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Development of a Biocompatible and Dissolvable Tympanostomy Tube

Permanent Link: http://ufdc.ufl.edu/UFE0024955/00001

Material Information

Title: Development of a Biocompatible and Dissolvable Tympanostomy Tube
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Sherman, Ethan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: alginate, biocompatible, dissolvable, ototoxicity, tube, tympanostomy, zebrafish
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Tympanostomy tubes (TTs) are designed to be implanted within the ear drum, facilitating pressure relief. TTs are commonly placed to treat recurrent acute and chronic otitis media with effusion in an estimated 2 million children yearly. The limitations of existing TTs include tube occlusion, premature extrusion, permanent hearing loss, and surgical removal. In addition, predictable and reliable ventilation is insufficient among existing tube designs due to variations in composition and geometry. As a result, the goal of this study was to develop a biocompatible tympanostomy tube (TT) capable of maintaining reliable ventilation as well as dissolving on demand. The criteria for developing an alternative TT is sufficient mechanical strength, occlusion resistance, dissolution on demand, and biocompatibility. To this end, TTs were created using calcium alginate hydrogels. The mechanical strength of the gels was enhanced by altering the gel tube composition and manufacturing constraints. Furthermore, occlusion resistance was improved via application of albumin coatings. One of the unique features of these alginate gel tubes is their ability to dissolve after implantation with the use of sodium, magnesium, or potassium solutions, minimizing the need for surgical removal. Furthermore, biocompatibility was maintained using clinically proven non-toxic ingredients and was corroborated using an in-vivo Zebrafish model. Results show that biocompatible TTs can be made using an Alginate:PEG solution crosslinked with CaCl. Alginate and PEG (polyethylene glycol) have been used extensively in medicine with few side effects. Additionally, it was discovered that the ensuing tubes were significantly stronger than commercial silicone tubes (tension and compression) extending implantation durations. Furthermore, alginate tubes (coated in albumin) are less prone to occlusion than un-coated commercial stainless steel tubes while having the unique ability to dissolve on demand within one week after exposure to NaHCO3 solution. Moreover, Zebrafish hair cell ototoxicity experiments indicate minimal toxicity levels after a 30 minute exposure to dissolved and filtered gel tubes. Therefore, dissolvable alginate gel tubes have the potential to limit complications associated with tube implantation. In conclusion, TTs can be developed using calcium alginate gels. The mechanical properties of these gels can be manufactured to exceed those of commercial silicone tubes while providing a minimally invasive removal technique. In addition, biocompatible dissolution solutions (NaHCO3) can be applied to dissolve the gel tubes on demand, reducing patient trauma. As a result, novel biocompatible alginate TTs can be developed that are capable of providing and maintaining reliable ventilation while dissolving on demand.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ethan Sherman.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Tran-Son-Tay, Roger.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024955:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024955/00001

Material Information

Title: Development of a Biocompatible and Dissolvable Tympanostomy Tube
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Sherman, Ethan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: alginate, biocompatible, dissolvable, ototoxicity, tube, tympanostomy, zebrafish
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Tympanostomy tubes (TTs) are designed to be implanted within the ear drum, facilitating pressure relief. TTs are commonly placed to treat recurrent acute and chronic otitis media with effusion in an estimated 2 million children yearly. The limitations of existing TTs include tube occlusion, premature extrusion, permanent hearing loss, and surgical removal. In addition, predictable and reliable ventilation is insufficient among existing tube designs due to variations in composition and geometry. As a result, the goal of this study was to develop a biocompatible tympanostomy tube (TT) capable of maintaining reliable ventilation as well as dissolving on demand. The criteria for developing an alternative TT is sufficient mechanical strength, occlusion resistance, dissolution on demand, and biocompatibility. To this end, TTs were created using calcium alginate hydrogels. The mechanical strength of the gels was enhanced by altering the gel tube composition and manufacturing constraints. Furthermore, occlusion resistance was improved via application of albumin coatings. One of the unique features of these alginate gel tubes is their ability to dissolve after implantation with the use of sodium, magnesium, or potassium solutions, minimizing the need for surgical removal. Furthermore, biocompatibility was maintained using clinically proven non-toxic ingredients and was corroborated using an in-vivo Zebrafish model. Results show that biocompatible TTs can be made using an Alginate:PEG solution crosslinked with CaCl. Alginate and PEG (polyethylene glycol) have been used extensively in medicine with few side effects. Additionally, it was discovered that the ensuing tubes were significantly stronger than commercial silicone tubes (tension and compression) extending implantation durations. Furthermore, alginate tubes (coated in albumin) are less prone to occlusion than un-coated commercial stainless steel tubes while having the unique ability to dissolve on demand within one week after exposure to NaHCO3 solution. Moreover, Zebrafish hair cell ototoxicity experiments indicate minimal toxicity levels after a 30 minute exposure to dissolved and filtered gel tubes. Therefore, dissolvable alginate gel tubes have the potential to limit complications associated with tube implantation. In conclusion, TTs can be developed using calcium alginate gels. The mechanical properties of these gels can be manufactured to exceed those of commercial silicone tubes while providing a minimally invasive removal technique. In addition, biocompatible dissolution solutions (NaHCO3) can be applied to dissolve the gel tubes on demand, reducing patient trauma. As a result, novel biocompatible alginate TTs can be developed that are capable of providing and maintaining reliable ventilation while dissolving on demand.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ethan Sherman.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Tran-Son-Tay, Roger.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024955:00001


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DEVELOPMENT OF A BIOCOMPATIBLE AND DISSOLVABLE TYMPANOSTOMY
TUBE





















By

ETHAN GLENN SHERMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009


































2009 Ethan Glenn Sherman


































To my wife Amy, and my parents, Amy and Robert









ACKNOWLEDGMENTS

The culmination of 30 years of my life has been quite journey. This journey could not

have been completed without the love, support, and guidance I have received from my parents.

Their commitment to my education and well-being through tough times has been unwavering

and it is this assurance that has propelled me to achieve my goals.

I would also like to thank my committee members Dr. Christopher Batich, Dr. Malisa

Sarntinoranont, and most importantly Dr. Patrick Antonelli who have all helped to steer and

motivate me to complete my PhD. I would also like to acknowledge Dr. William Ogle for filling

in for Dr. Batich on my committee for my dissertation defense. Their combined creative

guidance and technical support throughout this process has been second to none. Moreover, the

completion of my doctoral dissertation would not have been possible without the help and

guidance I have received from my advisor Dr. Roger Tran-Son-Tay. For more then 7 years he

has been a true supporter of my research and has helped me achieve my goals of completing my

doctorate. I would like to acknowledge all of their time, effort, and work propelling me

throughout my graduate studies.

Furthermore, I would like to acknowledge the support I have received from my fellow

graduate students. Without them this process would have been quite arduous. Jessica Cobb,

Chessy Fernandez, and Jennifer Jackson have been immensely helpful in working out difficult

problems and helping me to re-think design parameters. I would also like to thank Cecile

Perrault for her more than 12 years of support throughout my entire undergraduate and graduate

career.

Finally, I reserve my most heartfelt appreciation and love for my wife, Amy. The

magnitude of her devotion and her love of both my strengths and weaknesses has been one of the

most important driving forces in my life. Her desire for my happiness is evident by her never-









ending support and patience. The joy it has brought me has continued to be my beacon of hope

during this process.












TABLE OF CONTENTS


page

A C K N O W L E D G M E N T S ..............................................................................................................4

LIST OF TABLES .................................................................. ................. ........9

LIST OF FIGURES .................. .................................. 10

ABSTRACT .............. ................... ................................................................. 12

CHAPTER

1 RESEARCH PLAN ............... .................. ..... ....................... ..... .. .............. 14

Introduction............... ................... ............................. ......... 14
Rationale .............. ................... ................................................................. 14
Obj active .......... ..... .... ......... ...................................15
Specific Aim s....................................................15
Significance .................................................15

2 BACKGROUND AND SIGNIFICANCE.. ................... ...........16

Tympanostomy Tubes as a Cause for Sequelae ........................... ...... ...............16
Alginate Gel as a Non-toxic Degradable Gel Scaffold.....................................................17
Polyethylene Glycol (PEG) as a Dissolution Additive.......................... ...........19
Ototopical Solutions Used to Treat TT Sequelae ...................................... ........... ....20
Use of Zebrafish as a Screen for Ototoxicity...................................... .......................... 20
Absorbable Tympanostomy Tubes .................. ................. ........24

3 M ATERIALS AND M ETHODS ................................................. ...............27

Gel Preparation....................................................... ........27
In-Vitro Ear Cham ber ................... ............................................ ........ ..............28
E ar M odel C construction .............................................................28
Mucus Delivery .......................... ............................29
Air Delivery ...................... ......... ................... 29
Mucus Analog Preparation ..... .......... ......... ......... ........30
Tympanostomy Tube Testing Protocol ............................... ...............30
Zebrafish Preparation and H housing ......................................................................... .............31
Statistical Analysis......................................... ........ 32

4 RESEARCH DESIGN AND METHODS ................................................................... 34










Specific Aim 1: Develop an Alginate Gel Tympanostomy Tube.....................................34
R rationale ................. ................... .... ........... .... ........................34
Parameters Involved in Developing a Viable TT..................................34
TT Design ............. ...................... ... ................... ......... 35
Tube drying ......................................................36
M mechanical Testing .............................................. ..... ...37
Tension testing .................................................37
Data analysis ....................................................38
Tube compression ............................................... ..... ...39
Data analysis ........................................ ......... 40
Maximize Mechanical Strength................................ ...............41
Alginate:PEG concentration.................................. ......... 41
Ca concentration...................................... ........42
Drying time ........................................ ........42
Physiological Testing ...... ....................... ........... ...............42
Quantify mechanical strength after agar encapsulation ........................................ 43
Quantify mechanical strength from exposure to ototopical solutions....................43
Quantify plugging of the alginate gel tube...................................... .....................44
Specific Aim 2: Optimize the Gel Dissolution Components for Complete Dissolution........45
Rationale......................................................... ............... ........ 45
Dissolution Analysis................................................... ...............46
Image analysis...................................... ............................ ... .........46
Dissolution rubric ............... ....... .. ....... ...... ..... ....................46
Optimize the Dissolution Components for Complete Dissolution................................47
Dissolution additives .............. ......... .......... ...............47
Dissolution concentration.................................. ......... 47
Physiological Testing ....................................................... ..... ...47
D dissolution cham ber.............................. ... ... .. .... ..... ............... 48
Quantify the effect of dehydration on the dissolution rate of the gel tube.............49
Quantify the effects of human serum albumin coating on dissolution...................49
Specific Aim #3: Quantify Ototoxicity from Alginate Gel Dissolution using Zebrafish:......49
Rationale ................................ ..... ............................. ...............49
Quantify Ototoxicity from Dissolution Products .................................. .....50
Fluorescent analysis ..................................... .... .....52

5 RESULTS ............. ................... ...................................................... ........ 58

Specific Aim 1: Development of an Alginate Gel Tube......................................58
M maximize M mechanical Strength (Tension) ..........................................58
Maximize Mechanical Strength (Compression).................................................59
Physiological Experiments .................. ................................... ..... ........ 60
Quantify compressive strength after agar encapsulation.............. ....60
Quantify compressive strength from exposure to ototopical solutions....................60
M inim ize alginate gel tube plugging...................................... ...................61
Specific Aim 2: Optimize the Dissolution Components for Complete Dissolution............61
D issolution Solutions........................................... ...............61
Dissolution Concentration .................................................................. .. ........ 62









Physiological Experim ents ................................................................................................. 62
Effect of desiccation on dissolution ............................. ............... 63
Dissolution of H SA coated alginate tubes.......................................... .................63
Specific Aim 3: Quantify Ototoxicity from Exposure to Dissolution Products ..................64
Pilot Study Results .......... ... ...................... .............. 64
Ototoxicity after Treatment Exposure................................. ...................... ......... 64

