy exposure as well as the possibility of injecting HA into the salivary

glands to try and preserve them. The protocol for actual HYP analysis (excluding pilot studies)

differed from that of the Pilots in the following ways: The Pilot studies involved an initial acid

hydrolysis and subsequent baking/drying step. These steps, as well as the remainder of its

protocol can be found in the Appendix 9.3. The actual HYP analysis bypassed these steps and

instead involved the pulping of HA/DW-soaked irradiated/non-irradiated OM/PC samples (12

samples for each tissue type), as shown in Figure 36 and Figure 37. The soaked weight, as well as

the post-squeezed weight of the samples were recorded, to garner the weight of the extracted

pulp, which was used for HYP analysis. All of the pulped tissue remnants were then baked to

dryness and weighed. These weights were recorded by weighing the fluid-filled vial with the

tissue in it (The HYP content, especially in the irradiated tissue, would primarily exist in the

fluid, rather than still in the tissue). Then, an empty, unused vial was weighed and was subtracted

from the fluid/tissue-filled vial weight. The pulped tissue remnant was then analyzed for its post-

squeezed weight. Lastly, the pulped juice left over on the glass funnel used for pulping into the

vial was then combined with the vial juice (with gentle spraying of DW).

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Figure 36 - Funnel Dried with TechniCloth, Cleansed of Juice with Air Hose. Rinsed with DW

Figure 37 - OM HA IRR 1 Note the pink, cloudy OM pulp

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As evidenced in Figure 37, the irradiated specimens seemed to be able to be much more

easily pulped than their non-irradiated counterparts (this was especially apparent for OM

samples).

HA or DW left over from soaking will be included in these weights, but are insignificant

(even though they would comprise the majority of the filled-vial weight), because all non-

proteins will not be sensed in the HYP assay. From this point, the vial juice and pulp was

evaporated to dryness in an exhaust hood to yield the broken-down AAs (Step 4 in the modified

“actual” HYP protocol), and then proceeded with the protocol (detailed in Appendix 9.3) from

this step onward.

It should be noted that because this particular experimentation was at the conclusion of

the study, only a single trial was performed. When the samples were evaporated to dryness,

nearly all of the PC samples were near complete evaporation. This was due to inadequate pulping

of the PC samples. Because the tissue was so tough, it was extremely difficult for one to “pulp”

the tissue to harvest any collagen-rich “juice.” Therefore, there was no “standard” tissue to

compare the OM results to. A perfected and more uniform method to extract the “juice” from the

samples should be investigated in future studies. A use of a vise might be one method (instead of

pulping by hand).

3.10.1 HISTOLOGY AND LIGHT MICROSCOPY OF ORAL MUCOSA/PERICARDIUM SAMPLES, HYALURONIC ACID/DISTILLED WATER-SOAKED Visual confirmation of HA’s epithelial protective effects of PC/OM tissue was the motive

for this portion of the study. PC/OM tissue, left over from Static Friction Testing, was stored in

2.5% Glutaraldehyde from October 2014, to April 2015. Afterwards, samples were cut to only

encompass the arc of the sample’s wear tracking. The wear tracking was especially evident for

the PC and DW-soaked samples. There were 4 tissue specimens used: PC DW, PC HA, OM HA,

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and OM DW. Each tissue was then taken to the UB Histology Lab. The samples were placed in

wax and afterwards were cut, in cross-sections, in 5 micrometer increments. These slices were

then stained and placed on glass microscope slides, in order to be examined. Half of the samples

were stained via H & E (Hematoxylin and Eosin) Staining, yielding a pink-red color for the

tissue. The other half of the specimens was Gomori Trichrome Stained. The latter staining

method is a “one-step trichrome staining procedure that combines the plasma stain (chromotrope

2R) and connective fiber stain (fast green FCF) in a phosphotungstic acid solution to which

glacial acetic acid has been added [113].” The colors and corresponding tissue components are

listed in the following: nuclei: red-purple, normal muscle myofibrils: green-blue with distinct A

and I bands, intermyofibrillar muscle membranes and cytoplasm: red, and interstitial collagen:

green [113].

After staining, the samples were imaged, using a light microscope, at 63 x and 250 x

magnification. Digital images were taken of desired areas of the sample slides.

3.11.1 STATISTICAL EVALUATION OF DATA Statistical significance was established on a 95% confidence (p<0.05) for each portion of

data analyzed. For weighing analysis, a 2-way ANOVA test was used, comparing DW/HA and

OM/PC. For the Tensile Data, a 3-way ANOVA system was utilized, comparing the

aforementioned two groups, with the addition of irradiation/non-irradiation tissue comparison.

For the Cross-over Tensile Data, a 2-way ANOVA method was utilized (HA/DW and PC/OM).

Then, a Tukey HSD Multiple Comparison step was used in order to evaluate the order of the

soaking trials that the tissues underwent. Friction testing was not statistically evaluated due to the

clearly apparent differences and extremely small standard deviations throughout

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experimentation. Finally, HYP testing was evaluated using 2-way ANOVA (Irradiated vs Non-

irradiated and OM vs PC).

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4.0 RESULTS

4.1.1 CHEMOMECHANICAL TENSILE TESTING Figure 38-Figure 45 can be summarized by a few gleanings: HA-soaked specimens are

shown to have lower the Elastic Modulus (E, stress/strain) of the samples, as compared to DW.

Additionally, irradiation is shown to increase the E. this was accomplished primarily by

“embrittling” the tissue through cross-linking. Finally, PC was shown to have a much higher E

than OM.

In a past research study, the UTS (Ultimate Tensile Strength) of 19.1 (𝜎 = 2.2) MPa was

measured for adult glutaraldehyde-fixed PC [112]. In even earlier studies however, for unfixed

bovine pericardium, a UTS range of 25–29MPa [114, 115] was yielded and implied that the

fixed PC UTS would be even higher. The former forecast aligns with Figure 38’s data from the

present investigation. Although having the non-irradiated PC samples soak in water before

testing (had been previously glutaraldehyde-treated) could have slightly compromised the PC’s

strength, the UTS of 22.474 MPa still is in alignment with the literature sources.

Citing Figure 38: Grossly, non-irradiated PC and OM tissue had an average strain of 0.825

+/- 0.311 at failure and an average ultimate strength of 9.858 +/- 1.528 MPa. Irradiated PC and

OM tissue had an average strain of 0.485 +/- 0.186 at failure and an average ultimate strength of

13.597 +/- 4.480 MPa. The individual tissue averages are as follows: PC had an average strain of

0.726 +/- 0.124 and average ultimate strength of 21.818 +/- 6.398 MPa. OM had an average

strain of 0.586 +/- 0.326 and average ultimate strength of only 1.636 +/- 0.828 MPa. Thus, it is

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clear that the PC reference material was, biomechanically, proportionately stronger than the OM

tissue in both irradiated and normal (non-irradiated) conditions.

Figure 38-Figure 45 are expounded in the following:

As shown in Figure 38, these results were found:

Although not statistically significant (p=0.2574), irradiated tissue was sometimes seen to

gain strength by about 40%, and statistically significantly decreasing in strain to failure

by approximately 50% (became more brittle) (p=0.0160). These phenomena could only

be explained if collagen chain cross-linking outweighed that of simultaneous chain

scission when the specimen was irradiated. As previously noted, this has been well

documented by Inoue, et. al [22].

When comparing the tissues tested, PC and OM, the differences were mechanically

reflective of their relative collagen content. As previously noted, collagen is primarily

responsible for the mechanical strength and stability of a biological tissue. As seen in

Figure 38, PC had about 11x the ultimate strength of OM (p<0.001). PC was also shown

to be able to stretch about 25% farther than OM at failure, although this disparity was not

significant (p=0.2012).

Although the difference was not statistically significant (p=0.1818), HA-soaking of the

tissues (and not DW-soaking) showed to increase the tissues’ ability to stretch until

failure by approximately 30%. HA-soaked samples also insignificantly (p=0.4438)

lowered the tissues’ ultimate strength, a softening effect, by roughly 50%. A confirmation

of HA’s softening of tissue was therefore apparent from this study by noting the strain at

failure increasing simultaneously with a lessening of the tissues’ UTS.

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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

As denoted in Figure 42 and Figure 43, HA is seen to display more prominent effects when

comparing tissues soaked in it pre/post-irradiation, as compared to DW-soaked samples. While

HA was able to cause the samples to have a much higher strain at failure than DW-soaked

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samples pre-irradiation, HA’s mechanical effects were diminished dramatically post-irradiation.

Although HA and DW-soaked specimens shared similar post-irradiation strains at failure, HA-

soaked samples were able to possess a lower UTS (although not statistically significant).

Therefore, the Elastic Modulus was lower than for HA-soaked post-irradiation than DW-soaked

post-irradiation. Again, these differences were severely diminished as a result of the cross-

linking abilities of irradiation, therefore diminishing the ability for the irradiated tissue to retain

the large molecule of HA (and therefore its corresponding effects).

Citing Figure 44 and Figure 45, OM’s elastic modulus increased nearly solely due to a ≈35%

increase in UTS from irradiation’s “embrittlement.” This difference was not statistically

significant (p=0.4169). A change in strain at failure, stemmed from irradiation, was not observed

in OM samples (p=0.7224). Due to PC’s more collagenous composition, more drastic effects

were seen resulting from irradiation treatment. PC was seen to increase in UTS by ≈40% (not

statistically significant, p=0.5376), while a drastic decrease in strain at failure by ≈60% as an

outcome of irradiation (p<0.001).

Generally, the standard deviations of PC’s data were less than OM’s information. This was

because PC was more rigid tissue, possessed a near-homogeneous thickness (and tissue

composition) for each respective sample, and was much easier to work with (ease of cutting,

fastening, and positioning in CMT device).

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Figure 39 - Elastic Moduli of DW and HA-Soaked Samples of OM and PC, NonIrradiated vs. Irradiated Samples, n= 35 Irr, 19 NonIrr

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

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Figure 41 - Elastic Moduli of Irrad. and Non-Irrad Samples of OM and PC, HA vs. DW-Soaked Samples, n= 26 DW, 25 HA

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

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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

Figure 44 - Elastic Moduli of HA and DW-Soaked Samples of OM, Irr vs. NonIrr Samples, n= 17 Irr, 12 NonIrr

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Figure 45 - Elastic Moduli of HA and DW-Soaked Samples of PC, Irr vs. NonIrr Samples, n= 18 Irr, 12 NonIrr

4.1.2 Tensile Testing Crossover Study Citing Figure 46, for UTS, there was a significant tissue effect (p<0.001), and group effect

between HA and DW-soaked samples (p=0.0190). After multiple statistical comparison, both

HA x HA vs DW x HA and HA x HA vs. HA x DW had significant differences (p=0.0012 and

p=0.0010) in both OM and PC.

Additionally, for strain, there was a significant difference between HA and DW-soaked

tissues (p<0.001), but no difference between tissues (p=0.9889). After multiple statistical

comparison, HA x HA vs. DW x HA and HA x HA vs HA x DW had significant differences

(p=0.0012 and 0.0010 respectively).