6 DISCU SSION .................. ........................................... ................ ........ 87

Specific Aim 1: Development of a Calcium Alginate Tympanostomy Tube......................87
D developing the A lginate T T ................................................................................ 87
M axim izing M echanical Strength ...........................................................88
Physiological Mechanics .................. ........ ....................92
Alginate tube compressive strength after agar encapsulation................................92
Alginate tube compressive strength after exposure to ototopical solutions ............93
M inim izing plugging ..... .. .............. .. ... .. ...... .... .................. 94
Specific Aim 2: Optimize the Gel Dissolution Components for Complete Dissolution........96
Effect of Various Dissolution Solutions on Dissolution Rate.............................96
Effect of NaHCO3 Concentration on Dissolution Rate ...........................................100
Physiological Dissolution..................................... 101
Effect of desiccation on dissolution ............. ......... ......................102
Effect of H SA coatings on dissolution ............................................... ............... 103
Removal of retained tube components................. .......... .............. ..............103
Specific Aim 3: Quantify Ototoxicity from Exposure to Dissolution Products................ 104

7 FUTURE W ORK ............... ........................ ...................................108

8 CON CLU SION ................... ............................................ ... ......... ................

APPENDIX

A TENSION AND COMPRESSION TESTING FOR ALGINATE GEL SPECIMEN .........112

B NUMERICAL SOLUTION FOR TUBE COMPRESSION ...............................................114

C ZEBRAFISH SOLUTION CONCENTRATION ................................................................117

D NEUROMAST FLUROESCENT INTENSITY ..................................................... 119

E DISSOLUTION SOLUTION QUANTITY ................................................................... 121

L IST O F R E FE R E N C E S ...................................................................................................122

BIOGRAPHICAL SKETCH .............................................................................127









LIST OF TABLES


Table page

4-1 Dissolution rubric showing various stages of tube dissolution................ ............ 57

5-1 Dissolution Results Based on Treatment and Dissolution Time ....................................82

5-2 Survival and Ototoxicity Results Based on Treatment and Exposure Time...................82

6-1 Embryo media solution pH as NaHCO3 concentration is increased from 0.01M-1.0M..107

D-1 Fluorescent intensity, neuromast area, and hair cell number based on Zebrafish
treatm ent. ................... ............................................. ........................119









LIST OF FIGURES


Figure page

2-1 Surface structure of tympanostomy tubes (at ~ 500x magnification).............................26

2-2 Calcium alginate molecular structure. ................................. ............... 26

3-1 Schematic overview of the ear flow chamber............................................................32

3-2 Exploded view of the in-vitro ear chamber.................. .................................. ..... 33

4-1 Aluminum ear tube mold used for creating bobbin and T-shaped tubes. .......................53

4-2 M echanical testing device (tension).................................................. 54

4-3 Tension testing apparatus setup. ............................................... ............... 55

4-4 Tube com pression apparatus................................................................................ 56

4-5 Tube compression contact area schematic. ........................................... 56

4-6 Position of head and lateral line neuromasts.............. .............................57

5-1 Fresh calcium alginate/PEG T-tube on glass curing rod. ......................................66

5-2 Dried calcium alginate/PEG T-tube on glass curing rod. ......................................66

5-3 Dried calcium alginate/PEG T-tube removed from glass curing rod and placed in
dissolution well. .......................................................66

5-4 Fresh R euter B obbin tube. ............................................................67

5-5 D ried R euter B obbin tube. ............................................................67

5-6 Mechanical testing (tension) average modulus results for Alginate:PEG ratio and
CaCl concentrations when compared to commercially available silicone tubes. ..........68

5-7 Mechanical testing (tension) results for the effect of desiccation time on the
mechanical strength of the alginate gel when compared to commercially available
silicone. .............................. ................. ......... 69

5-8 Compressive strength of the Alginate:PEG gels based on alginate composition and
CaCl crosslinking concentration. ................................................ ......... 70

5-9 Compressive strength of the Alginate:PEG gels based on desiccation time. ..................71

5-10 Compressive strength of agar encapsulated alginate gel tubes after 15 and 30 days
compared to non-encapsulated alginate gel tube and silicone control..............................72









5-11 Compressive strength of alginate gels exposed to various ototopical solutions for 24
hours.............. ................... ....................................................... ........ 73

5-12 Occlusion percentage of the alginate gel tubes (HSA coated and uncoated) compared
to the uncoated stainless steel tubes using the in-vitro ear chamber..............................74

5-13 Nonparametric survival plot (Kaplin-Meier Method) for stainless steel, uncoated
alginate, and H SA coated alginate tubes.................................. ................. 75

5-14 Dissolution results for additives (NaCl and vinegar vs. NaHCO3) over 1 hour. ............76

5-15 Five hour dissolution results for additives (NaCl and vinegar vs. NaHCO3). .................77

5-16 Dissolution results based on NaHC03 concentration (0. 1M, 0.5M, 1.OM, 2.0M) over
1-5 hours. ............................. ..................... ......... 78

5-17 Effect of drying time (0, 15, 30 days dried) on the dissolution rate of the gel tube. .........79

5-18 Effect of HSA coating on the dissolution rate of the alginate gel tubes............................80

5-19 Dissolution within ear chamber over time (1-5 hours). ..................................... 81

5-20 Whole fish fluorescence after DASPEI stain............................................83

5-21 Fluorescent intensity for all three treatments (dissolution products, positive cisplatin
control, negative (no-treatment) control). .............................. ............... 83

5-22 Mean fluorescent intensity between the positive, negative, and dissolution product
treatm ents...............................................................84

5-23 Neuromast size based on exposure treatments........................... ...............85

5-24 Hair cell viability (number of hair cells per neuromast) based on treatment.....................86

A-i "Dog-Bone" specimen in clamps (time= 0).............................................112

A -2 Specim en pulled and elongated.. ....................................................................... .............. 12

A -3 Sam ple fractured w ith a clean even break. ................................................................ 113

A-4 Alginate gel tubes deformed between aluminum and PDMS platen............................113

B-i Compression of alginate gel tubes between two flat platens ................... .............1....114

D-i Zebrafish neuromast P4 hair cells for each experimental treatment.............................120










Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DEVELOPMENT OF A BIOCOMPATIBLE AND DISSOLVABLE TYMPANOSTOMY
TUBE

By

Ethan Glenn Sherman

August 2009

Chair: Roger Tran-Son-Tay
Major: Biomedical Engineering

Tympanostomy tubes (TTs) are designed to be implanted within the ear drum, facilitating

pressure relief. TTs are commonly placed to treat recurrent acute and chronic otitis media with

effusion in an estimated 2 million children yearly. The limitations of existing TTs include tube

occlusion, premature extrusion, permanent hearing loss, and surgical removal. In addition,

predictable and reliable ventilation is insufficient among existing tube designs due to variations

in composition and geometry. As a result, the goal of this study was to develop a biocompatible

tympanostomy tube (TT) capable of maintaining reliable ventilation as well as dissolving on

demand.

The criteria for developing an alternative TT is sufficient mechanical strength, occlusion

resistance, dissolution on demand, and biocompatibility. To this end, TTs were created using

calcium alginate hydrogels. The mechanical strength of the gels was enhanced by altering the

gel tube composition and manufacturing constraints. Furthermore, occlusion resistance was

improved via application of albumin coatings. One of the unique features of these alginate gel

tubes is their ability to dissolve after implantation with the use of sodium, magnesium, or

potassium solutions, minimizing the need for surgical removal. Furthermore, biocompatibility









was maintained using clinically proven non-toxic ingredients and was corroborated using an in-

vivo Zebrafish model.

Results show that biocompatible TTs can be made using an Alginate:PEG solution

crosslinked with CaCl. Alginate and PEG (polyethylene glycol) have been used extensively in

medicine with few side effects. Additionally, it was discovered that the ensuing tubes were

significantly stronger than commercial silicone tubes (tension and compression) extending

implantation durations. Furthermore, alginate tubes (coated in albumin) are less prone to

occlusion than un-coated commercial stainless steel tubes while having the unique ability to

dissolve on demand within one week after exposure to NaHCO3 solution. Moreover, Zebrafish

hair cell ototoxicity experiments indicate minimal toxicity levels after a 30 minute exposure to

dissolved and filtered gel tubes. Therefore, dissolvable alginate gel tubes have the potential to

limit complications associated with tube implantation.

In conclusion, TTs can be developed using calcium alginate gels. The mechanical

properties of these gels can be manufactured to exceed those of commercial silicone tubes while

providing a minimally invasive removal technique. In addition, biocompatible dissolution

solutions (NaHCO3) can be applied to dissolve the gel tubes on demand, reducing patient trauma.

As a result, novel biocompatible alginate TTs can be developed that are capable of providing and

maintaining reliable ventilation while dissolving on demand.









CHAPTER 1
RESEARCH PLAN

Introduction

Tympanostomy tubes (TTs) are commonly placed to treat recurrent acute otitis media and

chronic otitis media with effusion These tubes are designed to provide ventilation within the

middle ear. TT performance can be compromised by premature extrusion, occlusion, or

prolonged implantation in up to 25% of cases 2-4. These complications may result in patient

morbidity and increased expense, possibly requiring surgery. As a result, the development of a

reliable ventilation tube is critical for reducing complications associated with tube implantation.

Rationale

Prevention of TT occlusion, extrusion, and prolonged implantation has long been the focus

of commercial research 5-7. However, existing commercial tube designs have yet to successfully

eliminate these complications. Currently, decreasing premature extrusion promotes prolonged

implantation while minimizing occlusion increases the development of secondary infection 5'6

On the other hand, a dissolvable and biocompatible TT could potentially limit the development

of TT complications. Similar TTs have been created to reduce implantation duration and

ventilation times .11 However, unpredictable clinical conditions combined with a lack of

physician control limit their widespread use.

Optimal TTs should retain middle ear ventilation until clinical symptoms have resolved,

followed by simple removal. As a result, the focus of this research was on the development of a

biocompatible, dissolvable, tympanostomy tube capable of providing reliable ventilation while

limiting tube occlusions.









Objective

The overall objective of this project was to develop a biocompatible tympanostomy tube

capable of maintaining reliable ventilation while dissolving on demand.

Specific Aims

1) Develop a calcium alginate substrate that can be made into a tympanostomy tube:

a) Identify the parameters involved in developing a viable tympanostomy tube.

b) Develop a procedure for producing both reuter-bobbin and "T" shaped alginate
gel tubes.

c) Maximize the mechanical strength of the alginate gel tube and compare it to a
commercial Goode T-tube.

d) Quantify mechanical strength after agar encapsulation.

e) Quantify mechanical strength from exposure to ototopical solutions.

f) Minimize plugging of the alginate gel tube and compare it to a commercial
stainless steel reuter-bobbin tube.

2) Optimize the gel dissolution components for complete dissolution:

a) Optimize the dissolution components (additive solution and dissolution solution)
for complete dissolution.

b) Quantify the effects of dehydration on the dissolution rate of the gel tube.

c) Quantify the effects of human serum albumin coating on tube dissolution.

3) Quantify ototoxicity of alginate gel tube dissolution using a Zebrafish model:

a) Quantify ototoxicity from the dissolution products.

Significance

These studies have the potential to promote the use of minimally invasive biocompatible

techniques while controlling tube removal and limiting the expense and prevalence of surgery

associated with TT insertion.









CHAPTER 2
BACKGROUND AND SIGNIFICANCE

Tympanostomy Tubes as a Cause for Sequelae

Tympanostomy tubes (TT) are devices that are inserted into the tympanic membrane in

order to relieve pressure caused by the buildup of otitis media during an ear infection. They can

be composed of either metal (titanium, stainless steel) or plastic (fluroplastic, silicone) and can

be "T" or "Bobbin" shaped 5. Sixty to seventy five percent of TTs perform flawlessly.

However, some will develop a secondary complication. Three common and potentially

detrimental complications are tube occlusion (-25 %) 4, premature extrusion (-4%) 3, and

prolonged tube ventilation (>2yr (2.7%)) 2. These complications can lead to temporary or

permanent hearing loss and result in the superfluous implantation of tubes. In most cases tube

occlusion can be relieved medically with the addition of otic drops. However, some cases

involve the replacement of the tube. In addition, the majority of tympanic devices extrude

naturally as a result of the healing process but, premature extrusion prior to the resolution of

symptoms can lead to a resurgence of infection and additional tube implantation. Furthermore,

tubes implanted for extended periods of time require surgical removal, resulting in additional

trauma.

Two important factors involved in the development of these secondary complications are

geometric variability and composition. Currently there are >30 different styles of TT each

claiming to have advantages over the others. However, very little clinical data is available to

support these claims. For instance, larger diameter tubes have been shown to decrease the

development of an occlusion, but they increase the likelihood of chronic tympanic membrane

perforation and secondary infection 5. In addition, tubes designed to remain implanted for

prolonged periods decrease the prevalence of extrusion, but are subject to a higher incidence of









chronic perforation and surgical removal 6. Furthermore, tube composition plays a significant

role in cellular adhesion which can contribute to tube occlusion. Smoother surfaces such as

those impregnated with silver oxide, or albumin (Figure 2-1) have shown remarkable results in

preventing biofilm formation which leads to a decrease in occlusion frequency 5'7

Further studies conducted by Lentsch et al. 6 measured the correlation between elective

surgical tube removal and chronic perforation. His results show that 11% of the population who

voluntarily remove their tubes develop chronic tympanic membrane perforations. Therefore, the

surgical procedure of removing the tympanostomy tube may contribute to the development of a

perforation of the eardrum. At the very least it subjects the patient to an additional surgical

procedure. The ideal tube would simply spontaneously extrude from the membrane or become

dissolved after resolution of symptoms otitiss media) 12. In addition, there is evidence that shows

that the presence of granulation or cellular adhesions on the tube increases the healing capacity

of the membrane and decreases perforation development 6. This indicates that a biocompatible

environment surrounding the perforation will be useful in repairing postoperative complications

associated with tympanostomy tube placement.

Alginate Gel as a Non-toxic Degradable Gel Scaffold

Calcium alginate gel has been used as a scaffolding material for skin grafts, as well as a

successful drug delivery device for over 10 years. It is also biocompatible and has a very low

inflammatory response. In addition, calcium alginate has the unique ability to cure under

relatively inert conditions making it ideal for use in cellular encapsulation and growth 13-16

Alginate is one of the main components in most of the large brown seaweeds. It is

composed of alginic acid which consists of 2 monomers, 3-D-mannuronic acid and a-L-

guluronic acid. The individual monomers, as well as combinations of these two monomers, form

blocks which outline the basis for the resulting polymer gel. The G-Block is composed of L-









guluronic acid, and the M-Block is composed of D-mannuronic acid, with the MG-Block

composed of both L-guluronic acid and D-mannuronic acid. In terms of appearance the M-

Block is relatively straight and has the consistency of a ribbon, whereas the G-Blocks

stereochemistry aids in the formation of gels (Figure 2-2). It is the G-Blocks that are crucial

when creating gels using calcium ions 13,17. This is because the G-Blocks contain reactive

carboxylic acid sites whereas the M-Blocks are non-reactive. In order to create a gel, divalent

solutions including (Ca2+, Sr2+, Ba2+) are added to the gel. Magnesium (Mg2+), along with

monovalent solutions such as sodium (Na' ) or potassium (K +) do not form gels. However, in

the presence of calcium, the carboxylic acid site of the G-Block is ionically substituted with

calcium divalentt), and a link between two alginate strands can be made, forming a solid "egg-

box" structure (Figure 2-2) 13

The above procedure of adding calcium to alginate specifically in the form of calcium

chloride is one of the simplest ways to create a solid gel. The calcium content within the gel

rapidly increases within the first hour of gelation and levels off over time. Gel stiffness is

increased by diffusion of calcium ions from the liquid solution to the forming gel via the

carboxylic groups of guluronic residues. However, the resulting calcium chloride solution takes

several hours to diffuse smoothly into large quantities of alginate solution. On the other hand,

the use of low solubility solutions such as calcium citrate or calcium sulfate will form very

smooth gels quickly as a result of their slow steady release of calcium. Furthermore, this

addition of calcium ions to a solution of alginic acid will cause the viscosity to increase without

the addition of more alginate or by increasing the molecular weight17

A unique property of alginate gels is their ability to rapidly dissolve from a solid back into

solution. It has been shown that the chemical bond is reversible in calcium alginate gels, making









them ideally suited for rapid degradation in the presence of chelating or monovalent ions. The

chelating or monovalent ions can include sodium (Na ) or potassium (K ) 18. Moreover,

alginate gels can be degraded with the use of acidic hydrolysis or with the use of enzymes, along

with gamma radiation and ultraviolet light 19

Furthermore, calcium alginate has a proven biocompatible track record, which is a function

of the concentration and purity of the M and G blocks. In addition, natural alginate gels have

low cytotoxic and hemolysis effects. Moreover, calcium alginate has been used as a patch for

repairing tympanic perforations in the ear 20. Prior to the use of calcium alginate, paper patches

or a surgical tympanoplasty procedure were considered standard practice for repairing tympanic

perforations. However, recent developments have focused on the advancement of

implantable/injectable devices that act as a scaffolding over the perforation to promote healing.

Weber et al. 20 has successfully created a biocompatible calcium alginate plug with the use of an

injection-molding 3-D manufacturing technique. Histological results showed little to no toxicity

associated with gel implantation. This further validates the absence of cytotoxicity as a result of
20
graft placement and validates the use of alginate based implants for use as tympanic inserts 2

Polyethylene Glycol (PEG) as a Dissolution Additive

PEG is similar to alginate in that it is a common polymer used for tissue engineered

scaffolds and for drug delivery 21. PEG was added to the alginate gel solution to alter the final

gels properties and to facilitate dissolution. Alginate gel strength is a function of the available

alginate in solution. The substitution of PEG for some of the alginate prior to crosslinking

reduces the amount of alginate and can potentially limit the mechanical strength of the resulting

gel. As a result, the PEG encapsulated gels have the potential to dissolve faster then pure

alginate gels.









Ototopical Solutions Used to Treat TT Sequelae

There is a vast array of treatments available to treat tympanic complications ranging from

topical antibiotics to invasive surgical procedures. A large majority of these treatments consist

of the use of topical solutions. These include Hydrogen Peroxide (H202) 22-24, Sodium

Bicarbonate (NaHCO3) 2,23,25, Sodium Chloride 22, Vinegar 22,24, Ciprodex, and Ofloxacin26

Several of these topical solutions are unique in their ability to potentially degrade or assist in the

degradation of calcium alginate gels. These include sodium bicarbonate (NaHCO3), sodium

chloride (NaC1), and vinegar (CH3COOH, acetic acid) which are used with little clinical

evidence to clear tube occlusions 23. The first two compounds contain sodium (Na) which, as

stated previously, is one of the monovalent ions capable of dissolving calcium alginate gel back

into solution. Furthermore, sodium chloride has also been shown to be non-ototoxic 22. The

third compound, vinegar, does not contain any of the aforementioned monovalent ions.

However, it may behave as a catalyst accelerating the dissolution reaction with the production of

carbon dioxide gas, when in the presence of sodium bicarbonate. The advantages of the above

solutions are that they are widely available and have been used, with few side effects, in the

treatment of ear sequelae 22-25

There is however one known drawback associated with the use of these solutions. Sodium

bicarbonate 5% in solution has been associated with minimal pain. In a clinical study using

sodium bicarbonate to relieve tympanostomy tube occlusions, minimal pain was associated with

administration. However, the pain was mild in all patients and did not hinder the treatment

regime 23

Use of Zebrafish as a Screen for Ototoxicity

Hearing loss due to ototoxicity is commonly determined by measuring hair cell function.

Developments in alternative animal models have lead to the discovery of Zebrafish as a









promising candidate for measuring hair cell ototoxicity associated with medical treatments.

There are many advantages for using Zebrafish as a screen for ototoxicity. The most important

is that Zebrafish hair cells are structurally, and functionally similar to the inner ear hair cells of

mammals. However, unlike mammalian hair cells, these cells are located externally along the

lateral-line of the fish. Zebrafish also have typical vertebrate inner ears with both hearing and

vestibular organs along with the external hair cells 27. One advantage of using Zebrafish is that

these external hair cells can be observed within the organism as a whole rather then cultured

separately as in mammalian studies. Furthermore, Zebrafish embryos are optically clear, which

promotes hair cell observation via microscopy and facilitates fluorescent labeling. As a result,

the Zebrafish model allows testing of ototoxic samples, not just at the cellular level, but

metabolic level as well. Also, neuromast hair cells have been shown to undergo apoptosis after

injury using aminoglycosides in a similar manner to the way mammalian organ of Corti hair cells

undergo cell death. Moreover, mammalian studies frequently generate false negatives since

rodents can still respond to ambient sound after losing most of their high frequency hearing 28,29

Additionally, Zebrafish are inexpensive to obtain and can be rapidly analyzed making them ideal

for large sample sizes.

Hair cells convert mechanical sound vibrations into electrical signals that are carried by the

auditory nerve to the brain. The external hair cells in fish (Zebrafish) are organized into clusters

of 5-20 cells that are contained within neuromasts found along the lateral line and head. Each

neuromast is composed of a ring of support cells that surround the sensory hair cells within the

center. These neuromasts can detect frequencies of between 50-200 Hz and are primarily used to

facilitate schooling, and to avoid predators. Also, lateral line hair cells exhibit selective uptake

of fluorescent dyes including FM1-43 (via nompC TRP channel), DASPEI (2-(4-









(dimethylamino)styryl)-N-ethylpyridinium iodide) a fluorescent styryl dye, and Yo-PRO1,

another fluorescent dye, making them ideal for rapid ototoxicity analysis 28-31

The use of fluorescence to assess ototoxicity provides a quantifiable measurement of the

effect ototoxic chemicals have on individual hair cells. The fluorescent intensity of the hair cells

provides a measurement of hair cell viability. Furthermore, each fluorescent dye can selectively

label a different part of the hair cell allowing for a more specific cellular analysis. For example:

FM1-43 can selectively label only the cytoplasm of the hair cell while Yo-PROl will only label

the nuclei nucleicc acid, DNA) of the cell, and DASPEI is a voltage sensitive dye which can

label nerve fibers and neurons as well as hair cells of the lateral line neuromast and nasal

epithelium 27,28,30. More specifically, FM1-43, in Xenopus hair cell, labels organelles such as

mitochondria, and rough endoplasmic reticulum within the cytoplasm and is inhibited by cation

channel blockers such as neomycin and amiloride. On the other hand, Yo-PRO1 is a cyanine

monomer dye capable of entering cells through large non-selective channels such as P2X7

receptors 32. DASPEI cationicc) stains work by staining mitochondria within living cells. Under

specific conditions the stain can accumulate within the mitochondria due to the development of

negative potentials across the cell membrane. However, the DASPEI stain is not present (stored)

in adjacent supporting cells, only in the nucleus of hair cells. This is because there may be

differences in the ion channels and exposed carriers of both hair cells and support cells. Also,

hair cell transmembrane potential is higher than that of the surrounding support cells and it is this

membrane potential that has been shown to affect the uptake of other cationic dyes 33

Under fluorescence the hair cells glow depending on their viability and can be graded

based on florescent intensity. Typical results show a distinct decrease in intensity as toxicity

increases 30 and under increased magnification, individual hair cells within the neuromast can be









counted and compared. However, it is important to note that drug susceptibility is dependent on

cell maturation with 4 day and 5 day old fish showing marked differences. Younger (immature)

neuromast cells are less susceptible to ototoxic chemicals and therefore can give false readings

when compared to older samples. Studies have shown that individual hair cell maturation rather

than larval maturation is the determining factor when observing stage-dependent differences.