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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

4.2.1 STATIC FRICTION TESTING

4.2.2 Static Friction Testing Oral Mucosa Figure 47-Figure 50 depict the static friction testing results for OM in the main study.

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Figure 47 - OM DW vs OM HA, PreIrrad, n= 4 DW, 3 HA

Figure 48 - OM DW vs OM HA, PostIrrad, n= 4 DW, 3 HA

105

Figure 49 - PreIrrad vs. PostIrrad, OM HA, n=3

Figure 50 - PreIrrad vs. PostIrrad, OM DW, n=4

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Figure 47-Figure 50 show that OM HA had about half the μ (friction coefficient) as OM DW

before irradiation (.35 to .7). After irradiation, OM HA and DW CoFs merged to ≈ 0.45.

4.2.3 Static Friction Testing Bovine Pericardium Figure 51-Figure 54 depict the static friction testing results for PC in the main study.

Figure 51 - PC DW vs PC HA, PreIrrad, n= 3 DW, 3 HA

Figure 52 - PC DW vs PC HA, PostIrrad, n= 3 DW, 3 HA

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Figure 53 - PC DW, PreIrrad vs PostIrrad, n=3

Figure 54 - PC HA, PreIrrad vs PostIrrad, n=3

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Figure 51-Figure 54 reveal that PC HA had about half the μ (friction coefficient) as PC DW before

irradiation (.45 to .9). After irradiation, PC HA (.5) and DW (1) CoFs did not nearly see the same

effect with μ change as with OM, as they remained generally constant.


Hyaluronic Acid/Phosphate-Buffered Saline Application as a Lubricant As depicted in Figure 55, immediate lubrication relief was observed, both before and

after irradiation treatment of 70 Gy. However, as differing from other present salivary substitutes

[8], persistent lubrication is also seen for HA. A possible mechanism for this action could be that

the long, straight molecules of HA can embed themselves between the long, straight fibers of

collagen. Penetration through the epithelium into the collagenous Lamina Propria can be

observed, for example, in Figure 90 and Figure 94.

Figure 55 - OM Pre/Post Irradiation, HA/PBS Application, n=3

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4.3.1 WEIGHT TESTING

Figure 56-Figure 57 are given below and depict a comparison of tissue weights initially,

after HA/DW soaking, after baking, and after Dry Room storage. These data were statistically

evaluated. When observing initial weights, there was only a significant difference between OM

and PC (p<0.001), but no significant difference for HA/DW soaking groups (p=0.6819). This

was surprising because of the high molecular weight of HA, it was expected that HA-soaked

samples would weigh significantly more than DW-soaked samples. This trend of only having

PC/OM have significant weight disparities (and not DW/HA soaking) continued after soaking

and baking as well.

A point to be made regarding the weighing measurements not being statistically

significant is that the MW of HA used was 1.37 * 109 Da (Daltons). With one Da = 1.66*10-21 g,

one molecule of HA is equal to 2.27*10-12 g. The Mettler AE 100 is accurate to the thousandth of

a gram. Therefore, there would require 4.40*108 HA molecules in a sample in order to be sensed

by the weight balance. Therefore, for future work, a balance capable of weighing much more

miniscule weights would be recommended!

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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

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

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4.3.2 Weight Testing Crossover Study of Previously Soaked/Baked Oral Mucosa/Pericardium Figure 58-Figure 61 depict the weighing data for samples soaked again after Dry Room

storage.

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

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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

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Figure 60 - X-Study Weight Replenishment/2nd Bake, n= 6 OM DW, 6 OM HA, 5 PC DW, 4 PC HA

Figure 61 - Change Proportions For Each X-Weight Study Abscissa, n= 6 OM DW, 6 OM HA, 5 PC DW, 4 PC HA

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It should be noted that the reason for some of the crossover study data groupings for

weight analyses having 4 groups (OM DW, OM HA, PC DW, PC, HA), and not 8, is because

each category was condensed from 2 others corresponding, for ease of developing results and

conclusions (for example, OM DW-DW and OM DW-HA were averaged to make OM DW

AVG).

Figure 62 is a depiction of a dried OM samples after baking. Each sample originally was

≈2 cm in length or more. Therefore, considerable volumetric shrinkage was observed (confirmed

in Figure 63 and Figure 64).

Figure 62 – OM Baked-To-Complete-Dryness

4.4.1 VOLUME TESTING (Figure 63 and Figure 64)

Figure 63 and Figure 64 are given below and depict a comparison of tissue weights

initially, after HA/DW soaking, after baking, and after Dry Room storage.

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

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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

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SPECTROSCOPY

Figure 65-Figure 76 are the MAIR-IR percent-transmittance (%T) of each of the PC/OM

DW/HA-soaked samples (4 groups). All samples were baked and thus completely dry, a

necessary step to be tested on KRS-5 prisms. These dried tissue samples were saved from the

bulk weighing analysis experiment. Each sample (OM freshly extracted) was weighed initially,

soaked for a uniform period, and then baked to complete dryness. A few conclusions gleaned

from Figure 65-Figure 76 can be made. First, no discernable remnant of HA or DW was found.

Also, OM’s fatty composition, and PC’s proteinaceous make-up were clearly apparent.

Figure 65 - OM DW Post-Bake

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Figure 66 - OM HA Post-Bake

118

Figure 67 - PC DW Post-Bake

119

Figure 68 - PC HA Post-Bake

120

Figure 69 - PC DW, PC HA, OM DW, OM HA Post-Bake (TopBottom)

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Figure 70 - PC HA vs. PC DW Post-Bake (TopBottom)

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Figure 71 - OM DW vs OM HA Post-Bake (TopBottom)

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Figure 72 - OM HA - OM DW Post-Bake SUBTRACTION

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Figure 73 - OM HA - PC HA Post-Bake SUBTRACTION

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Figure 74 - PC HA - PC DW Post-Bake SUBTRACTION

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Figure 75 - OM DW - PC DW Post-Bake SUBTRACTION

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Figure 76 - Hyaluronic Acid MAIR-IR Spectrum

4.6.1 HYP ASSAY TESTING

Standard samples with known HYP concentration were used in the study to assemble a

standard curve to then develop corresponding HYP concentrations to the PC, OM, and BAT

(Bovine Achilles Tendon) samples. 15 samples were tested, in addition to 14 standard HYP

samples, ranging from 0-65 ppm (5 ppm increments). The samples evaluated were 3 OM Irr

(Irradiated), 3 OM NonIrr (Non-Irradiated), 3 PC Irr, 3 PC NonIrr, 2 BAT Irr, and 1 BAT

NonIrr.

As previously cited [58], non-irradiated PC is approximately 67% collagen. Because the

raw data percentages were too high, each of these percentages (and corresponding standard

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deviations) were scaled down to align PC NonIrr being 67% collagen. Human OM collagen

content has not been researched in the past. This study suggests that the OM was ≈ 23% collagen

(Figure 77 and Table 5). The irradiated and non-irradiated versions of these tissues were not

statistically different. However, it was shown that the non-irradiated samples were able to be

broken down more and yield more AAs. This revealed, once again, that irradiation causes

collagen chain crosslinking more so than chain scission.

Figure 77 - Pilot Study Relative Collagen Concentration Graph, n= 3 OM Irr, 3 OM NonIrr, 3 PC Irr, 3 PC NonIrr

AVG % Collagen

StDev

OM Irr 21.66% 1.76%

OM NonIrr

24.42% 2.12%

PC Irr 65.08% 8.55%

PC NonIrr

67.00% 3.78%

Table 5 - Pilot Study Relative Collagen Concentration Chart, n= 3 OM Irr, 3 OM NonIrr, 3 PC Irr, 3 PC NonIrr

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4.7.1 HISTOLOGY AND LIGHT MICROSCOPY OF ORAL MUCOSA/PERICARDIUM SAMPLES, HYALURONIC ACID/DISTILLED WATER-SOAKED (Figure 78-Figure 103) From Figure 78-Figure 103, a conclusion can be made that there are drastic histological

differences in the amount of friction-induced wear/damage of the samples when comparing

PC/OM soaked in DW to HA. Through these images, HA is visually shown to protect against

epithelial damage, which therefore could lead to minimal deep tissue disruption, causing less

pain to the xerostomic patient.

Figure 78 - OM DW 1 L 1 63 x Middle H and E

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Figure 79 - OM DW 1 L 1 63 x Middle TriChrome

Figure 80 - OM DW 1 L 1 250 x Middle H and E Rubbed-Off Epithelium

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Figure 81 - OM DW 1 L 1 250 x Middle H and E Shredded Epithelium

Figure 82 - OM DW 1 L 1 250 x Middle TriChrome

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Figure 83 - OM HA 1 L 1 63 x Middle TriChrome

Figure 84 - OM HA 1 L 1 250 x Middle H and E

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Figure 85 - OM HA 1 L 1 250 x Middle TriChrome

Figure 86 - OM HA 1 L 1 63 x Middle H and E

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Figure 87 - OM DW 1 L 1 400 x LEFT trichrome_basement membrane-collagen junction

Looking at Figure 88 (OM soaked in DW), the entire epithelium of the tissue was worn off

due to friction wear (on the right side of the image), leaving the previously-underlying

collagenous Lamina Propria exposed.

Figure 88 - OM DW 1 L 1 63 x LEFT H and E

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Figure 89 - OM DW 1 L 1 63 x LEFT trichrome

Figure 90 - OM DW 1 L 1 400 x LEFT H and E

136

Figure 91 - OM DW 1 L 1 400 x LEFT trichrome

In Figure 92 (OM soaked in HA), the structures of the OM and underlying tissue are

clearly apparent (and therefore can be applied when viewing the other histological photographs

as well). The epithelial layer of the OM is on the right of the image. It is approximately 6-7 cells

thick and was in direct contact with the rubbing pinned tissue during friction testing. This

explains for the minor wear-induced damage towards the lower-right portion of the epithelium.

Underlying this layer is the collagenous Lamina Propria (dense, more lightly-colored region on

the right of the photograph). Beneath this layer (moving to the left in the image of Figure 92) is a

layer of vacuoles, possibly vacant (with empty space). Most deep in the image (furthest to the

left of Figure 92) is the underlying muscle fibers.

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Figure 92 - OM HA 1 L 1 63 x LEFT H and E

Figure 93 - OM HA 1 L 1 63 x LEFT trichrome

138

Figure 94 - OM HA 1 L 1 400 x LEFT H and E

Figure 95 - OM HA 1 L 1 400 x LEFT trichrome

139

Figure 96 - PC HA 3 L 1 63 x Middle H and E

Figure 97 - PC DW 2 L 1 63 x Middle H and E

140

Figure 98 - PC DW 2 L 1 63 x Middle TriChrome

Figure 99 - PC DW 2 L 1 250 x Middle H and E

141

Figure 100 - PC DW 2 L 1 250 x Middle TriChrome

Figure 101 - PC HA 3 L 1 63 x Middle TriChrome

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Figure 102 - PC HA 3 L 1 250 x Middle H and E

Figure 103 - PC HA 3 L 1 250 x Middle TriChrome

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5.0 DISCUSSION

5.1.1 CHEMOMECHANICAL TENSILE TESTING

Although the PC and OM samples, both irradiated and non-irradiated, exhibited relatively

linear stress-strain curves, a nonlinear increase in stress relative to strain was seen briefly,

initially, for every sample examined. This observation can be explained because of the

realignment of collagen fibers when stretched. According to Tanaka et. al. [116], collagen has a

wavy structure under unstrained conditions. When a viscoelastic specimen is deformed, the water

is squeezed out of the specimen and the orientation of the collagen fibers is rearranged. The

wave pattern of the collagen fibers gradually disappears as the PC is strained and completely

disappears at 1-1.5% strain level. Beyond 2-2.5% strain level, the stress-strain relationship

becomes linear because the collagen fibers become parallel and are oriented in the direction of

the applied load.