Therefore, it is important to maintain strict control over the treatment dosage and hair cell

maturation when comparing across samples 32

Prior to the use of Zebrafish, in-vitro cell lines cultured from mammalian and avian

isolated hair cells were used. These in-vitro methods, however, are not capable of assessing the

metabolism of the drugs by the organism or the potential causes of toxicity in that animal. As a

result, many cell based assays fail expensive animal studies. More importantly, the function of

hair cells depends on not just the cells themselves but the surrounding support cells, which are

not present in in-vitro cell culture. Furthermore, cells cultured in-vitro have a limited number of

target sites when compared to whole organisms and can not represent the diversity available in

whole animals (ex. fish). In-vitro cell culture also requires the use of "fresh" primary cell lines

with short longevity times 34. In addition, up to this point, cell based assays have been based

more on availability and not on specific function. Moreover, isolated cells are less sensitive to

individual chemicals than whole fish, reducing their use in obtaining absolute safety levels 35

Therefore, in-vitro cell culture may be inadequate for reproducing in-vivo conditions.

There are, however, some drawbacks to the use of Zebrafish as a model for studying hair

cell loss. The hair cells contained in the neuromasts ofZebrafish do not contain stria vascularis

or spiral ganglia, which are known targets of ototoxic drugs, nor do they contain perilymph and

endolymph. In addition, unlike mammals, Zebrafish hair cells can regenerate after exposure to









ototoxic chemicals after 12-24 hours 28. Also, lateral line hair cell death may be accelerated in

Zebrafish when compared to mammalian ototoxicity results 32. Furthermore, the morphological

and physiological characteristics of neuromast hair cells are more similar to vestibular rather

than mammalian cochlear hair cells 30. Despite these limitations, the optical clarity of embryonic

Zebrafish, along with the selective uptake of fluorescent dyes and reduction in cost due to rapid

analysis of dose response assays, paves the way for measurable in-vivo assessment of hair cell

death 29,30

Absorbable Tympanostomy Tubes

The majority of commercial TTs are composed from either stainless steel, titanium,

silicone, or fluroplastic and do not have the ability to non-toxically degrade within the body.

Therefore, the duration of implantation is limited by the geometry of the tube and healing rate of

the patient. However, several new techniques have been developed for manufacturing tubes out

of absorbable polymers. Two polymer choices are poly (D, L-lactide-co-glycolide) (PLGA) and

poly-bis(ethylalanate)phosphazene (P-PHOS) 8-1. Both are biodegradable polymers that have a

defined resorption rate. The potential impact of these devices is to eliminate or at least reduce

the need to surgically remove displaced tubes or to help control retained tubes 8

However, the main drawback associated with these absorbable polymers is the inability to

control the rate of dissolution in-vivo. Once implanted, absorbable tubes have a pre-set

resorption time, which cannot be altered. As a result, after tube absorption any incidence of

recurrent otitis media with effusion will require tube re-insertion, increasing trauma to the

patient3

The development of these degradable TT has been spurred by the often unpredictable

nature of middle ear ventilation using conventional tubes. This is due to the extremely high rate

of spontaneous extrusion (up to 90%) among commercial tubes resulting from the natural healing









process. Currently the most common solution to combat premature extrusion and promote

extended ventilation is the use of "long-term" tubes. These tubes are designed to resist extrusion

while maintaining ventilation over an extended period of time (> 2 years). Usually, patients

requiring "long-term" ventilation have chronic or recurrent otitis media with effusion and require

repeated tube implantation. The development of the Goode T-tube in the 1970s provided a

device that resisted extrusion and offered long term ventilation. The Goode tubes and other "T"

shaped tubes achieve this with use of an extra wide flange on their medial side 36,37

There are, however, complications associated with the implantation of long-term

ventilation tubes which limits their widespread use. These complications include the need to

surgically remove the tube in up to 90% of retained cases, and the increased incidence of

tympanic membrane perforation post extrusion 37. Furthermore, tympanosclerosis (membrane

thickening) or granulations surrounding the tubes can form and cause bleeding. Also,

cholesteatomas (abnormal skin growth) can develop as a result of epithelial entrapment due to

localized atrophy of the tympanic membrane 9,37. On the other hand, the development and

implantation of a biocompatible and dissolvable TT will eliminate the need for surgery and can

further reduce the complication rate.

The significance of our approach is that it focuses on attacking one of the most prevalent

postoperative complications associated with tympanostomy tube implantation (reliable tube

ventilation). The use of dissolvable calcium alginate gel implants may reduce patient trauma by

reducing the need for additional surgical procedures. Also, with more effective implant devices,

the frequency of hospital visits for post operative care will decrease, and therefore the amount of

anesthesia administered to children will decrease. As a result, the recovery time will be

substantially quicker than with previous techniques.






















Figure 2-1: Surface structure oftympanostomy tubes (at ~ 500x magnification). (A) Uncoated,
large deep cracks in the tube surface. (B) Human serum albumin coated, small
surface cracks.


Guluronic Acid Group Mannuronic Acid Group
OH COO- OH
Coo- 0f7n 0O
0 OH O HO 00
fO H 1-10 0 O C0
C OH 0 i
I 0 0 COO- OH
OH COO-


Liquid alginate


Addition of Ca++ ions




+ V /
-^^^ 0

I) *-


^-^


Gelled alginate

0/*

0-v


Figure 2-2: Calcium alginate molecular structure. (A) Guluronic and Mannuronic acid groups.
(B) Egg-box structure for gelled calcium alginate.









CHAPTER 3
MATERIALS AND METHODS

Gel Preparation

For the following experiments, alginate (gel) was prepared by mixing 3 grams low

viscosity alginate powder (Sigma, A-2158, Macrocystis pyrifera (Kelp), M/G ratio -1.5) into

100ml deionized water (DI) using a magnetic mixer (Coming Stirrer/Hotplate, PC-420) at 500C

for 1.0 hour. The alginate gel has an M/G ratio of 1.5-1.67 and a molecular weight of 50 x 103

38,39. This mixture was allowed to rest over night resulting in a 3% concentration of alginate 39

Calcium chloride (curing solution) was prepared by mixing 1.1 grams of calcium chloride

(Sigma, C-2661, Calcium Chloride Anhydrous), with 100ml of DI water at 500C using the

magnet mixer. This mixture resulted in a 0.1M solution of CaCl. 0.5M, l.OM, and 2.0M

solutions were created by adding 0.266 grams, 0.599 grams, and 1.265 grams of calcium chloride

to 6ml of 0.1M CaCl prepared solutionl3'40

Polyethylene Glycol (PEG) (dissolution additive) was prepared by mixing 0.335 grams of

PEG (Sigma, P-3640, MW 3350) with 100ml of DI water at 500C. This mixture resulted in a

0.001M PEG solution 41

Sodium bicarbonate (Baking Soda, NaHCO3) (dissolution solution) was prepared by

mixing 0.1008g NaHCO3 into 12ml of DI water at 370C. This mixture resulted in a 0.1M

NaHCO3 solution. 0.5M, IM, and 2M solutions were created using 0.504g, 1.008g, and 2.016g

ofNaHCO3 in 12ml of solution 22,23. The above concentration ratios were chosen due to their

use in previous studies as well as their ability to rapidly crosslink each other into a solid gel 18

Prior to mixing, the solutions were filtered using a 0.22um filter (Fisherbrand 09-719A). The

filter also strains out any particulate matter contained within the alginate gel, PEG, NaHCO3, or









CaCi and creates a smooth homogeneous solution 39. All remaining solution concentrations were

described as necessary.

In-Vitro Ear Chamber

An in-vitro ear chamber was used to quantify tube plugging. The chamber was designed to

simulate the flow of a fluid (mucus simulant) and air through a tympanostomy tube.

Ear Model Construction

A model ear chamber was machined from commercially available acrylic (Lexan). This

included ports for the delivery of mucus and air into a larger chamber overlying a replaceable

latex diaphragm that held the TTs (Figures 3-1, 3-2). Figure 3-1 (section view of the ear

chamber) demonstrates the flow of mucus and air within the chamber and through the tube.

Figure 3-2 describes each of the ear chamber components. Figure 3-2C-D-E-F-G are shown in

section view in Figure 3-1. Figure 3-2B (foam seal (Darice Foamies Sheets, 1144-13, 2.2mm x

2.2mm x 2mm thick, Strongsville OH)) provides a seal for the front surface of the tympanic

membrane apparatus (Figure 3-2C, 3-2E) and prevents air/mucus from leaking. Figure 3-2A

(compression plate and machine screws) maintains chamber integrity while providing a port for

visual correlation of TT status with chamber pressure. Figure 3-2H corresponds to a fenestration

on the side of the air delivery port (Figure 3-2F) allowing for measurement of chamber pressure

via a pressure transducer.

A replaceable diaphragm, tympanicc membrane," consisted of a thin latex membrane

(Fisherbrand powder free latex exam glove, Fisher Scientific, Pittsburgh, PA) (Figure 3-2C, 3-

2E) supported by a rigid metal washer (Steel, 15.875mm outer diameter, 12.7mm inner diameter,

0.254mm thick). The membrane was glued to the surface of the washer (Elmer's Multipurpose

Spray Adhesive, E452, Columbus, OH) and then a tympanostomy tube was inserted through a

fenestration in the center.









Mucus Delivery

Flow of mucus was assumed to result from both production of mucus by the middle ear

mucosa and influx of air from the Eustachian tube during swallowing, sneezing, and nose

blowing. The rate of mucus production was assumed to be linear. However; it is "unknown" in-

vivo. Mucus flow rates (volumes) were based on observations from patients with post-TT

otorrhea. Rates of TT occlusion were measured over a wide range of mucus flow rates. Higher

flow rates (e.g., 1 ml / hour) resulted in nearly universal occlusion rates regardless of tube length,

composition, and diameter. At much lower flow rates (i.e., 50 [l / hour) TT occlusion was not

seen. Mucus delivery was increased until a consistent mid-range of occlusion was achieved with

Goode "T" TTs (i.e., 50% occlusion of TTs over 2.5 hours with egg white delivered at 80 [l /

hour or roughly 1 V2 drops / hour, total volume delivered was 0.22ml).

Mucus delivery was achieved using a custom built syringe pump consisting of a 5 led

unipolar stepper motor driver kit (Jameco Diy Kit 109 Stepper Motor, 139109, Jameco

Electronics, Belmont, CA) attached to a 24V stepper motor (Sanyo, 15837 MS,

www.MPJA.com). The rate of mucus injection into the chamber was half that of the air injection

(every 4 minutes) and was modeled to occur as a bolus, such that a fraction of the total mucus

volume was delivered at each episode. This system allowed for computer control of the mucus

delivery rate.

Air Delivery

Air flow was modeled to occur as a bolus, such as occurs with swallowing, nose-blowing,

and sneezing. Air was delivered to the ear chamber via an air compressor which was controlled

with a pressure regulator and a solenoid valve (Clippard, EVO-3, 12VDC, Cincinnati OH). A T-

valve was used to divert part of the air flow to a pressure transducer (Validyne, DP 15-46,

1546N1S4A, Northridge, CA) before entering the chamber (Figure 3-2F&H). The pressure









transducer was then connected to a digital-to-analog signal conversion board (Measurement

Computing, PMD-1208FS, Norton, MA). Flow was controlled and pressures recorded using

commercially available software (LabView version 6, National Instruments, Austin TX).

Boluses of air were delivered every other minute, corresponding to normal human

swallowing 42. Air was delivered at a volume twice that of hourly mucus flow. Air pressure was

determined by titration in pilot experiments. Pressures set to 25 mm H20 resulted in 100%

occlusion rate, independent of TT design. Pressures were gradually increased until the occlusion

rate decreased to 50%. This pressure value was (- 110 mm H20 20mm H20) and served as the

standard pressure for comparing TTs.

Mucus Analog Preparation

A human mucus analog consisting of chicken egg white (albumin) was used. Egg white

was chosen due to its very stretchy (stringy), non-homogeneous viscous nature (~4500cp non-

homogenized) 43 along with its ability to form a solid crust-like material after drying, similar to

human mucus 44. These characteristics facilitate rapid experimental analysis, providing

occlusion results within a matter of hours as opposed to months for in-vivo experiments. The

egg white was carefully separated from the yolk, homogenized using a long thin blade, and

stored at 50C until use. Egg white contaminated with yolk was discarded. The resulting egg

white samples had a physical consistency of serious mucus 44 and a viscosity of 388 5.8cp,

similar to homogenized human mucus (402 9. Icp). For each experiment a one ml syringe was

filled from the plunger port with 0.3 ml of albumin to prevent bubbles and reduce waste.