Some samples, instead of failing in the middle of the sample as would be expected, failed

right at the bottom clamp. This occurred because of minor heterogenic torsion of the material as

it was being stressed by the clamp, whose edge was not positioned completely perpendicular to

the direction of stress vector. Because of unequal loading of the sample at these fixtures, they

failed before true failure would have occurred at the middle of the sample (in-between the top

and lower clamps. If this mode of failure occurred, another piece of the same material would be

loaded and another test would be conducted. The samples that failed at the “grips” instead of at

the middle of the notched portion of the sample were not included in the experiment’s results.

The most ideal (although not perfect) mode of failure for each sample was used in the final

results and tables for the CMT.

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Regarding the reason for PC strength, the maximum strength of a tissue composed of

collagen would be aligning with direction in which the collagen fibrils are oriented. Thus, when

the collagen is more aligned in the direction in which force is applied, the tissue will have higher

UTS. Strength was found to be due to the sum of the components of the fibrils that lie in the

direction of force in addition to a component due to the other matrix materials [112].

Concerning the nature of the stress-strain curves for both OM and PC, in a previous study

[116], an apparent escalation in the modulus of bovine TMJ disks was observed during strain rate

observation, especially in the lower strain region. This trend was also apparent in our tensile data

for OM and PC. The lower strain region, deemed the “toe region,” correlates with the

reorganization of the wavy pattern of collagen into an aligned fiber pattern while the higher

strain region, called the “the linear region,” relates to the strain of the aligned fibers [112].

5.1.2 Tensile Testing Crossover Study Eighteen (18) total samples of PC and OM (12 PC, 6 OM) were evaluated. After

statistical analysis, it was gleaned that DW x HA vs HA x DW did show significant difference

for UTS (p=0.0157). Therefore, the original soaking solution well-embedded itself into the

PC/OM tissue and inhibited cancellation of the original soaking solutions’ effects when exposed

to the second soaking liquid (when evaluating the tissues’ UTS).

As demonstrated in the bulk tensile testing, it was again demonstrated that OM and PC

did not differ statistically significantly for strain (p=0.9889), but only for stress. Additionally,

HA x HA vs DW x HA and HA x HA vs HA x DW had significant differences (p=0.0012 and

0.0010). These findings suggest that when HA was used as the original soaking solution; it more

permanently embedded itself into the PC/OM tissue than DW (as the original soaking solution).

This conclusion can be drawn because there was no significant disparity in similar groups with

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DW as the original soaking agent. Furthermore, there was no significant difference for DW x HA

vs HA x DW (unlike for UTS evaluation) (p=0.9976). This means that the initial soaking

solution’s strain effects could be reversed when exposed to the other soaking solution.

It was expected that the secant moduli of PC and OM (data taken from bulk tensile study)

would fall in between that of the cross-over tissues. This, however, was not the case. As shown

in Figure 46 and Figure 104 (where strain, ultimate stress, and elastic moduli are all group

averages), the average cross-over values for PC did reveal a difference from the PC HA and PC

DW values derived from the previous bulk PC tensile testing study. An average of PC DW x HA

and PC HA x DW at failure yielded a stress of 25.69 MPa and strain of .305 (an E of 97.032

MPa). Citing the previous bulk study, the expected stress and strain of Average X PC (Average

of PC DW and PC HA) was 1.0625 MPa and 18.347 strain at failure. These values expressed

were different in that the crossed-over specimens had an elastic modulus that was about five

times higher, exhibited about one-third less strain at failure and exhibited about a 40% increase

in UTS. These preliminary results suggest that a possible strengthening and “embrittling” of the

tissue could have been accomplished with the cross-over specimens. A comparison to the

statistical analysis of these groups is imperative to determine what differences are worth noting.

However, this “embrittling” due to solution transfer s is likely not the proper conclusion.

Possible errors in this study could have arisen with the fewer samples tested as compared to the

bulk study. Other possible errors (although only clean-breaking samples were used for data

compilation) include imperfect PC tissue alignment. Also, as this cross-over study was

conducted after the bulk tensile study, experience in the tensile testing procedure and familiarity

with the tensile instruments were higher (naturally) with the cross-over study. Therefore,

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possible operating errors during the crossover study could have been less than in the bulk tensile

study.

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

5.2.1 STATIC FRICTION TESTING As suggested by prior studies [17], HA has been thought to penetrate past the superficial

layers of the OM, to foster greater persistence of lubricity.

HA soaked into OM/PC tissue has been shown to be able to protect collagen fibers

against cross-linking. It also has been shown that there is a low-friction outcome for both

pre/post irradiation samples and that HA can provide lasting positive low-friction characteristics.

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PC, because of its high collagenous make-up, was more rigid in structure and therefore

not able to uptake HA as well as OM. Therefore, PC, when friction tested in a dry environment,

dried much more quickly. For this same reason, OM samples were seen to lose much more

volume when dried/baked when compared to PC. It should be noted that the intermittent addition

of 50 μl of Phosphate Buffered Saline (PBS) acted as a moisturizing agent for the PC/OM

tissues. PBS acted when applied to the surface of the tissue, while being tested, and was

gradually absorbed. PBS then mobilized any prior tissue-soaking medium that had been

embedded in the tissue (DW or HA), therefore bringing its lubricious effects to the surface of the

material. It was observed that when — for some samples — PBS was added before tissue

dryness was complete (in order to preserve consistency of protocol of interval of PBS

application), liquid “pooling” on the tissue surface occurred. This caused plowing to occur with

the pinned tissue sample having to move through this pool of liquid. Therefore, the CoF was

shown to anomalously increase for these better-wetted samples.

Additionally, irradiation effects were able to be noted during testing. The irradiated tissue

was observed to not absorb PBS as well (additional “pooling” of excess liquid on surface, rather

than being absorbed at the rate shown in non-irradiated tissue). This would be explained by

irradiation cross-linking so; the toughness of the tissue increased and became more rigid. Thus,

the collagen fibers were less able to expand to accommodate liquid absorption between them.

Referencing Figure 47, before irradiation, HA was able to halve the CoF (≈ .35) as

compared to DW (about .7) for OM. However, after irradiation, as seen in Figure 48, there was

less disparity between the DW and HA-soaked samples for OM, as the CoFs merged to about

.45. This phenomenon could be explained by crosslinking between collagen fibers occurring

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during irradiation, so that HA’s effects lessened. Due to HA’s large molecular size, “fused”

collagen fibers could prevent entry of some of the HA into the OM tissue.

A drastically similar (relating to OM) effect of halving the CoF to about .45 when

comparing HA to DW-soaked samples, as evidenced in Figure 51, was observed for pre-irradiated

PC. However, as seen in Figure 52, unlike OM, HA was able to lower the CoF by about ½ after

irradiation. This could indicate fiber rigidity variances between OM and PC. PC, being rigid in

structure because of its 67% glutaraldehyde-tanned collagen make-up, is barely further affected

by irradiation treatment because of the high cross-linking it already possesses. However, OM, at

approximately only 23% un-cross-linked collagen (as determined in the HYP Assay Section of

this study), was more affected by irradiation because of its initial lower tissue rigidity. This

reflected the fact that its collagen content was only about 1/3 that of PC.

Saliva and 0.5% HA both act as lubricants and a comparison is made in Figure 105-Figure

106 and Figure 130-Figure 131. As depicted in Figure 105-Figure 106, the PC-PC lubricated friction

interface was decreased to about .1 for both unstimulated saliva, as well as HA. This confirms

that HA mimics saliva and is therefore capable of providing the lubricious properties lost from

the lack of salivary production in oral cancer patients that have undergone

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radiotherapy.

Figure 105 - CoF vs Time plot for unstimulated saliva from a female control [8]

Figure 106 - 0.5% HA in Normal Saline_Three Different PC Tissue Couples [17]

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In Appendix 9.4, a test was performed to determine if HA’s lubricious effects could

mirror those of saliva even after 70 Gy of irradiation. Figure 130 and Figure 131 verify that HA’s

effects are preserved, even after a clinically-used total radiotherapy dosage. In these figures,

tissue properties were kept consistent by the utilization of PC for testing, rather than OM. In

detail, Figure 130 showed that the CoF decreased to ≈0.14 after 64 minutes of testing in dry-room

conditions. Figure 131 revealed that the CoF decreased to ≈.12 after 45 minutes of the procedure.


Hyaluronic Acid/Phosphate-Buffered Saline Application as a Lubricant

As recorded in Figure 55, after irradiation, HA’s lubricious integrity is preserved. This

was demonstrated by HA halving the CoF of irradiated samples. Also, Figure 55 showed that the

increase in CoF, although slight, was significantly demonstrated with the tissue as-is, when PBS

was applied, and when HA was applied. Additionally, HA’s lubricating effects are seen to

provide lasting frictional relief even after irradiation, whereas PBS application resulted in only

≈15 minutes-worth of frictional relief before returning to prior condition.

5.3.1 WEIGHT MEASUREMENTS Referencing Figure 57, HA uptake for PC over a 24-hour span was higher than that of DW

uptake (4.59% +/- 5.63% vs. -3.65% +/- 9.97%) when compared to the tissues’ original weights.

However, for OM, DW uptake was actually greater (11.44% +/- 12.96% vs. 5.88%). High

standard deviations might skew both of these statements’ findings. For the PC samples after

baking, the DW-soaked samples were able to retain their weight better than the HA-soaked

samples (-68.22% +/- 1.28% vs. -76.06 +/- 3.07%) when compared to pre-bake soaked weights.

For the OM sample post-bake, the weight loss was more for DW than HA-soaked samples (-

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91.61% +/- 1.41%, -86.36% +/- 2.69%). Also, an extremely minute amount of weight was lost

during the 3.5 month storage, confirming that the samples were baked to complete dryness.

Citing the same figure, it can be said that OM retained HA better than DW after baking to

dryness. This difference was statistically significant. This therefore implied that about 5% more

HA than DW, of the samples’ soak weight, was permanently integrated into the HA tissue. This

confirms HA’s native tissue invasiveness. PC, on the other hand, retained water better than HA.

Because of the cross-linked rigidity of its fibers, it was less open to accommodate the large HA

molecules.

5.3.2 Weight Testing Crossover Study of Previously Soaked/Baked Oral Mucosa/Pericardium Citing Figure 58, it can be observed that HA was not able to be absorbed as well as DW

upon re-soaking after initial bake, both for OM and PC. This difference was statistically

significant for PC. These facts can be attributed to the fact that baking condensed the collagen

fibers of the tissues during evaporation of water content. Simultaneously, the tissue became more

rigid, with a semi-crystalline structure, Upon re-baking, all tissues, both of OM and PC, returned

to nearly the same weights as were recorded in the original baking step.

5.4.1 VOLUME MEASUREMENTS Citing Figure 64, HA-induced volume increase for OM over a 24-hour span was higher

than that of DW uptake (47.29% +/- 20.69% vs. -22.78% +/- 10.74%) when compared to the

tissues’ original volumes. However, for PC, DW uptake was greater (26.17% +/- 43.62% vs.

12.85%). A high standard deviation might skew the latter statement’s findings however. OM,

with less collagen content, was more able to accommodate the large molecules of HA and

therefore increased in volume more than soaking in DW as a result. Due to the rigidity of PC, it

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was not able to accommodate the larger HA molecules as well as the DW and therefore increased

in volume more for DW.

For the PC samples after baking, the DW-soaked samples were able to retain their

volume better than the HA-soaked samples (-51.55% +/- 7.27% vs. -64.15 +/- 12.10%) when

compared to pre-baked soaked volumes. Whereas for the OM sample post-bake, the volume lost

was about even for both DW and HA-soaked samples (-81.6% +/- 3.35%, -80.02% +/- 6.68%).

These findings suggest that as PC was not able to accommodate HA as well, it lost HA upon

baking just as easily and therefore retained DW better. Although OM was able to gain more

volume with HA than DW, it was able to lose DW more easily than HA and therefore the

volumes were about even after baking.

Citing Figure 107, the tissue irregularities (not rectangular) of both the PC and OM tissues

contributed to the fact that these findings (for volume) are not reliable. A three dimensional

analysis was attempted using a goniometer, but the simple geometry used to estimate the

samples’ volume is simply not reliable enough to be factually substantiated in this study.

Figure 107 - Baked to Dryness PC Samples

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5.5.1 MULTIPLE-ATTENUATED INTERNAL REFLECTION INFRARED (MAIR-IR) SPECTROSCOPY Referencing Figure 69, hydrocarbon peaks were observed for all tissues at ≈2853 cm-1 and

≈2923 cm-1. These peaks were slightly more significant for the OM tissues. This symbolized the

composition disparity between the OM skin-like tissue and PC heart-sack tissue. The next

significant peaks were seen at ≈1742 cm-1 and ≈1160 cm-1. These peaks represent a –COOR

group (ester). These peaks were evident especially in OM tissue, indicating the oils existent in

skin-like tissues. A very subtle ester peak was also seen in PC DW, but probably was indicative

of oils of skin placed on sample during procedural handling by the laboratory user. No peak at

this location was observed for PC HA. Additionally, peaks at ≈1629 cm-1 and ≈1527 cm-1

indicate amide peaks, making an existence of protein in all the tissues. Because PC is more

proteinaceous, these peaks were more apparent for PC tissues (vs. OM). Additional hydrocarbon

peaks were observed for all 4 tissue types at ≈1457 cm-1 and ≈1377 cm-1. Carbon-oxygen and

carbon-nitrogen peaks were existent in all samples at ≈1070 cm-1. These peaks were more

apparent in the OM tissue, again indicating compositional variances from PC tissue.

Regarding Figure 76, the primary HA peaks to be addressed were at ≈3351 cm-1 (N-H

bond), ≈1610 cm-1 (N-H bond), and ≈1020 cm-1 (-OH group). These peak remnants were not

existent for the OM and PC dried tissue, therefore indicating that the soaking and baking process

completely alleviated these two tissues of any HA or DW remnant able to be sensed by the

MAIR-IR Spectrometer (only bulk weighing analysis was able to develop quantifiable

conclusions about permanent HA’s effects on the OM/PC tissue after DW/HA soaking/baking).

As depicted in Figure 108 and Figure 76, unstimulated saliva and HA share several

properties, reaffirming that HA could chemically suffice as a salivary substitute. The primary

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peaks of ≈3351 cm-1 (N-H bond), ≈1610 cm-1 (N-H bond), and ≈1020 cm-1 (-OH group) were

apparent in both the saliva and HA readings (in Figure 108 and Figure 76).

Figure 108 - Unstimulated saliva from P3, Black – Water leached and air dried, Blue – Water rinsed and air dried [8]

5.6.1 HYP ASSAY TESTING The mechanical strength and rigidity of a biological tissue directly corresponds to the

amount of collagen within it. The mechanical strength advantages of PC over OM can be briefly

explained by the fact that PC contains about 3x more collagen than OM (p<0.001). Additionally,

there was a slight decrease in the amount of HYP made available after irradiation, although not

statistically significant (p=0.8860). Although not statically significant, it is believed that with a

larger sample size tested, that cross-linking outweighing the level of chain scission caused by

irradiation would be validated (by having this decrease in irradiated sample HYP be statistically

significant).

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5.7.1 HISTOLOGY AND LIGHT MICROSCOPY OF OM/PC SAMPLES, HA/DW SOAKED To summarize Figure 78-Figure 103, a few conclusions can be made:

When analyzing the epithelial layers of the OM tissue, the epithelial cells were nearly

rubbed completely away due to friction testing, as seen in Figure 78-Figure 82, and Figure 87-Figure

91. From specimen inspections, ≈90% of the epithelium was worn-away. This is a visual

representation of how this “cushioning layer” is removed, and therefore underlying nerve

endings are more easily affected, possibly resulting in pain sensation to the patient. In Figure 78

specifically, in the lower right-hand corner, the epithelial layer is shown to be worn-away, by not

occupying the remainder of the bottom of the image. The collagenous tissue located more deep

to this epithelium is exposed. Beneath that, at the top-left of the image, striated A-band muscle

fibers are seen to be torqued and twisted. This observation could possible signify the visual

evidence of how muscle-embedded nerves become irritated because of lack of sufficient

lubrication in the mouth, therefore causing patient pain. This similar occurrence, also in OM

DW, is seen in Figure 88, with the exception being that the entire epithelial layer is removed

(observed on the right side of the image). Contrasting to Figure 88, Figure 92 (soaked in HA

instead of DW) is shown to have an intact epithelial layer of 7-8 cells in thickness (although

exhibiting some wear from friction testing), ≈5 μm thick. We can also discern that the even

deeper striated muscle fibers retain their linear, parallel orientation. This signifies the lack of

disruption of these fibers, implying minimal nerve irritation (and therefore diminishing pain to

the patient).

When comparing these OM DW figures to OM HA (OM HA: Figure 83-Figure 86 and

Figure 92-Figure 95), OM HA had very little of its epithelium removed due to friction testing,

implying HA’s protective effects during wear examination.

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This disparity, although not as polarizing, was also seen in PC HA vs PC DW samples.

Especially when comparing Figure 98-Figure 101, or Figure 100-Figure 103, the wear differences are

clearly apparent. Barely any damage had been done to the PC HA tissue. The serosal side of the

PC (smooth side, which was friction tested) was seen to be much more ragged when comparing

PC DW to PC HA after friction wear.

Further conclusions can be made from Figure 78-Figure 103. Such findings include that in

the DW-soaked samples, the collagen bundles, colored green in the Gomori TriChrome, were

more tightly-packed. This finding suggests that the HA was able to embed into the collagen

fibers and push them apart. This intrusion could contribute to HA’s lasting effects. Also observed

was the fact the PC had no cells existent, but rather collagen fibers constituting its composition.

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6.0 CONCLUSIONS

Possible HA clinical applications are diverse. For example, HA can mechanically (by its

lubricating effect) obstruct the transfer of energy from the collagenous basement membrane to

the underlying nerve termini, which consequently reduces pain sensation relayed to the brain.

When comparing HA to other xerostomia remedies, speculation has been offered that the

HA provides its unique lubrication effect by a novel mechanism: insertion between the high-

friction collagen fibrils to provide macro-molecular—rather than water-based—friction

reduction. This friction-relief mechanism, as evidenced in the friction experiments, is applicable

to both natural and radiation-treated human mucosa, both prophylactically applied prior to

irradiation or afterwards.

HA has been revealed to be imbibed more easily into OM than PC. Irradiation’s effects

on tissues’ level of uptake were confirmed with HYP pulp remnant tissue weight testing.

However, through the tensile testing, HA’s effects were seen to have greatly diminished as a

result of irradiation.

HA has shown to lower the elastic modulus for both PC and OM. However, these effects

are diminished due to irradiation-induced collagen chain cross-linking. OM, due to it possessing

≈3x less collagen than PC, has been proven to be more mechanically weak by having a much

lower elastic modulus.

As far as HA’s prospective use by the public is concerned, because HA’s effects of

softening tissue, collagen impregnation, and lubricious quality are entirely reversible (as

especially evident by the light microscopy and MAIR-IR results), HA, in any application to the

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body, can be classified as a medical device, rather than a drug. This is because there are no

permanent metabolic or chemical effects resulting from HA application.

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7.0 LIMITATIONS OF THE STUDY

7.1.1 LACK OF INVESTIGATION WITH HOW BLOOD FLOW AFFECTS LIVING OM IN XEROSTOMIC PATIENTS Because the study dealt with OM obtained from cadavers, the variable of blood flow was

not involved in the research. Citing Hikichi et. al. [117], degradation of HA can be caused by

bilirubin and Red Blood Cell (RBC) exposure (bilirubin is created upon RBC death). Irradiation

generates free-radicals (a highly-reactive uncharged molecule with an unpaired valence electron)

via the photon reactions it creates. The radical-induced changes in HA have been recorded and

depicted as follows: MW of HA (original MW of 2*106) decreased by 60% after 12 hours of

radiation exposure and 75% decrease after 24 hours with bilirubin present. Without bilirubin

present, there was no MW change in the 24 hours of radiation exposure. It should be noted that

the iron metal ion is released from hemoglobin in RBCs upon irradiation exposure and also has

been reported as inducing HA degradation [118].

Some of the specifications of the experiment are as follows: light-irradiated samples were

placed on a white light irradiation system that had two fluorescent tubes (F15T8/CW) and a

small cooling fan to counteract heat generated during irradiation. The light intensity was 22,000

lux, approximately one-quarter the intensity of bright sunshine at noon during the spring in

Boston, MA, USA. The temperature on the irradiation stage was controlled at 23-25°C.

It would be beneficial to determine, as well, if and how there is any correlation between

the total degradation/degradation rate of collagen and HA when both undergo irradiation.

7.2.1 ALTERATION OF RADIATION DOSAGE A ɣ-irradiation dosage of 70 Gy was used for this study. This dosage, according to

previously-cited literature, is on the high-end of the standard range of total irradiation exposure

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for a head/neck cancer patient. The purpose of applying the highest clinically-used dose found in

the literature was to make the effects of irradiation all the more apparent. In the future, more

common clinically-used dosages of irradiation (with consequent diminished adverse effects)

should be tested.