Tympanostomy Tube Testing Protocol

TTs of each design were inserted into latex membranes and tested in a random sequence.

A temperature controlled chamber was placed over the testing device and maintained a constant

370C environment. The chamber was pre-filled with 0.02-0.03ml of egg white. The chamber









pressure was recorded every second and stored in an Excel (Microsoft, Redmond, WA) file.

Raw pressure data were compiled and normalized. A plug was defined as a peak in the chamber

pressure equal to the input pressure (-110 cm H20). Pressure spikes below this input pressure

were considered partial occlusions.

Zebrafish Preparation and Housing

Zebrafish were obtained from the Aquatic Toxicology Facility at the Center for

Environmental and Human Toxicology (Building 118 Mowry Road). Fish were transferred at 4

dpf (days post fertilization) into 24 well plates (-10 fish per well). The fish were maintained in

embryo medium (13.7 mM NaCl, 540 tM KC1, pH 7.4, 25aM Na2HPO4, 44aM KH2PO4,

300aM CaCl2, 100lM MgSO4, 420 aM NaHCO3, pH 7.4) at 28.50C 29. After hatching, 5dpf fish

were exposed to dissolved and filtered gel tubes. Following exposure, a ImM DASPEI

fluorescent dye solution was created using embryo medium and the larvae were incubated within

this solution for 15 minutes. After exposure to the fluorescent dye, the fish were washed in

embryo medium and anaesthetized with 10[lg/ml MESAB (0.5mM 3-amino-benzoic acid ethyl

ester, 2mM Na2HP04). Following this, the fluorescent intensity, neuromast area, and number of

viable cells were measured. After experimentation, the fish were euthanized using MESAB

(250mg/L MESAB buffered with 200mg NaHCO3/L) for 5 minutes or until all respiration had

ceased28

Power Analysis: The number of fish required to achieve significant results for

experimentation was determined by performing a power analysis. The power analysis was

completed by consulting previously published work concerning the assessment of ototoxic and

otoprotective agents 29. Using results obtained from their research a delta of 1.0 was detected

with a power of 0.8, an alpha level of 0.05, and a sample size of n=17 was obtained using









Minitab (Minitab 13 Statistical Analysis Software). The value of delta constitutes a modest

change in toxicity that is both biologically, as well as statistically significant.

Statistical Analysis

Statistical analysis was conducted on all experimental results using a one-way un-stacked

ANOVA test (Minitab 13, Statistical Analysis Software Package). P-values below 0.05 were

considered statistically significant (95% confidence interval).





Mucus Input


Air Flow Input


Tympanostomy
Tube


Figure 3-1: Schematic overview of the ear flow chamber.















-.GH

B -. :j:- ,D.















Figure 3-2: Exploded view of the in-vitro ear chamber. (A) Front compression plate. (B) Foam
seal. (C) Vent tube. (D) Ear chamber. (E) Latex membrane (diaphragm) with ring. (F)
Input for air. (G) Input for mucus. (H) fenestration for measuring chamber pressure.









CHAPTER 4
RESEARCH DESIGN AND METHODS

Specific Aim 1: Develop an Alginate Gel Tympanostomy Tube

Rationale

TTs are described as any flanged tube manufactured out of biocompatible components that

is capable of providing reliable ventilation and limiting the development of secondary

complications. Critical parameters involved in developing a viable ear tube are mechanical

stability and consistent ventilation (low occlusion rate). When retained, commercial tubes are

surgically removed and are incapable of dissolving in-situ with the use of non-ototoxic

techniques. On the other hand, alginate TTs can be developed using biocompatible/dissolvable

ingredients, providing a non-toxic non-surgical removal method. Mechanical strength can also

be optimized to meet or exceed that of commercial tubes. Furthermore, design modifications can

be directly incorporated into the alginate gels limiting the occlusion rate.

Alginate was chosen as the tube material due to its unique ability to rapidly cure and its

high biocompatibility within the body 45. Furthermore, alginate has low cytotoxic and hemolysis

effects. Alginate gel can also be inertly crosslinked into a solid calcium-alginate gel with the use

of calcium chloride (CaCI) and dissolved back into solution with the addition of sodium (Na).

Moreover, alginate can conform into any shape allowing it to be molded into various tube

diameters and geometries. In addition, alginate is an abundant substance in the food industry and

is available in large quantities, making it inexpensive to produce.

Parameters Involved in Developing a Viable TT

The development of a reliable tympanostomy tube includes 4 main parameters: mechanical

strength, geometry, dissolution, and biocompatibility.









Mechanical Strength: Maintaining a compressive strength above that of commercial tubes

provides an environment for increased ventilation performance. Exposure to ototopical

solutions/in-vivo environments can decrease mechanical strength (ventilation performance)

leading to an increased incidence in occlusion formation. Parameters that may affect gel

strength include alginate/PEG ratio, Ca concentration, and desiccation time.

Geometry: Tube geometry, specifically barrel surface roughness, can contribute to

increased occlusion rates. Alginate surface roughness can be minimized using topical

treatments. Parameters that may affect smoothness include manufacturing techniques, and

albumin coating.

Dissolution: Commercial tubes that become retained require invasive surgical removal

procedures. However, alginate tubes have the ability to dissolve in-situ using minimally invasive

techniques. Parameters that may affect dissolution include alginate/PEG ratio, Ca concentration,

desiccation, dissolution solution concentration, and dissolution additives.

Biocompatibility: Biocompatibility associated with alginate tube dissolution reduces

patient trauma and provides an environment for healing. Parameters that may affect

biocompatibility include PEG concentration, Ca concentration, dissolution solution

concentration, and dissolution additives. Each of the above parameters was investigated and

optimized in order to develop a reliable tympanostomy tube.

TT Design

A negative aluminum mold was machined to produce several types of tubes (Figure 4-1)

(2mm long x 2mm wide bobbin-tubes and 6mm long x 3mm wide T-tubes). The bobbin- tubes

have an inner diameter of 1.02mm and the T-tubes have an inner diameter of 1.14mm. T-tubes

were used in testing mechanical strength (tension and compression) while Reuter Bobbin tubes

were used in testing occlusion rates. The inner lumen of the tube was developed using a glass









rod (a) which provided a smooth crosslinking surface and aided in tube removal. Depressions (b)

on one/both sides of the mold created the tube flanges. Final tube dimensions were increased

-80% to account for shrinkage during drying. The barrel wall thickness, after drying, was set at

~0.1-0.2mm.

Tube construction consisted of injecting Alginate:PEG (< 0.lml) solution into the mold

followed by the application of CaCl along the top flange surface of the gel. Twenty to thirty

minutes later, additional CaCl solution was injected through the top flange of the gel and into the

bottom (barrel) portion of the mold, curing the remaining alginate gel. Thirty minutes later,

(total curing time -60 minutes) the T-tube was removed by pulling the glass rod and gel out

through the top of the mold.

Reuter Bobbin tubes were created by injecting Alginate:PEG (< 0.lml) solution into each

mold using a 25.5 gage syringe with a 450 bend at the end. Following this, CaCl (< 0.lml) was

injected using a 27.5 gage syringe with a 900 bend. The syringe needles were bent in order to

completely inject the solutions into the bottom "hidden" flange of the mold. The CaCl was

injected first into the bottom flange of the mold followed by additional CaCl solution along the

top surface of the mold facilitating curing of both the flanges and barrel. After 45-60 minutes

(total curing time) the mold was separated gaining access to both flanges. Using tweezers, one

flange was pushed through the opening of the form and out of the mold. Care was taken to

prevent the glass rod from falling out during tube extraction. During extraction one of the two

flanges was damaged requiring repair. The damaged flange was rendered functional after

manipulation and application of additional CaCl curing solution.

Tube drying









Fresh alginate gel tubes were prepared for implantation via desiccation. Fresh tubes were

very flexible, large, and soft making them difficult to manipulate for implantation. Desiccation

removes water and dries the tube, potentially increasing strength, while preventing additional

shrinkage after implantation. Prior to drying, 0.01ml CaCl solution was applied to the gel tubes

prolonging desiccation time. Measured, deliberate drying first at 230C (24 hours) followed by 7-

10 days at 370C, removed water from the gel matrix, prevented additional shrinkage after

implantation, and produced a rigid strong tube. Tubes were suspended using the glass rod over

the wells of a 24 well plate preventing the flanges from flattening. After drying, tubes were cut

to a specific length and slid off their glass supports. Glass rods were removed prior to

experimentation and were used to position the tube for tympanic insertion.

Mechanical Testing

The mechanical properties of the alginate gels (T-tube configuration) were measured by

quantifying the modulus of elasticity and compressive strength of alginate gel according to

ASTM standards (D 638, D 882, D 3575, F 2064) 38,46-48. These results were compared with

those obtained from commercially available silicone tubes. The silicone tube (Goode T-tube,

Silicone, 1.14mm ID, 12mm length, Medtronic-Xomed Inc. Jacksonville FL) was used as a

benchmark for comparison with alginate gel tubes due to its relatively low mechanical strength

and widespread use.

Tension testing

Samples of alginate gel were molded into "dog-bone" specimens, dried, and clamped at

either end to a support (n=10 for each concentration). A dog-bone mold was cut into a 2.5mm

thick PDMS plate using an x-acto knife placed over a dog-bone pattern. Each specimen was cast

to a width of 2.0mm (at the thinnest section) and a length of 20mm. Alginate:PEG solution was

injected into the molds followed by CaCl curing solution. Glass microscope slides were placed









over the curing gel to prevent gel movement. Gels were cured for 30 minutes, removed from the

mold and additional CaCl curing solution (0. Iml) was added to the gels (groups of 5) to prolong

desiccation and promote crosslinking. Gels were kept in the incubator until testing. The dry gel

thickness and width were measured using a digital caliber (Mitutoyo Corp, 500-196, CD-6"CS).

Silicone samples were cut into "dog-bone" specimens from existing silicone tubes (12mm long,

0.25mm thick, center section width of 1-3mm, and an overall width of 5mm).

Samples were loaded into a mechanical loading device (Figure 4-2, 4-3) (Appendix A) and

a camera system was used to record the deformation against a calibration chart. Each specimen

was placed into the tension grips (Figure 4-2B) starting with the top grip, then lowered into the

bottom grip. Soft foam inserts were glued to the insides of the aluminum clasps increasing

friction and preventing gel damage. Samples were pulled with a constant strain rate using a

manual screw drive (Figure 4-2C) while a load cell (Honeywell-Sensotech, 34, 5 lbs, Columbus

OH), attached to the bottom grip, recorded the stress on the sample. The control arm (slide)

Figure 4-2D prevented rotation of the sample during loading. A Labview (Labview National

Instruments v8.0, Austin TX) data acquisition system along with a DAQ board (Measurement

Computing, PMD 1208FS, Norton MA) recorded and analyzed the results to determine the

modulus according to Hooke's Law Equation 4-1. Prior to experimentation samples were pre-

loaded with -5-10 grams (0.022 pounds) preceding stretching. The alginate gel samples used for

mechanical testing were assumed to be isotropic.


E= (4-1)


E= Young's Modulus, a = stress (Force/Area), F = strain (AL/L), L = length of gel sample,

Area = cross-sectional area

Data analysis









Video from each sample was digitized and opened (7 in long by 6 in high) in QuickTime

(QuickTime, Apple Inc.). The corresponding Excel file, obtained from the Labview program,

was also opened and graphed. The video was then cued up to the frame prior to failure. This

frame corresponded to the point of maximum tension on the Excel file and was used as the

starting point for analysis. The conversion factor between the screen image and actual

dimension was determined. Stress/Strain values were obtained every second (corresponding to

10 data points (data acquisition 10 Hz)) and recorded on the excel sheet starting from the max

load and decreasing until the load measured Og. This entire procedure was repeated for the each

of the remaining tension experiments.