7.3.1 VARIANCE OF HA/DW TISSUE SOAKING TIME For the entirety of the study, HA/DW soaking time was conducted consistently with the

amount of samples for both DW/HA, ranging anywhere from 24 hours to one week. In future

studies, select soak times should be used for the same data groupings in order to observe the

effects of soaking times on the tissue. Especially in weight studies, a complete saturation level

could be measured after recording the weight of a specimen daily while it continues to soak. It

was assumed, in this study that both for 105˚C baking and fully-immersed soaking, that complete

saturation/dryness could be accomplished within 1-2 days.

7.4.1 MINOR TISSUE SLIPPAGE (UNSENSED) MAY HAVE LED TO SKEWED TENSILE TESTING DATA The vast majority of samples tested were seen to exhibit pure elastic deformation when

stretched, as seen in Appendix 9.6. However, this deformation was often not perfectly linear,

meaning that the samples could have exhibited some minor plasticity, or that there might have

occurred minor slippage in the “grips” anchoring the tissue as it was pulled. Because of PC’s

strength and high-collagen composition, possible slippage was likely culprit for any plasticity

shown erroneously in the stress-strain curve.

Machined steel grips with polymerized sand bound to its surface, which could be

bolt/screw-tightened should be the future route for a more ideal tensile testing apparatus.

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7.5.1 ASSUMPTION OF ISOTROPIC STRAIN OF ORAL MUCOSA TISSUE WHILE TENSILE TESTING As previously cited, OM composition was said to be isotropic [25]. Because of this, the

assumption was made that when stretched, OM would deform uniformly throughout the sample

area (between the fastened sections of tissue). However, perfect isotropy might not have

occurred for our samples because of minor underlying fatty tissue/OM imperfections that arose

during cadaver extraction. Traditionally, pins or pen marks have been uniformly distributed

throughout the sample to monitor each respective portion’s displacement of the tissue when

stretched. This technique was not utilized due to possible damage that these markers could have

made on the tissue samples, thus possibly weakening the samples. In future studies, another

avenue that could be utilized for observing the fractionated strain biological tissue such as OM

would be to use a camera with markers on a piece of glass/lens in front of the sample being

stretched. This way, fractionated stretching could be monitored without compromising the

structural integrity of the tissue sample.

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8.0 FUTURE DIRECTIONS

8.1.1 PROSPECTIVE BODILY APPLICATIONS FOR HA OUTSIDE OF THE ORAL

CAVITY

8.1.2 Vagina Lubricant for Menopausal Women

HA, naturally found within the body and extremely lubricous, could help both with the

lubrication during sexual intercourse by easing subsequent pain (due to prior lack of sufficient

lubrication), as well as aiding in the restoration of the vaginal tissue that had previously been

irritated/inflamed because of lubrication deficiency during intercourse.

8.1.3 Stem Cell Research

Stem cell proliferation and specialization is attributed to differing mechanical properties,

which HA might be able to influence. For example, the more stiff stem cells become osteocytes

and turn out to be more invasive. Studies in progress are presently concerned with engineered

tissues of collagen and cells. Treating these micro-tissues with HA, by injecting it into the tissue

for 60 seconds, modifies contractility of fibroblasts into a dog bone shape. Force sensor pillars

are being utilized to measure force changes when influx/efflux of HA occurs.

A practical application of HA to OM tissues might decrease patient pain, in that HA

induces contraction of cells at about 1/3 of the magnitude of cells not treated with HA, as

depicted in Figure 110. This could potentially diminish the impact at underlying nerve endings.

Additionally, because of HA’s large size, it could act as a physical obstruction for physical

changes to be spread to nerve endings, possibly lessening pain as well.

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A detailed depiction of this experimental process, as well as the findings, is assembled in

the following. The following protocol and resultant data was compiled by SUNY at Buffalo

School of Biomedical Engineering Professor, Dr. Ruogang Zhao, with the assistance of graduate

student, Yan Li [119]:

Fabrication of Microtissue Devices: The microtissue arrays (10 rows by 13 columns)

devices were cast from polydimethylsiloxane (PDMS) by replica molding from stamps made

using a multilayer microlithography technique. The elastic modulus of cured PDMS was 1.6

MPa. The micropillars had an effective spring constant k = 0.9 μN μm-1.

Microtissue Seeding and Cell Culture: The devices were sterilized in 70% ethanol for

fifteen minutes before cell seeding. Additionally, to prevent cell adhesion, the devices were

treated with 0.2% Pluronic F127 (BASF) for one minute. Un-polymerized rat tail collagen type I,

3-4 mg ml-1 (Corning Life Sciences, Tewksbury, MA, USA), was mixed with NIH 3T3

fibroblasts and then introduced into the apparatus wells by centrifugation. Cell culture was

maintained up to three days in high-glucose DMEM-containing 10% bovine serum, 100 units

mL-1 penicillin, and 100 mg mL-1 streptomycin (all from Invitrogen, Life Technologies, Grand

Island, NY, USA). The media were changed every two days.

Pharmacological Treatment of the Microtissues: Microtissues were grown for 2-4 days,

and then washed with phosphate buffered saline (PBS) before treated with 0.5% HA solution for

thirty seconds or one minute separately. After HA treatment, HA solution was extracted and

culture media was added before imaging.

The preliminary results of the experiment’s findings are depicted in Figure 109 and Figure

110:

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Figure 109 - Images of Microtissue, Before and After HA Treatment

Referencing Figure 109, barely any evidence is present that reveals any kind of

dimensional effect on HA. As depicted, there is an extremely slight shrinkage that occurs after

HA soaking, as compared prior to HA implementation.

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)

It should be noted that further investigation is required to determine the mechanism for

why HA-exposed cell-collagen matrix samples contract with ≈1/3 of the force of non-HA-

exposed samples. It is suggested that somewhere in the collagen + cell construct, “slippage”

occurs after the HA addition (because of its lubricious nature). This sliding (which translates to

less contractile force of the cell-collagen matrix) could potentially reside between the cells and

collagen, or between the collagen fibrils. It can most likely be assumed that the contractile force

of the cells remains the same, even after HA-injection and extraction. The possible source of this

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contractile disparity could be that these contractile forces are more weakly transmitted to the

connecting pillars when the HA has been added, due its lubricating effect.

With the current data assembled, it is difficult to know exactly where the slippage

occurred in the microtissue. A further investigation to determine the slippage location would be

to disrupt the cell cytoskeleton or kill the cells to produce a “pure collagen” structure. HA

treatment could then be administered to conclude if HA lubrication occurs in the fibrils of pure

collagen.

This experimentation, observing the effects of HA uptake into the cell-collagen matrix,

was performed by Dr. Rhuogang Zhao and graduate student Yan Li.

8.1.4 HA’s Prospective Usage for Amputees Suffering from Phantom Limb Syndrome

Scar tissue accumulates around nerves adjacent to amputation sites. This scar tissue

proliferation causes cells to infringe upon the neighboring nerve endings, causing pain to the

patient. Scarless wound healing via HA could be utilized to help limit such scar proliferation.

HA’s tissue regeneration properties could be used to help prevent scar tissue formation anywhere

on the body where the scar would be undesired.

8.2.1 VOLUMETRIC ANALYSIS OF PC AND OM AS A RESULT HA/DW SOAKING Computerized 3-Dimensional Analysis of Formalin-preserved tissue should be

accomplished to obtain more accurate readings.

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8.3.1 MEASUREMENT OF XEROSTOMIC PAIN RELIEF WITH HA APPLICATION

Additionally, a way to measure the amount of pain that a xerostomic patient must endure,

both with and without xerostomia, needs to be evaluated. Possible animal models of this have

been suggested, such as utilizing the tail-twitch model of a mouse.

8.4.1 CONFOCAL INFRARED IMAGING OF TESTED TISSUES

Confocal Infrared Imaging (CII) is another test that needs to be addressed in the future to

observe occurrences inside the PC and OM tissues (preserved in 2.5% Glutaraldehyde) as a

result of tensile and friction testing, as well as the effects of HA/DW soaking and 70 Gy of ɣ-

irradiation. The break-down of collagen (which primarily will be relevant for irradiation’s effects

on the tissue) will be evident by dark and brighter “spots” in the confocal imaging. The darker

spots, which represent more “water-filled” areas of tissue, should signify regions of more

broken-down collagen. The brighter spots could indicate less broken-down collagen.

8.5.1 TESTING OF IRRADIATED OM AND PC, HA-SOAKED VS DW-SOAKED, WITH GRAFTED ANTIMICROBIAL AGENT Via glycoprotein attachment, post-irradiated oral cancer patients become increasingly

susceptible to fungal oral infection [28]. Recently, antimicrobial agents have been added to HA

solution with hopes of preventing related infections such as these, and have proven success [97].

The antimicrobial agents with the most advantages are antimicrobial peptides (AMPs). They are

secreted by many living organisms such as micro-organisms, vegetables, insects, fish, and

mammals, and amphibians to protect themselves from invading microbes. AMPs function by

“permeabilizing” the bacteria cell membranes through pore formation or other structural flaws

[97]. Compared with conventional agents, AMPs are advantageous in that they can operate at

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very low concentrations of merely a few μg/mL and to have versatile antibacterial activities.

Therefore some AMPs, like Nisin [120, 121] have been immobilized on the substratum surface.

It has been shown that AMPs retained their antimicrobial activity post-immobilization on a

substratum. Nisin is a small peptide that is cationic and hydrophobic [100]. Nisin acts against

Gram-positive bacteria, oftentimes involved in bacterial infections. Due to isolated HA (with no

added antimicrobial agents) possessing antimicrobial properties that are dependent on its

concentration and bacterial species, it is essential to add more reliable antimicrobial agents to

HA to ensure antimicrobial versatility.

Nisin-supplemented HA is intriguing for antibacterial applications, such as wound

dressings, contact lenses, cleaning solutions for contact lenses and cosmetics formulations.

Because of these prospects, it is useful, as a future study, to conduct the same experiments as this

study, but instead substituting HA with HA + several antimicrobial agents (such as Nisin).

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) It has been suggested that free radical creation could possibly be more for small dosages

of irradiation over a one-two month treatment period vs. a one-time mass-dosage (used in this

study for time convenience, but not according to clinical application methodology. It would be

interesting to determine if these dosage treatment discrepancies would affect the tests

investigated in this study, or if the PC/OM tissues react differently in any way.

8.8.1 CHEMOMECHANICAL TESTING ALTERATIONS SEEKING MORE ACCURATE RESULTS Several alterations to the tensile testing protocol can be made with hopes of improving

the accuracy of the data. First, a more accurate means to determine strain can be the utilization of

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a Linear Variable Differential Transformer (LVDT) strain gauge. LVDT strain gauges can be

fastened to the middle of each tissue sample, and can measure the strain observed on each

specimen. Such strain gauges are commonly used today for measuring strain of steel samples

[110]. However, because soft tissues were used in this study, and were therefore not rigid and

easily measured, another avenue was selected for measuring strain. Imaging equipment could

also be used in the future to analyze the samples as stretching progresses during tensile testing.

Digital measurements could be taken of strain and compared. This apparatus could be used as a

compliment to the load cell conversion process used in the chemomechanical tensile testing

protocol.

Another area that was not addressed was the necking that occurred in each sample when

pulled. Necking, as observed in Poisson’s Ratio [122], would be a useful measurement in

developing more accurate values for ultimate tensile strength (UTS). As each sample was

deformed vertically, the cross-sectional area decreased horizontally, evidencing necking.

Because the original pre-test cross-sectional area was used to compile UTS, the UTSs given in

this study are indeed less than what they theoretically would be if necking was taken into

account. Necking could be measured by interrupting the experiment (pausing the loading cell)

intermittently to record cross-sectional area changes. These measurements could be confirmed by

having two cameras (situated 90° from one another), in order to measure the sample width and

thickness.

8.9.1 IN-DEPTH ANALYSIS OF FOOTBALL LEATHER UPON HA TREATMENT To this point, all results from the HA-application of leather footballs were subjective and

judged by the football handlers themselves. Surface chemistry analysis, MAIR-IR, and friction

169

wear studies can all be utilized in future experiments to help better discern the effects, both

physically and chemically, that HA application had on the collagenous leather footballs.

8.10.1 Observation of Remnant Cancer Cell Motility in the Oral Mucosa After Radiotherapy As previously discussed, HA acts as cancer cells’ means to spread to other tissues of the

body by serving as a “wedge.” Cancer cell motility in the OM of living oral cancer patients

should be monitored for change in motility of remnant malignant cells after HA application to

treat xerostomia. An increase in motility could prove detrimental to patient health.

170

9.0 APPENDICES

9.1.1 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue Introduction

The figures in Appendix 9.1 relate to a pilot study conducted, observing the effects of

differences in tissue rehydration liquid on friction and tissue wear characteristics both

before/after drying. This pilot study was conducted out of curiosity to determine if HA could

provide improved and extended lubrication (as compared to DW) even after rehydrating a

previously rigid dried tissue.

The structure of PC is unique (as compared to dermis); it possesses a basement

membrane on both sides, meaning it “not only is a smooth yet porous surface for cellular

attachment and proliferation, but also in sufficient density for soft tissue exclusion [124].” This

tissue is almost entirely made up of collagen. Histologically, HA has been demonstrated to show

wear protection during friction testing both in cell-rich tissues (OM epithelium) and collagen-

rich tissues (PC).

Summarizing the results of this pilot study, friction was even lower for the dry-then- HA

soaked samples than the original PC tissue. For dry-then-DW-soak, the CoF increased slightly as

compared to original PC samples.

Minor tissue tearing was apparent on all of the rehydrated samples (from the tissue-tissue

friction), causing skidding on the recorder pen. This was MUCH more evident for DW-

171

rehydrated samples than for HA-rehydrated samples. Concerning “mucositis”, this frictional

phenomenon may be responsible for epithelial cell shedding and subsequent pain & dysfunction!

9.1.2 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue Materials and Methods Four Samples of PC were taken, all of which were stored in propylene oxide-filled

polypropylene containers. The samples were then cut into smaller segments for testing in a DW-

filled dissection tray. The fibrous side of the PC-pin segment was glued to the furniture tack.

After being lightly blotted with a TechniCloth®, the samples were then friction tested. Three 4”

x 4” squares of TechniCloth®, moist with DW, were placed on the Parafilm®-lined cardboard

square before placing the base tissue sample on top (and being pinned around its perimeter while

taut) (Figure 111). 50 μl of DW was added at the onset of friction testing, as well as every 5

minutes thereafter for 25-minute trials for each sample.

Figure 111 - Friction Testing Apparatus at Initial Testing (Pre-Dry/Soak)

172

The samples were then taken off the friction device and left to dry on a sheet of TechniCloth®,

on a cardboard box in a low-humidity room for two days (Figure 112 and Figure 113).

Figure 112 – PC 1 Pinned to Cardboard for Drying (After Initial Friction Testing)

Figure 113 - PC 1 on left, PC 2 on Right

Notice PC 1 has been drying for half hour; while PC 2 has just begun drying (shriveling is clearly apparent after just a half hour)

173

After drying, the four PC samples were re-hydrated, sample 1 and 2 in HA, and 3 and 4

in DW (completely submerged in Low-Density Polyethylene, LDPE, bags). After rehydration for

6 days, they were re-friction tested (Figure 114-Figure 120), using the same as previous protocol. It

was pivotal that the same area of the tissue sample was friction tested. This implies that the base

sample was “rubbed” by the pinned sample at the exact “arc” that was examined in the initial

friction testing trial. This was accomplished by denoting a diagram of the sample’s orientation,

as well as the arc of the pinned sample on the base sample.

Figure 114 - Decrease in Friction Apparent from PC 1 Test Sample (On Left) to PC 1 Test-Dry-HA Soak-Test (On Right)

(Same Tissue Sample Before/After)

174

Figure 115 - Minimal Skidding/Tearing of Base Tissue on HA-Rehydrated Samples (PC HA 1)

Figure 116 - Skidding/Tearing of Base Tissue Very Apparent on DW-Rehydrated Samples (PC DW 3)

175

Figure 117 - PC 4 DW-Rehydrated Post-Test

Figure 118 - PC 3 DW-Rehydrated Post-Test

176

Figure 119 - PC 2 HA- Rehydrated Post-Test

Figure 120 - PC 1 HA- Rehydrated Post-Test

177

The tissues samples, after testing, were stored in 2.5% Glutaraldehyde-DW solution.

Then, they were taken to SUNY at Buffalo’s Pathology Lab where the arcs (as indicated by the

figures’ black encompassment) were analyzed for differences between the HA and DW-restored

samples (Sections 4.7.1 and 5.7.1 of this thesis).

9.1.3 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue Results Observing Figure 121 and Figure 122, friction was approximately halved for the dry-then-

HA soaked samples compared to the original PC tissue tested. For dry-then-DW-soak, the CoF

increased slightly as compared to original PC samples.

Figure 121 - Static CoF for PC HA, Bone Resurfacing Study

178

Figure 122 - Static CoF for PC DW, Bone Resurfacing Study

9.1.4 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue Discussion

Minor tissue tearing was apparent on all of the rehydrated samples (from the tissue-tissue

friction), causing skidding on the recorder pen. This was MUCH more evident for DW-

rehydrated samples than for HA-rehydrated samples, as evidenced by the latter 4 figure pictures

(Figure 117-Figure 120). Even by rehydrating the “rubbed” surface every five minutes (which was

made to simulate a patient taking a drink of water), there was only a very brief reprieve from

friction increase. As a result, this tissue tearing occurred. When discussing mucositis, this

frictional phenomenon (lack of sufficient lubrication, as evidenced by the DW-rehydrated

samples) may be responsible for epithelial cell shedding and subsequent pain & dysfunction.

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9.1.5 Static Friction Testing of DW/HA Rehydrated Bovine Pericardium Tissue Conclusion HA not only restores the natural lubrication properties of PC after rehydration, but

actually improves the tissues’ lubricious properties. This could prove pivotal in the safety and

effectiveness of this prospective implant.

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9.2.1 IRB EXEMPTION NOTICE Robert Baier Alyssa Astran <[email protected]> Tuesday, March 26, 2013 9:27 AM Robert Baier IRBNet Board Action Please note that SUNY University at Buffalo Health Sciences IRB (HSIRB) - Committee A has taken the following action on IRBNet: Project Title: (442641-1) Examination of human oral mucosa for image quality before and after needle puncture Principal Investigator: Robert Baier, PhD Submission Type: New Project Date Submitted: March 13, 2013 Action: EXEMPT Effective Date: March 26, 2013 Review Type: Exempt Review Should you have any questions you may contact Alyssa Astran at [email protected]. Thank you, The IRBNet Support Team www.irbnet.org

http://www.irbnet.org/

181

9.3.1 HYP PROTOCOL USED FOR PILOT STUDY

Tissue Collagen Analysis by Hydroxyproline

Author: Mark Lauren, Revised by Aaron Huber

SUNY at Buffalo Biomaterials Graduate Program

November, 2014

Overview

a. Digest samples in HCl while heated

b. Add color reagent that reacts with HYP in the samples/standards

c. Dilute them and add the developing reagent

d. Insert and Read %T/Absorbance in Visible Spectrometer

e. Compare Results to Curve created from Standard Samples (set amounts of ppm of

pure collagen)

Production of Solutions:

1. Solution A

Materials:

20 ml H2O

30 ml n-propanol

50 ml citrate-acetate buffer pH 6 (the following ingredients produce approx. 51

ml)

182

o 16.67 ml H2O

o 1.67 g citric acid

o 0.4 ml GA (Glacial Acetic Acid)

o 4 g Sodium Acetate

o 1.13 NaOH mixed with 33.33 ml of DW FOR SOLID NaOH

For Liquid NaOH (1 N), protocol calls for 0.8 N NaOH: 26.67 mL

NaOH, 6.67 mL H2O

Must be mixed with 0.845 g Chloramine T (acts as an oxidizing agent [60]) to 100 ml

of Solution A DAILY for proper reaction to occur. In order to extend life of Solution A,

only use enough of Solution A for 1 ml for each sample tested.

Example: 12 vials to test (6 test samples, 6 standards), therefore only 12

ml of Solution A, mixed with Chloramine T is needed. Therefore, take 15

ml of Solution A (to have a little extra) and mix with 0.0563 g Chloramine

T.

2. Solution B

29.75 mL of Perchloric Acid, 3M, and dilute to 100 ml with DW. Use 25

ml graduated cylinder (multiple times) for this step.

3. Solution C

Mix 1.96 g P-DABA with 50 mL of n-propanol. P-DABA is light-

sensitive, NEEDS DARK STORAGE. This solution can remain stable

for up to two months in darkness [61].

4. Standard Solutions: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 ppm (µg/ml) HYP

183

Prepare Main Standard (MS) Solution (100 ppm HYP in 100 ml of DW,

and then dilute from there to produce each standard solution) in sealed

Ehrlenmeyer/ Florence Flask.

Materials:

5 or 10 ml pipette for DW extractions

Micropipettes with 20-200 µl, 100-1000 µl capabilities, 6

syringe tips (amount = # of Test Standards)

13 x 10-ml sealed cuvettes (amount = #of Test Standards)

Procedure:

o Main Standard (MS): 10 mg (.01g) in 100 ml DW (100 ppm)

o Each Test Standard (TS): 10 ml

o Use micropipette (with new syringe for each sample) to extract

from MS

o Use 10 mL pipette (or 5) to add DW to Test Standards to yield 10

ml for each.

5 ppm: 50 µl MS 35 ppm: 350 µl MS






65 ppm: 650 µl MS

184

Equipment for Sample Testing

Spectrophotometer, Balance, Oven, Water bath (or hot plate), pH meter

Screw Capped test tubes (13 x 100 mm), or ones that can fit in a rack (will

be needed for TSs, samples, and one for DW sample.