Tube compression

Gel samples were also manufactured in a tube configuration and their compressive strength

was compared with commercially available silicone tubes. During the healing process, the skin

of the tympanic membrane compresses the barrel of the tube and in many cases can extrude it.

However, if the tube is too weak (mechanically), then the encroaching skin will compress the

barrel of the tube and occlude it. Therefore, the amount of force required to compress the barrel

of the alginate tube was measured and compared with the commercial silicone tube.

Compression testing consisted of deforming the barrel of the tube between two flat platens

while a camera system (Sony, ExwaveHAD Color Video Camera, DXC-190) attached to a zoom

lens (Edmund Industrial Optics, VZM 450i) observed the distance between the top and bottom

platen. The top platen was composed of aluminum, while the bottom platen was composed of

PDMS. A funnel composed of paper was attached to the aluminum platen and extended up into

a set of support arms providing stability (Figure 4-4). Prior to testing, part of the T-tube flange

was removed to allow the gel to lie flat on the testing surface. Red dye was applied to the front









surface of the tube increasing visibility (normally the gel tube is clear). Tube symmetry was

maintained after coloring with the re-insertion of the glass support rod. A calibration ruler was

placed on the video screen and initial measurements were taken of the diameter of the tube and

its placement on the bottom platen. The tube was not moved from this point on. The top platen

with the funnel was then carefully placed on top of the tube (1mm from front edge) and

adjustments were made to align the ruler on the video screen. Sugar was then slowly added to

the funnel while the distance between the platens was monitored on the screen (Appendix A,

Figure A2. After compression (25%), the funnel (sugar, top platen, and funnel) was removed

from the tube and weighed. The compression strength 46 of the tube was obtained from Equation

4-2 below.


CD = (4-2)
A

CD = compression deflection compressivee strength) force per unit of specimen area, (kPa). F =

force required to compress the specimen 25% outer diameter, (N). A = specimen compression

contact surface area, (m ).

Data analysis

The contact surface area (A) from the above equation was calculated using the

conservation of volume and Equation 4-3 (undeformed condition) and Equation 4-4 (deformed

condition) after 25% compression (Figure 4-5). The mechanical properties of the alginate tubes

were modified based on the ingredient concentrations while maintaining uniform dimensions

(inner diameter, outer diameter, wall thickness) across all tube types. As a result, only the force

(F) required to deform each of the tubes (25%) will differ. Therefore, assuming that the contact

area of each tube was uniform along with uniform wall thickness (h = constant before and after

deformation) the contact area (2b) can be determined by setting the front surface area of the









tubes equal to each other (Equation 4-3 equal to the top portion of Equation 4-4) and solving for

b (Equation 4-4 bottom). In 3D the contact area was described as a rectangle with sides equal to

2b and a length of L (tube length) (Appendix B). Each experiment was performed 10 times to

ensure accuracy.

Area(Aj) = A, A, = 7rX r, (4-3)



A reA2(2 h

4hb 2b-h

2b 2 (0.75)(d) (4-4)
h = thickness (cons tan t)
d = tube outer diameter


4h

Maximize Mechanical Strength

Using the methods described above, the mechanical properties of the gel tubes were altered

by changing the alginate/PEG ratio, Ca concentration, and desiccation time. Unless otherwise

noted, tubes were dried for 7 days prior to testing. Each parameter was tested separately via

tension and compression experiments to isolate their effect on the mechanical strength. Tension

and compression results were compared to commercial silicone Goode T-tubes.

Alginate:PEG concentration

The mechanical strength of the gel was enhanced by increasing the alginate ratio within the

gel. Alginate bonds with Ca from the crosslinking solution and creates a solid gel. Therefore,

the more alginate present in solution the more bonds will be created. PEG promotes dissolution

by limiting the amount of alginate available for crosslinking. Gel samples with Alginate:PEG

ratios (3% Alginate, 0.001M PEG determined from literature) ranging from 4:1 to 2:1 and finally









1:2 were created and mechanically tested. The mechanical strength of each gel configuration

was determined via tension and compression experiments. Initial experiments were crosslinked

with l.OM CaCl solution.

Ca concentration

In addition to the Alginate:PEG concentration, the Ca concentration was maximized to

promote mechanical strength. Higher quantities of Ca within alginate gels have been shown to

make stronger gels 49. Therefore, the concentration of Ca within the curing solution was

increased from 0.5M to 1.OM and finally 2.OM. The calcium concentration range was

determined from previous studies 13,40. The mechanical strength of each gel configuration was

determined via tension and compression experiments. The Alginate:PEG ratio used for testing

was determined from the above Alginate:PEG concentration experiments.

Drying time

As the gel tubes dry, moisture was removed, increasing rigidity. The effect of moisture

loss on gel tube strength was measured after drying over several days. Prior to desiccation

"fresh" tubes contain the highest concentration of water and are mechanically weak. Therefore,

desiccation time (370C incubator) was increased, while measuring the mechanical strength, from

1, 2, 3, 4, 5, 15, and 30 days, holding the Alginate:PEG ratio and CaCl concentration constant

(max values) from the above experiments.

Physiological Testing

The above experiments were designed to quantify mechanical strength based on tension

and compression experiments. Additional experimentation was conducted on samples subjected

to physiological conditions including agar encapsulation, ototopical solution exposure, and tube

plugging. These conditions simulated environments that may be encountered during in-vivo

implantation.









Quantify mechanical strength after agar encapsulation

TTs are designed to be implanted within a warm moist environment tympanicc

membrane) for several months until clinical symptoms resolve. During implantation it is

possible for the tubes to become encapsulated by the surrounding tissue. In-vivo tissue

encapsulation was simulated by encapsulation within an in-vitro agar substrate (30% w/v

Pluronic F-127 added to 125ml 0.85% NaCl, Sigma P2443). The solution was allowed to rest

for 3 days at 50C until homogeneous, followed by autoclaving for 15 minutes at 2500F. After

cooling, (50C) the agar solution was mixed with antibiotics (Antibiotic Antimycotic Solution,

A9909, Sigma) in a 10:1 ratio (agar : antibiotic) and poured into culture plates. Pluronic solution

is unique in that at 370C the gel forms a solid, while at 50C the gel is a liquid, making tube

insertion and extraction straightforward. After submersion for 15 and 30 days, tubes were

removed from the agar, washed in phosphate buffered saline (PBS) several times to remove

remaining surface agar, and compression tests were conducted. Results were measured and

compared to both a non-encapsulated alginate control and silicone commercial tube.

Quantify mechanical strength from exposure to ototopical solutions

During treatment, solutions may interact with the TT that can become detrimental,

prematurely degrading the gel and limiting ventilation. These ototopical solutions include pool

water, soapy water, salt water, hydrogen peroxide, blood serum, vinegar, ear mucus, and

antibiotic treatments or ear drops (Ciprodex, Ofloxacin). Children are the primary benefactors of

TT placement and periodic exposure to the above solutions is possible. Therefore, tubes created

using the strongest ingredient ratios (determined from above experiments) were submerged in the

above solutions for 24 hours, at 370C.









Solutions were prepared as follows. Chlorinated pool water (1-3 ppm) was applied (0.5ml)

to dried alginate gel tubes and the mechanical strength was recorded. Furthermore, soapy water

(hand soap (0.5ml)), salt water (35g Na/kg water), hydrogen peroxide 3%, vinegar (1:1 dilution),

human ear mucus (pooled samples), as well as FDA approved over the counter ear drops (0.5ml)

(Ciprodex, Ofloxacin) 26 were also exposed to the gel tubes. In addition, blood serum 0.5ml was

added to the gel tube enabling measurement of the effects wound healing might have on

mechanical strength. Serum was collected by centrifuging whole blood at 1,500rpm for 10-15

minutes followed by aspiration of the top "clear" serum layer above the red blood cell layer. The

concentration of salt water (35 g Na/kg water) was chosen to be similar to the sodium

concentration present in ocean sea water. Alginate gel samples remaining intact after

submersion for 24 hours were mechanically tested (tube compression). Results were compared

to a no-treatment control as well as commercial silicone tubes.

Quantify plugging of the alginate gel tube

The performance of the above ear tube was further characterized by comparing the

plugging frequency of the reuter-bobbin-shaped alginate tube (1.02mm ID, 2 mm long, alginate,

with and without human serum albumin) to a commercial stainless steel reuter-bobbin tube

(1.02mm ID, 2 mm long, stainless steel). Among commercial tubes, stainless steel TTs have the

highest occlusion rate and were therefore chosen for comparison with alginate tubes. Successful

gel tube designs should minimize plugging at or below commercial levels.

Plugging was minimized with the application of albumin (0.01% Human Serum Albumin

(HSA) (Sigma, A6909 30% in 0.85 NaCl, diluted in phosphate buffered saline (PBS))) along the

surface of the tube. Albumin has been shown to decrease surface roughness and limit occlusion

7. Albumin application was achieved after removal of the Alginate TTs from their glass support









rods and submersion in the albumin solution overnight at room temperature. Tubes were then

rinsed (2 times) in 10mM phosphate buffered saline (PBS) and dried with a Kim-wipe. Glass

rods were re-inserted after HSA application to reform the tube lumen. Gel tube samples were

tested, with and without human serum albumin, against commercial stainless steel tubes and in-

vivo clinical trials. Uncoated alginate tubes were submerged overnight in PBS only (no HSA).

All experiments were performed using an in-vitro ear chamber 50 outlined in Chapter 3. The

strongest tube composition as determined from mechanical testing experiments (Alginate:PEG

ratio, CaCl concentration, drying time) were developed into Reuter Bobbin tubes and tested

against the stainless steel tube. Stainless steel commercial tubes were not coated with albumin.

Comparisons were made between un-coated alginate tubes and un-coated commercial stainless

steel tubes as well as with albumin coated alginate tubes. Plug formation was measured by

comparing the experimental pressure to the known input pressure. Occluded tubes have the

same pressure reading as the input pressure. Each experiment was performed 10 times.

Specific Aim 2: Optimize the Gel Dissolution Components for Complete Dissolution

Rationale

Dissolvable TTs provide a minimally invasive method for removing ventilation tubes.

Currently, retained commercial tubes are surgically removed leaving the patient with an

increased risk of complications and trauma. Absorbable tubes have been developed to eliminate

or at least reduce the need to surgically remove displaced tubes. However, their rate of

dissolution, in-vivo, cannot be controlled. Once implanted, absorbable tubes have a pre-set

resorption time.

On the other hand, dissolvable ear tubes composed of calcium/alginate/PEG have the

potential to dissolve only in the presence of a dissolution solution (ex. sodium bicarbonate

(NaHCO3), NaC1, or vinegar), providing uninterrupted function if additional complications arise.









These solutions were chosen as potential dissolution solutions due to the presence of Na

(NaHCO3, NaC1), one of the ions capable of dissolving alginate, as well as their use in previous

studies treating TT sequelae (complications).

Since mechanical strength is directly related to tube ventilation, priority was given to

mechanically superior tubes over rapidly dissolving ones. Altering mechanical strength may

facilitate rapid dissolution, but at the cost of functionality. Therefore, dissolution was optimized

with regard to dissolution additives and dissolution concentration only. (If not otherwise stated

the gel tube composition (T-tube shape) for the following experiments was 3% Alginate in a 4:1

ratio with 0.001M PEG, 0.5M CaCl, and 0.5M NaHCO3 as the dissolution solution.)

Dissolution Analysis

Image analysis

Dissolution images were obtained using a CCD camera (Hamamatsu, C2400) attached to

a macro lens (Sigma Macro f=50mm 1:2.8). Images were taken at 0, 1, 2, 5 hours. Photographs

were adjusted for light intensity (Microsoft Word, Format Picture (Brightness-Contrast)) to

highlight the dissolution products.

Dissolution rubric

Tubes were prepared for dissolution after removal from their glass support rods and placed

within a 24 well plate. Images were taken prior to dissolution at t=0 hours and again at 1, 2, and

5 hours. Dissolution rate was determined using a dissolution rubric (R value) (Table 3). The

rubric consists of 6 stages: R=l (NO dissolution) through R=6 (complete dissolution). Images

were adjusted for light intensity bringing out the color and contrast of the gel products in

solution. R-value averages were compiled and graphed.