Plastic/Metal Container

50 ml beakers

Small Magnetic Stirrers

Filter Paper (No. 1 Whatman suggested)

Micropipettes, pipettes

Procedure for Sample Testing

1. Weigh accurately 50-80 mg (65 mg avg) dried sample, record weight. Baking of 3-4

days is recommended

For OM (human oral mucosa): dries by 75% after bake samples need to

weigh 260 mg (.26 g) wet (after dabbing slime remnant with Texwipe)

For PC (bovine pericardium): dries by 70% after bake samples need to

weigh 216.67 mg (.216 g) wet (after dabbed with Texwipe)

BAT (Bovine Achilles Tendon): dries minimally, take 80 mg (.080 g) for

testing (per sample)

**Must tare each sample! Each vial weighs slightly different!

2. Hydrolyze samples in 5 ml 6M HCl for 24 Hours (or over weekend) at 105˚C in

screw capped test tubes (sealed to prevent oxidation and evaporative loss of sample

[61]). Placement in metal test tube rack is preferable for proper handling. **Anything

polymeric will melt! Biological tissues (such as OM and PC) contain high quantities

185

of GAGs (primarily, chondroitin sulfate), which are un-degradable even under severe

acid hydrolysis environments. This causes them to be “left behind” after acid

hydrolysis; therefore the remnant is able to be examined in the HYP Assay Test.

3. Transfer hydrolyzate to clean 50 ml beaker, rinse tubes with water into each own

respective beaker. Shaking tubes before dumping into beaker will get contents on

walls of container out. Then, rinse them with DW after dumping to get the remainder

of the tissue remnants out and into the beaker.

4. Evaporate to dryness, yielding broken-down AAs (via hydrolysis in the 6M HCl),

under exhaust hood.

a. If the experiment needs to be paused at this time, samples can be left at this

stage, covered in Parafilm at 4 °C for 2-3 days (in refrigerator).

5. Add 10 ml of water to dissolve residue (a few hours would work best). After

dissolving in H2O, samples must be analyzed within 24 hours.

6. Make up to 50 ml by adding DW and then filter contents of each using own

respective filter paper. Make sure to rinse funnel with DW in between every filtration.

7. Adjust to pH 6 (pH of the acetate-citrate buffer between 6.0-6.5 yielded maximal

absorbance values for all samples. pH of the reaction components plays a crucial role

during HYP oxidation [21]) (using pH meter in 308 Squire) using a drop or two of

Ammonium hydroxide (NH4OH). Use a drop or two of HCl to lower the pH if too

high. Make sure that both of these are diluted! 1 M NaOH and 12 M HCL, even

with 1 drop can alter pH value by 4! Refrigerate samples, if chosen to save for

future testing, in order to prevent contamination (such as mold growth).

186

8. Take 2 ml of filtered solution and add 23 ml of DW (use 25 ml graduated cylinder to

pour into beaker). Take old containers (used to pour contents into filter paper down

into new container) and wash and dry in fume hood. Use for final dilution (Step 8)

USE AIR OUTLET FROM FUME HOOD TO ACCELERATE

DRYING OF VIALS/BEAKERS

HYP ANALYSIS

*Ensure that the tip of the pipette does not contact liquid in each sample’s container

and keep contact with walls of container to a minimum in order to prevent

contamination.

**For Steps 1-4, use a 2 ml pipette to ensure exact 1 mL dosage. A 1 ml pipette might

have some liquid in tip left over (and therefore will not be exactly 1 ml).

***In between Steps 1-4, make sure that solutions are capped in order to prevent

evaporation.

1. Transfer 1 ml of sample to a screw-capped test tube using a different pipette for each

sample. For reagent blank, use 1 ml of H2O.

2. Add 1 ml of Solution A using the same pipette for each sample, mix well by shaking

the tube once sealed, and wait 20 min at room temp. The purpose of Solution A is to

neutralize any remnant of the HCl that was involved in the hydrolysis step at the

beginning of the test [61].

3. Add 1 ml of Solution B using the same pipette for each sample, mix well and wait 5

min at room temp.

187

4. Preheat Oven to 60°C (takes 10 mins to get to desired temp). Add 1 ml of Solution C

(to create the chromophore Ehrlich’s reagent is responsible for creating the color

that is able to be sensed by the visual spectrometer [21]) (previously stored in

darkness) using the same pipette for each sample, mix well and heat at 60°C for 20

min (chromophore is reliant on oxidation time. Further investigation of this relation

indicated that ideal results are garnered in 20-25 min of incubation [21]). Then, cool

to room temp by immersing in cold water (within large container).

5. Read absorbance (after allowing spectrometer to warm up for 15 mins) at 555 nm

[60] (or peak absorbance if different 510 nm has been observed to be peak in past)

with reagent blank as reference. Test the standards first (to establish a standard curve

determining %T’s (or Absorbance’s) corresponding ppm value of HYP)), followed by

test samples. This HYP Assay Methodology involves preliminary chromatographic

separation of AAs, which enables the subsequent spectrophotometric detection of

HYP, and gives the most accurate results of any HYP detection technique [60]. The

success of spectrophotometric determination of HYP in tissues containing GAGs

depends on the completeness of GAG hydrolysis.

6. The use of spectrophotometric techniques for HYP analysis:

Please reference operating instructions for spectrophotometer in Appendix

9.3.3.

188

Hypothetical Curve with measured Absorbance (Therefore, %T would have a

negative slope) (Figure 123):

Figure 123 - HYP Standard Curve Example

Bioacoustics Research Laboratory, University of Illinois, Urbana, IL 61801 U.S.A.

Clinica Chimica Acta (Impact Factor: 2.85). 07/1980; 104(2):161-7. DOI: 10.1016/0009-

8981(80)90192-8 Source: PubMed

CALCULATIONS

1. Plot Absorbance (or %T) at 555 nm (or peak absorbance, if different) vs. (HYP) (1

μg/ml = 1 ppm)

a. (HYP) of standard = (HYP) curve

b. (HYP) of sample = (HYP) curve * (25/2) * 50 * 10-6

c. %HYP = (HYP) * 100 / sample dry weight (g)

d. %Collagen = %HYP * 8.44

http://www.researchgate.net/journal/0009-8981_Clinica_Chimica_Acta

http://www.ncbi.nlm.nih.gov/pubmed/7389130

189

9.3.2 HYP Equipment and Reactive List/Locations (Table 6)

Reactive Quantity Needed for Protocol Do we have it? Location Equipment Quantity Needed for Protocol Do we have it? Location

ammonium

hydroxide,

NH4OH (30%)

10 mlYes (350 ml of 28-

30%)

308 Squire

Box under

fume hood

Container,

Plastic/Metal2 Yes 308 Squire

Bovine Achilles

Tendon, Pure

Type I

dehydrated

200 µg Yes500,000 µg

(.5 g)

Filter paper,

No. 1

Whatman

15 sheets

Yes, but much

thicker (Grade

415 VWR)

308 Squire,

in

surfactom

eter

supply

drawer

Chloromine T,

.03 M .845 g Yes Pilar

Test tubes,

Screw

Capped

13 x 100 mm Yes 308 Squire

citric acid (1

H2O)50 g Yes, 1 lb

B30 Closed

black

cabinet,

shelf #2

Auto-pipetteMANY (at least 20 each) of 1,2, 5,

10 mLYes

Pilar, 308

Squire, in

pipette

drawer

glacial acetic

acid (GA)30 ml

Yes (200 ml of

Acetic Acid)

308 Squire

Box under

fume hood

Balance 1 YesPilar and

308 Squire

HCl, 10M 0.1 ml in 100 ml H2O Yes Pilar Beakers, 150

mL4 Yes Pilar

Hydroxylamine

Hydrochloride

(HONH2-HCl)

70 g Yes

308 Squire,

under

Chememe

ch. Tester

Beakers, 50

mL5 Yes

Pilar and

308 Squire

HYP, 0-30 ppm

(μg/ml) 10 grams Yes

308 Squire

under

chemomec

h. Tester

Beakers, 50

mL5 Yes Pilar

Methyl Orange

Solution8 mL of 0.1% in 20% EtOH Yes

308 Squire,

under

Chememe

ch. Tester

Burets Several Yes Pilar

n-propanol 830 ml Yes, 1 L

behind

clean

room, 3rd

floor

Squire,

Flam.

Cabinet

Cuvettes, 10

mL~# of standards Yes 308 Squire

p-DABA (5% p-

dimethylamino

benzaldehyde)

50 ml Yes (25 g)

308 Squire

Box under

fume hood

Ehrenmeyer

flask, 125 ml 1 Yes Pilar

perchloric acid

(HClO4), 3M 43 ml Yes (400 ml, 60%)

308 Squire

Box under

fume hood

Ehrlenmeyer

flask, 50 mL5 Yes Pilar

sodium acetate

(3 H2O)120 g Yes Pilar

magnetic

stirrers, small2 Yes 308 Squire

sodium

hydroxide,

NaOH

34 g Yes (1 lb)

B8 Squire,

in black

cabinet

Oven 1 Yes B30

pH meter 1 Yes 308 Squire

Reference

Pure

Collagen

Material (to

establish

maximum

amt. of HYP ~

30% of

sample

weight

100 mg Yes

308 Squire,

@ base of

Chemome

ch. Tester

Spectrophoto

meter1 Yes

308 Squire,

@ base of

Chemome

ch. Tester

Water Bath

(or hot plate)1 Yes

Pilar and

308 Squire Table 6 - HYP Equipment and Reactive List/Locations

190

9.3.3 pH Meter Operating Instructions pH Testing Using Beckman Φ (Phi) 43 pH Meter (by Beckman Coulter Inc.

Fullerton, CA) (Figure 124)

Figure 124 - pH Meter Depiction

TO CALIBRATE:

Remove black rubber cover from electrode tip

pH desired: 6, therefore use buffers 4 and 7 (2-point method calibration)

Use enough of each buffer to submerge the tip of the electrode

Use small magnetic stir bar

When “Auto” begins to blink on the screen, the pH value is soon approaching

Ensure electrode doesn’t touch bottom of container

191

WHEN TESTING:

Rinse stir bar (can be taken out of test beaker by using other stir bar as a

magnet) and electrode with DW into a waste container

After using NaOH and HCl solutions to alter sample pH, remove tip from

pipette and place in respective NaOH or HCl solution’s beaker in order to

prevent contamination of the pipette itself

Press for a continuous reading of #s (if machine has locked on to a

constant #)

9.3.4 Visual Spectrometer Protocol used

Operating the Bausch and Lomb Spectronic 20 Spectrophotometer (Figure 125)

192

Figure 125 - Visual Spectrometer Depiction

The Bausch and Lomb Spectronic 20 spectrophotometer is a line-operated single-beam

instrument. Radiation from the source (a tungsten filament lamp) is dispersed by a replica

grating, and the desired wavelength is focused upon an exit notch. The emergent beam passes

through the cell compartment; that portion which is not absorbed is measured by a phototube.

The response of the phototube is recorded in terms of transmittance (upper scale) or of

absorbance (lower scale).