Optimize the Dissolution Components for Complete Dissolution

Dissolution additives

Ototopical solutions, including sodium chloride, vinegar and NaHCO3, have been used

clinically to treat TT sequelae and may potentially accelerate tube dissolution. Sodium (Na)

within the NaCl and NaHCO3 may degrade alginate TTs by replacing Ca within the crosslinked

gel. Vinegar along with sodium bicarbonate is highly reactive producing carbon dioxide gas

which may also be used to degrade alginate gels. Tubes were exposed to 0.5ml of sodium

chloride (0.85%) along with 0.5ml of vinegar (1:1 dilution, household distilled) and NaHCO3

[1.OM]. In addition, 50/50 ratios were created of NaCl and vinegar with sodium bicarbonate

solution.

A range of tube compositions, consisting of high and low concentrations, were developed

based on the results from Specific Aim 1. Priority was given to those solutions that dissolved

mechanically superior tubes since mechanical strength was conserved over rapid dissolution.

Tubes were tested against the above dissolution agents and those solutions providing complete

dissolution (R-value >3) within 1-5 hours were used in further dissolution experiments.

Dissolution concentration

Adjusting the quantity of Na present in solution may regulate the rate of alginate

dissolution. The optimal Na concentration designed to minimize dissolution time was obtained

by increasing the concentration of dissolution solution (ex. NaHCO3) from 0. 1M, to 0.5M, IM,

and 2M. Results were quantified using the rubric in Table 3.

Physiological Testing

Above experiments were dissolved by complete submersion within the various dissolution

solutions. However, in-vivo, dissolution solution is unable to completely envelop the tube due to









physiological limitations. Therefore, a dissolution chamber was created to simulate in-vivo

dissolution conditions.

Dissolution chamber

After determining the optimal dissolution solution and concentration from the above tests,

the remaining experiments were dissolved within an in-vitro ear chamber capable of simulating

the in-vivo tube conditions. After in-vivo implantation, access to the tube for dissolution was

limited. Therefore, an ear chamber was designed that allowed dissolution solution to be applied

only to one side of the tube (as seen in-vivo). Tubes (T-shaped) were suspended within a latex

membrane (flange side out) and supported by a thick metal ring. The thickness of the metal ring

(3mm) above the latex simulated the external ear canal and provided a holding area for the

dissolution solution. The barrel portion of the tube was suspended over a 24 well plate

representing the middle ear space. This implantation method differs (inverse) from that of in-

vivo implantation where the flange portion of the tube is implanted facing the middle ear space

and the barrel portion is implanted facing the external ear canal. Tube implantation was

modified in order to simulate a "worst-case" scenario. Access to the thicker flange portion of the

tube during removal (rather then thinner barrel portion) prolonged dissolution and represented

the maximum time frame (worst case) necessary for tube removal. Furthermore, mounting tubes

using this inverse method minimized tube extrusion during dissolution. Allowing the flange to

face the external ear canal prevents the gel tube from extruding from the latex membrane. The

tubes were suspended vertically allowing solution to flow through the tube. T-shaped tubes were

chosen due to their larger size (worst-case) and ease of manufacture. 0.5ml of dissolution

solution was applied to the external ear canal and the tubes were dissolved from the flange side

to the barrel side. Dissolution times were recorded and compared among samples.









Quantify the effect of dehydration on the dissolution rate of the gel tube

Throughout implantation, moisture was continuously removed and the tubes became

increasingly rigid. Increased mechanical strength and decreased dissolution may result from

increased rigidity. As a result, tubes were dried for 0, 15, 30 days at 370C 51 and the dissolution

rate was compared. Dissolution was performed within the in-vitro ear chamber.

Quantify the effects of human serum albumin coating on dissolution

Coating tubes with 0.01% human serum albumin (HSA) (Sigma, A6909, diluted in

phosphate buffered saline (PBS)) reduces the occlusion rate and limits patient morbidity

However, HSA coatings may alter the dissolution rate of the gel after application. Therefore,

HSA coatings were applied to alginate tubes overnight and dissolved using the in-vitro ear

chamber. R-values were obtained after 1-2 hours and compared to non-coated (PBS only)

alginate controls.

Specific Aim #3: Quantify Ototoxicity from Alginate Gel Dissolution using Zebrafish:

Rationale

Minimizing the ototoxicity associated with alginate gel tube implantation and especially

dissolution is critical to the acceptance of dissolvable alginate tubes as a viable alternative to

commercial tubes. The purpose of these ototoxicity experiments was to measure and quantify

toxicity associated with the dissolution of the alginate gel tubes. High toxicity can lead to patient

trauma including permanent hearing loss. Ototoxicity can be measured using an established

Zebrafish model. The resulting fluorescent intensity is directly related to hair cell ototoxicity

and can be compared to known ototoxic agents (Cisplatin). Viable hair cells within the

neuromasts of Zebrafish uptake fluorescent dye and glow. Non-viable cells do not uptake the

dye. This assay provided a quantitative analysis of the ototoxic effect of alginate gel tube

dissolution.









Quantify Ototoxicity from Dissolution Products

Fluorescent Staining: Zebrafish were fluorescently stained with ImM DASPEI solution

made from embryo medium for 15 minutes. The Zebrafish were then rinsed in fresh embryo

medium three times (5 minutes each), and anesthetized with 10tg/ml MS-222 for 15 minutes.

Fish were then transferred using a transfer pipette to a microscope slide with a depression, and

analyzed under a fluorescence microscope equipped with a DASPEI filter (450-490nm). Excess

embryo media was siphoned from the microscope depression using a kim-wipe. Neuromast hair

cells are located along the later line of the Zebrafish as well as along the head region (Figure 4-6)

28. A fluorescent intensity map of the target neuromast (P4) was measured followed by specific

localized analysis (neuromast area and number of hair cells labeled with DASPEI dye) (Figure 4-

6). Fluorescence intensity is directly related to the number of hair cells present within the

neuromast and is a function of the area stained using fluorescence. After experimentation

Zebrafish were euthanized using 250mg/L MS-222 buffered with ~200mg/L NaHCO3.

Pilot Study: Ototoxicity testing began by limiting morbidity/mortality associated with the

dissolution concentration and treatment exposure duration. Zebrafish are a freshwater fish and

are comfortably maintained at NaHCO3 concentrations at or below 0.004M in embryo media

(Appendix E). However, the NaHCO3 concentrations used to dissolve alginate gels varied

between 0.1M and 0.5M NaHCO3. Therefore, a pilot study was conducted to determine the

concentrations necessary to maintain life while providing measurable results. To this end,

NaHCO3 concentration was reduced along with the exposure duration (24hours, down to 1 hour,

30min, 15min, 10min, and 5min) in the hopes of achieving a non-toxic threshold. Five day old

embryonic Zebrafish (n = 3) were exposed to 0. lml of the dissolution products alginatee gel

tubes dissolved in NaHCO3 and filtered (0.22[tm)). The concentrations of the solutions were

prepared using embryo media as the "base" mixing solution. Analysis consisted of









morbidity/mortality outcomes associated with decreasing the concentrations and exposure times

of the dissolution products.

Once a survival threshold was determined, a positive control consisting of 50tM ImM

Cisplatin (Sigma-Aldrich, P4394) 30 and a negative control consisting of embryo media were

used to calibrate the UV analysis. The positive control has a well documented history of

Zebrafish hair cell ototoxicity 29,30 and provided a baseline reading for toxicity. Positive control

dosage was determined empirically in a pilot study identifying high dosages of Cisplatin that

caused hair cell death without mortality within relatively short time periods. The concentrations

of the solutions were prepared using embryo media as the "base" mixing solution. Fish were

individually separated into 96 wells plates and incubated at 280 C for testing. After treatment

exposure, fish were removed from the solutions and allowed to rest in embryo media for 3 hours

prior to fluorescent analysis.

From this, a survival and ototoxicity chart was created. Those concentrations and exposure

durations yielding measurable ototoxicity results (noticeable comparisons between treatments)

while maintaining survival were used for experimental analysis.

Experimental Protocol: After determining the threshold values for Cisplatin, NaHCO3,

and exposure duration, a power analysis was performed to determine sample size (n = 17)

(Chapter 3). Five day old embryonic Zebrafish were then exposed to 0. ml of the positive

Cisplatin and negative embryo media control along with dissolved and filtered alginate tubes

(calcium alginate gel tubes dissolved in NaHCO3 and filtered (0.22[am)). Once again the

concentrations of the solutions were prepared using embryo media as the "base" mixing solution

(Appendix C). Fish were individually separated into 96 wells plates and incubated at 280 C

during testing. After treatment exposure, fish were removed from the solutions and allowed to









rest in embryo media for 3 hours prior to fluorescence. Comparisons were made between the

controls and samples. Based on the ototoxicity results, dissolution was re-calculated using the

concentration and exposure times determined from the Zebrafish experiments.

Fluorescent analysis

Fluorescent intensity was measured using Adobe Photoshop (Adobe Photoshop v7.0,

Adobe Systems Incorporated). Neuromast P4 was isolated from each image and a fluorescent

intensity map was created. DASPEI stain glows green under fluorescent light. A range of

individual pixels representing the full range of green fluorescence (low to high) was selected

using the "color range" feature in Photoshop. After selection, the histogramm" feature was used

to quantify the average "green" luminosity (intensity). Neuromast area (in pixels) was also

determined via the histogram results. Pixel area was converted to nanometers via a micrometer

calibration. Individual hair cells were represented by circular globes of green light within the

neuromast and were counted microscopically.















i a) Glass Rod


Short
Reuter-
Bobbin
Tubes


"T" Shaped
~Tube
(Long)

---I
v~-






Figure 4-1: Aluminum ear tube mold used for creating bobbin and T-shaped tubes. (A) Glass
rods. (B) Flange groove.


































Figure 4-2: Mechanical testing device (Tension). (A) Dog-Bone testing specimen between
clamps. (B) Sample clamps with foam inserts. (C) Hand screw drive. (D) Control
arm (slide). (E) Load cell. (F) Stand base.










































Figure 4-3: Tension testing apparatus setup.

































Figure 4-4: Tube compression apparatus.




F=O F






P. d 0.75(d) r
2b r2b







Figure 4-5: Tube compression contact area schematic. (A) Undeformed silicone and alginate
gel tubes. d = distance between the two platens. d = outer diameter of tube. ri = inner
radius. r2 = outer radius. F = force (load) applied to the barrel of the tube. (B)
Deformed tube (25%) showing contact area of 2b. h = tube wall thickness











Table 4-1: Dissolution rubric showing various stages of tube dissolution.


R=l : Tube remains in tact R=2 : Tube remains in tact, R=3 : Tube dissolved, large
little to no change swollen, bloated, soft pieces remain


R=4 : Tube dissolved, small R=5 : Tube dissolved, only
pieces remain fine particles remain


R=6 : Tube dissolved, tiny
particles remain, clear


oc b


M-T


1mm


Figure 4-6: Position of head and lateral line neuromasts. Position names are based on body
position. D = dorsal trunk, IO = infraorbital, M = mandibular, MI = middle, 0 = otic,
OC = occipital, OP = opercular, P = posterior, PO = preoptic, SO = supraorbital.









CHAPTER 5
RESULTS

Specific Aim 1: Development of an Alginate Gel Tube

Both T-shaped and bobbin shaped alginate gel tubes were created using an aluminum

mold. The results show that after 24 hours (at 230C) the gel tubes had begun to dry and had

retained most of their original size (90% original size). After 6 additional days (at 370C) the gels

were completely dried and could be moved freely on the glass rod. On the other hand, rapid

desiccation (370C for 5 days) proved to be detrimental, resulting in shriveled unusable tubes.