Operating Controls

The wavelength control is located on the right side of the top. Once it has been set to the

desired wavelength, this control is left undisturbed. The effect of stray radiation is electronically

eliminated with the dark current control, located to the left. This control is adjusted until the

193

instrument records zero transmittance (or infinite absorbance) when the cell compartment is

vacant.

The light control, located beneath the wavelength control, is adjusted until the instrument

records 100 percent transmittance (or zero absorbance) with the blank in the light path.

The sample holder accommodates special test tubes (available at the stockroom). The hinged

top of the compartment is raised, and the tube is gently but firmly seated inside. Care must be

taken to align the etched mark on the tube with the mark on the cell compartment. The top must

be closed before readings are taken.

Operating Instructions

A. Set the desired wavelength with the wavelength control.

B. Turn on the instrument by clockwise rotation of the dark current control. Allow the

instrument to warm up for about 15 minutes before attempting to take readings.

C. Adjust the dark current control so that the instrument indicates infinite absorbance.

Adjust left knob so the needle reads 0 %T with NOTHING IN SAMPLE HOLDER

D. Place the test tube containing the blank (DW) in the cell compartment; adjust the light

control so that zero absorbance is indicated. Adjust right knob so needle reads 100 %T with

DW Sample in holder

194

E. Replace the blank with a tube containing the sample and read its absorbance. Use matched

pairs of test tubes. When switching samples, make sure that λ is set to 555 nm, not Peak

Absorption λ

* Steps B, C, and D should be repeated several times for each sample to minimize the effect of

fluctuations in response.

** Read %T approximately 10-15 seconds after inserting vial into spectrometer (in order to

allow needle to settle)

9.4.1 STATIC COEFFICIENT OF FRICTION RESULTS WITH INTERVENTION INDICATED OVER TIME 9.4.2 Static Coefficient of Friction Results With Intervention Indicated Over Time PC (Figure 126-Figure 131)

Figure 126 - PC DW 1_Abscissa

195



196

Figure 129 - PC HA 1_Abscissa


197


9.4.3 Static Coefficient of Friction Results With Intervention Indicated Over Time OM (Figure 132-Figure 138)

198

Figure 132 - OM DW 1_Abscissa

199


200


201


202

Figure 136 - OM HA 1_Abscissa

203



204

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)

Figure 139 - OM 1 Pre/Post Irrad_HA/PBS Application


205


9.4.5 Static Friction Testing: Bone Resurfacing Study Intervention Indicated Over Time (Figure 142-Figure 145)

Figure 142 - Bone Replenishment PC 1 HA-Rehydration

206

Figure 143 - Bone Replenishment PC 2 HA-Rehydration

Figure 144 - Bone Replenishment PC 3 DW-Rehydration

207

Figure 145 - Bone Replenishment PC 4 DW-Rehydration

9.5.1 0.5% HA SOLUTION’S MANUFACTURING

9.5.2 Certificate of Analysis

Product Name: Sodium Hyaluronate

CAS# 9067-32-7

Starting Material: Streptococcus Zooepidemicus

Country of Origin: China

Batch Number: HA12044

Manufacture Date: Nov. 24, 2012

Pure Bulk Lot Number 20131105-04

Test Date: Aug. 20, 2013

Retest Date: Aug. 19, 2015

208

9.5.3 Test Results Obtained by PureBulk

ITEMS SPECIFICATIONS RESULTS

Sodium hyaluronate (Enzymatic-HPLC) ≥95.0% 99.00% (dry basis)

Glucuronic acid (UV-Vis) ≥42% 42.98% (dry basis)

Loss on Drying (LOD) ≤10.0% 6.71%

Identification (ATR FT-IR) Positive Confirmed

Aerobic Plate Count ≤1,000 cfu/g 56 cfu/g

Yeast & Mold ≤100 cfu/g Not Detected

Coliforms Report Only Not Detected

E. Coli Negative Absent

Salmonella Negative Absent

Staph aureus Negative Absent

Pseudo. Aeruginosa Negative Absent

The following information is an indirect translation of information provided from the

Manufacturer's Certificate of Analysis, and should not be used solely as an

instrument for strict quality control.

ITEMS SPECIFICATIONS RESULTS

Appearance White or Off-White Powder Conforms

Odor Odorless Conforms

pH 6.0 - 8.0 (0.1% solution) 6.3

Particle Size Thoroughly pass through 60 mesh Conforms

Protein ≤0.1% (on the dried substance) 0.04%

Molecular Weight ≥1.0 x 106 Da 1.37 x 10

6 Da

Ash ≤13.0% 11.8%

Iron (Fe) ≤80 ppm <80 ppm

Heavy Metals ≤20 ppm <20 ppm

Arsenic (As) ≤2 ppm <2 ppm

Cadmium (Cd) ≤1 ppm <LOD (0.01 ppm)

Lead (Pb) ≤2 ppm 0.09 ppm

Conclusion: This products conforms to enterprise standards

9.5.4 Storage Conditions

Keep in sealed container under 25°C.

Keep away from strong light, heat and moisture.

Purebulk.Inc verifies that the information contained herein is true and correct to the best of our knowledge.

Verified By: Candace Gibson/Brenda Bristow

( This document was produced electronically and is valid without signature. )

Purebulk Inc., [email protected] Ph: 1-541-679-1500 • Toll Free: 1-888-280-0050

1640 Austin Rd, Roseburg, OR, USA 97471 Fax: 1-541-393-9005

mailto:[email protected]

209

9.6.1 ORIGINAL TENSILE TESTING CHARTS (Figure 146-Figure 154)

Figure 146 - PC Cross-over STUDY_(1) DW x HA PC_(2) HA x DW PC_NonIrrad

210

Figure 147 - OM DW vs OM HA NonIrrad

211

Figure 148 - PC DW Irrad

212

Figure 149 - OM HA Irrad

213

Figure 150 - PC HA vs OM DW_scan

214

Figure 151 - PC HA vs OM DW_photograph

215

Figure 152 - Cross-Over Study_OM

216

Figure 153 - OM HA

217

Figure 154 - OM DW/HA_PC HA/DW/PO

218

9.7.1 PILOT STUDY OF TANNED COLLAGEN-BASED LEATHER (UB’S UNUSED GAME BALLS, Figure 155)

Figure 155 - Photographs of Footballs Tested (via www.eastbay.com)

Severe hydrolysis of the leather of footballs when exposed to precipitation, mud, and dew

has caused accelerated break-down of that leather. A cloth was used to scrub HA into untreated

leather footballs (new, in packaging), graciously provided by Dave Borsuk, Head Equipment

Manager of the UB Football Team.

The HA worked as a treatment to "break-in" leather footballs, as well as to preserve the

integrity of the leather over time (especially when confronted with rainy conditions); Hyaluronan

is known to prevent hydrolysis, as well as aid in tissue preservation (extended life of football

use) [129]. Both of these facts could prove to be advantageous when applied to footballs, by

ensuring the continued integrity of the leather/grip.

HA, which like perspiration and saliva, naturally exists in the body, performed well as an

organic collagen treatment to aid in the conditioning, preservation, and grip of the bovine leather

footballs. HA, in trace amounts, even exists in saliva (Table 7), which is often used by active

athletes for this same purpose.

HYALURONAN (HYALURONIC ACID) IN HUMAN

219

Table 7 - Concentration of HA in Human Saliva [77]

Noting that HA does not give the leather any “unnatural” characteristics, but rather

accelerates the conditioning process (as well as increasing the collagen’s resistance to

hydrolysis), there would be no discernable effect given to the ball that would render is being

deemed illegal for game usage by inspecting officials prior to a game [130]. Pilot experiments

were conducted in this fashion: 10 ml of 0.5% HA solution was applied to four new leather

footballs, all inflated to 13 PSI (NCAA required pressure range: 12.5 PSI to 13.5 PSI [130]), two

Wilson GST 1003 balls and two Nike Vapor 1 balls. Observing HA’s initial effects, HA made

each ball like a bar of wet soap for about 5 minutes after initial HA scrubbing (with a minimally-

absorbent 85% nylon, 15% elastane cloth Under Armour Heat Gear, Under Armour,

Baltimore, MD, USA). The then-conditioned leather, caused each ball to become easily gripped

(sticky, but not unnaturally tacky), as well as rendering the ball apparently softer. This HA

application (a “natural” conditioning method) appeared to be far superior and able to exhibit

favorable results much faster than current ball treatments in these pilot studies, as noted by

experienced quarterbacks.

220

Investigating the mechanism by how HA acts when scrubbed onto (and into) the pebble-

grained leather of the footballs, HA impregnated into the tissue, therefore maximizing its

effectiveness over time by not simply being "wiped-away" by sweat, rain, or other moisture.

Contact-angle liquids were absorbed into belt leather used in another pilot study (Spinneybeck

Enterprises Inc., Getzville, NY, USA), predicting an “intrusive” molecule like HA could also

embed itself in tissue, as evidenced in this study’s Pilot experiments.

Citing Peramo, et. al., biomaterials with regenerative capacities, such as the natural

biopolymer HA are associated with wound regeneration [129]. HA is the primary GAG species

in skin, particularly in the dermis [131, 132]. HA also exists in the epidermis, although is not a

major GAG species there [133].

Increased temporary expression of HA and sulfated glycosaminoglycans (GAGs) has

previously been demonstrated in skin injuries [134], with HA contributing to scarless wound

healing [135]. The most likely mechanism for HA’s regenerative effects can be derived from the

collaboration of the GAG with the cell, controlling receptor signaling without receptor

expression. This idea was first proposed by Gallo and Bernfield [136], as a mechanism that could

create inhibition and/or modulation of growth factors participating in wound repair.

HA turnover may be instrumental in skin regeneration. In skin, HA turns over every one-

two days in the epidermis [137]. The epidermis is renewed every 2-4 weeks [131].

HA, in vivo, may also contribute to fibroblast activity, which is responsible for the

production of the dermal structure. In vivo, HA’s presence increases during the first three days

post-injury and then diminishes after wound closing/scarring [81].

As a result of other testing, the skin’s basement membrane, treated with HA, became

stronger than in non-treated samples. Peramo, et. al. [129], hypothesized that HA contributed to

221

this effect by helping basement membrane formation, while also being present between

keratinocytes of the epidermis in vivo [138]. HA is on the surface and between the basal and

spinous cell layers of the human epidermis [139]. Further testing has revealed that the dermal–

epidermal junction is better preserved in HA-treated samples. This indicates that the increased

HA in the region may prove beneficial to strengthening keratinocyte–keratinocyte contacts,

especially in the basal layer.

For these and other reasons, HA has become the most widely-used medical application

used to treat skin [140]. HA is involved in keratinocyte proliferation [141]. HA-treated skin

samples show a reduction of apoptosis and proliferation. This leads to the hypothesis that the

presence of HA may maintain a better epidermal architecture, while aiding in tissue regeneration

and wound healing. These attributes are crucial in reducing complications stemming from the

chronic protruding of percutaneous devices through the skin [142]. All of these attributes support

the potential ability to aid in the preservation of collagenous leather and other collagen-based

tissues.

222

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