Alginate T-tubes (Figure 5-1, 5-2, 5-3) have a flange on one end to prevent the tube from

expiating after insertion. Alginate T-tubes have a dried barrel outer diameter = 1.5-1.6mm, a

wall thickness = 0.1-0.2mm, a length = 5-6mm, and an inner diameter = 1.1-1.2mm. Reuter

Bobbin tubes (Figure 5-4) have a flange on both sides with a short 1-2mm barrel between them.

Reuter Bobbin tubes have a final dried barrel diameter of 1.6mm, a wall thickness of 0.2mm,

and a length of 2mm (Figure 5-5). Furthermore, dimensional constraints appear to be conserved

after drying. However, dimensions can be altered depending on physician specifications and use.

Maximize Mechanical Strength (Tension)

Alginate:PEG Raito: Alginate gel strength was measured first in tension (dog-bone

configuration) followed by compression (tube configuration) (See Appendix B). Dog-Bone

specimens were created, dried for 7 days, and tested for each gel concentration and ratio starting

with Alginate:PEG [4:1], [2,1], [1,2] crosslinked with [1.OM] CaCl solution. Experimental

results show ratios lower than 1:2 (ex. 1:3, 1:4...) fail to produce solid gel tubes. The dried dog-

bone dimensions were 10-12mm long, 1-2mm wide and 0.3-1.0mm thick. From these tension

experiments it was determined that an Alginate:PEG ratio of [4:1] (Figure 5-6) yielded the

highest average modulus (~20,000kPa).









CaCI Concentration: Based on the results above, CaCi concentrations ([0.5M], [l.OM],

[2.0M])(Figure 5-6) were varied using gels composed of an Alginate:PEG ratio of "[4:1]".

Statistical comparisons were made between the experimental results and a commercial silicone

tube control. Alginate:PEG ratios of [4:1] along with the [0.5M] and [1.OM] CaCl crosslinking

solutions were all significantly stronger then the commercially available silicone tubes in tension

with [0.5M] CaCl achieving a maximum value (~50,000kPa) (*P-value < 0.05).

Drying Time: Gels composed of an Alginate:PEG ratio of [4:1] crosslinked with [0.5M]

CaCl yielded the strongest overall modulus (from above) and were used to determine the optimal

gel drying time. Results show drying times greater then 4 days produce gels that are

significantly stronger than commercial silicone tube controls in tension (*P-value <0.05) (Figure

5-7). Mechanical strength levels off after 4 days with minimal increases up to 30 days. Based

on these results, the strongest gel combinations were achieved using an Alginate:PEG ratio of

[4:1] crosslinked with a CaCl concentration of [0.5M] that had been dried for at least 4 days.

Maximize Mechanical Strength (Compression)

Compression Testing: Alginate gel tube compressive strength was determined by

measuring the force required to compress the gel tubes 25%. The results follow a similar trend

to those from the tension experiments (Figure 5-8, 5-9) where the strongest gel tube in

compression was composed of an Alginate:PEG ratio of [4:1] crosslinked with a CaCl

concentration of [0.5M] (Figure 5-8) and allowed to dry for at least 1 day (Figure 5-9). All

ingredient concentrations and combinations were significantly stronger than the commercially

available silicone tube in compression (Figure 5-8) (*P-value < 0.05). Furthermore, in

compression, gel samples dried for longer then 1 day (Figure 5-9) were all significantly stronger

then the commercially available silicone tube (*P-value < 0.05).









Physiological Experiments

After determining the mechanical properties of the alginate gels in tension and

compression physiological experiments were conducted.

Quantify compressive strength after agar encapsulation

In-vivo tissue encapsulation was simulated by encapsulation within an in-vitro agar

substrate. Mechanical compressivee) strength of alginate gel tubes ([4:1] Alginate:PEG

crosslinked with [0.5M] CaCl and dried for 7 days), after encapsulation within an agar substrate

(Pluronic F-127), was measured over 15 and 30 days. Results show a maximum of 50% loss

(Day 15) in mechanical compressive strength over the 30 days compared with a non-

encapsulated control (Figure 5-10). Fifteen day encapsulation results were significantly weaker

than the alginate control (*P-Value = 0.014). However, 30 day results were statistically similar

to those of the control (*P-Value = 0.299). Fifteen day results are not significantly lower than

(Day 30) results (*P-Value=0.087). However, all tubes encapsulated within the agar were

significantly stronger than the silicone tube in compression (*P-Value < 0.05)

Quantify compressive strength from exposure to ototopical solutions

Children are the primary benefactors of TT implantation and occasional contact with

ototopical solutions can occur. Alginate gel compressive strength after 24 hour exposure to these

ototopical solutions was determined. Tubes were composed of Alginate:PEG [4:1] crosslinked

with [0.5M] CaCl, and dried for 7 days. Ototopical solutions included pool water, soap water,

salt water, H202, Blood Serum, Vinegar, Ear Mucus, Ciprodex, and Ofloxacin. Results show

that exposure to Ofloxacin (antibiotic) yields the largest drop-off in compressive strength of

(73%). Compressive strength was conserved in alginate tubes exposed to blood serum (47%

decrease from control) (Figure 5-11). Ear Mucus showed a decrease in compressive strength of

58%. All ototopical treatments reduce the compressive strength significantly (*P-Value < 0.05),









from the no treatment alginate control. However, none are below the compressive strength of the

commercial silicone tube which is more than 90% lower than the alginate control and greater

then 60% lower than the most detrimental treatment (P-value < 0.05).

Minimize alginate gel tube plugging

The plugging rate of the alginate reuter-bobbin tube was measured using an in-vitro ear

chamber. Ten commercially available stainless steel reuter-bobbin tubes and 20 Alginate:PEG

[4:1], CaCl [0.5M], 7 day dried gel tubes (10 uncoated and 10 coated with human serum albumin

(HSA)) were tested in the chamber. Occlusion results were compared to a previously published

report showing the in-vivo plugging rate of a stainless steel reuter-bobbin tube to be 74% 52. The

experimental outcome showed uncoated alginate gel tubes with a plugging rate of 50%, while the

HSA coated alginate tubes had a plugging rate of 30%. Results indicate that the process of

submerging/coating alginate tubes in HSA limits the formation of an occlusion. Glass rods were

re-inserted after HSA application to reform the tube lumen due to swelling resulting from re-

hydration. Uncoated stainless steel reuter-bobbin tubes had a plugging rate of 70% using the in-

vitro ear chamber (Figure 5-12). HSA coated alginate tubes were 50% less likely to occlude than

the stainless steel tubes (30% plugging vs. 70% plugging) while un-coated alginate tubes were

20% less likely to occlude. Survival distribution function curves (Kaplin-Meier Method) show

that the HSA coated tubes resist occlusion over a significantly longer time period than

commercial stainless steel tubes (*P-value = 0.047) (Figure 5-13).

Specific Aim 2: Optimize the Dissolution Components for Complete Dissolution

Dissolution Solutions

Removal of alginate gel TTs from the tympanic membrane can be achieved with the use of

a dissolution solution. The rate of dissolution was measured after exposure to several treatments

(NaC1, Vinegar, NaHCO3). Gel tubes, of various compositions, were exposed (complete









submersion) to each treatment for several hours (1-5 hours) and given an R-value based on the

amount of dissolution that had occurred. Results were averaged and graphed (Figure 5-14).

Analysis shows that while dissolution is maximized (R-value = 4.5) in gels composed of an

Alginate:PEG ratio of [1:2], mechanical strength is limited (1 hour). Tubes composed of an

Alginate:PEG ratio of [4:1] (high mechanical strength) achieved an R-value of 3.5 after only one

hour, using a 50/50 mixture of NaCl and NaHCO3 [1.OM] and NaHCO3 alone [1.OM] (Figure 5-

14). Dissolution continued to increase, while solution was present, from 1 to 5 hours (Figure 5-

15). Vinegar and vinegar+NaHCO3 proved to be minimally effective in removing alginate tubes.

Although promising, NaCl+NaHCO3 did not perform (dissolve after 1 hour) as well as the

NaHCO3 alone across all samples. On the other hand, sodium bicarbonate's (NaHCO3)

dissolution performance as well as its use in previous clinical studies provides a viable method

for removing TTs after insertion. Therefore, sodium bicarbonate was used as the dissolution

solution for the remaining experiments.

Dissolution Concentration

The quantity of NaHCO3 present in solution may alter the rate of tube dissolution. As a

result, NaHCO3 concentration was increased from [0.1M], to [0.5M], [1.OM], and [2.0M] while

measuring dissolution (R-value). Results show that dissolution is not linearly related to NaHCO3

concentration, rather dissolution peaks at [0.5M] (R=5, 5 hours) followed by decreased

dissolution (Figure 5-16). Over time (1-5 hours), dissolution increases significantly (P-Value <

0.05) across all treatments.

Physiological Experiments

After determining the optimal dissolution concentration and ingredients, remaining

dissolution experiments were performed within an in-vitro ear chamber which limited dissolution

access to one side of the tube.









Effect of desiccation on dissolution

Throughout implantation (drying in an incubator at 370C) moisture was removed from

the tube. The effect of increased drying time (0, 15, 30 days at 370C) on dissolution was

measured within an in-vitro ear chamber (See Figure 5-17). The dissolution conditions within

the in-vitro ear chamber were similar to those encountered during in-vivo dissolution. Results

show that the alginate tubes dissolve in two steps: first the barrel portion dissolves away from the

flange, followed by swelling and breaking apart of the flange portion. After one hour, the flange

portion of the each of the tubes had only partially dissolved and incomplete dissolution was seen

in the majority of the tube barrels. Then, additional dissolution solution was applied to the tubes

to replace solution lost to evaporation. After two hours the fresh tubes (flange and barrel) had

dissolved to R=4.2 while the 15 and 30 day tubes (flange and barrel) had dissolved to an R=5.3

and R=5.5 respectively (Figure 5-17). All dried tubes dissolved within two hours based on the R

value. Analysis shows that fresh tubes or tubes with a higher percentage of internal moisture

dissolve at a slower rate then those dried for extended periods of time (*P-Value < 0.05).

Dissolution of HSA coated alginate tubes

Previous experiments have shown that HSA coated tubes limit tube plugging (Figure 5-

12) and increase performance. However, it was not known what effect HSA coatings have on

dissolution. As a result, tubes coated in HSA solution were dissolved in an in-vitro ear chamber

using [0.5M] NaHCO3 for 1-5 hours and compared to un-coated alginate tubes. Results show

that HSA coatings do not negatively affect dissolution (Figure 5-18). After 5 hours all tubes

coated in albumin were completely dissolved (R=5.5). No significant difference was measured

between HSA coated and un-coated alginate tubes when dissolved in [0.5M] NaHCO3 (*P-Value

> 0.05). Dissolution results are summarized in Figure 5-19 and Table 5-1.









Specific Aim 3: Quantify Ototoxicity from Exposure to Dissolution Products

Pilot Study Results

Dissolution solution concentration and treatment exposure times were optimized based on

morbidity changes. NaHCO3 + (dissolution solution along with dissolved alginate gel tubes)

concentration was reduced from 0.5M to 0. 1M while reducing the exposure time (24 hours, 1

hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes). Cisplatin concentration was

simultaneously increased to obtain measurable results within short exposure times. Survival and

ototoxicity results based on treatment and exposure time was recorded (Table 5-2). Results show

that the highest concentration tolerated by the Zebrafish was 0. 1M NaHCO3 + for a duration of

30 minutes. The Cisplatin concentration necessary to elicit a measurable result after 30 minutes

was ImM.

Ototoxicity after Treatment Exposure

After optimization, ototoxicity experiments based on exposure to the above treatments

(dissolution products and controls) were conducted. Zebrafish hair cells were isolated and

analyzed via fluorescence under a UV microscope (Figure 5-20). Fluorescent intensity,

neuromast area, and hair cell quantity were measured and compared, at neuromast (P4) between

all three treatments (dissolved and filtered alginate gel tubes, Cisplatin solution, and embryo

media solution). Figure 5-21 and Figure 5-22 show the mean fluorescent intensity between all

three treatments (after normalization to the no-treatment control) when compared to the negative

(No-Treatment) embryo media control. Analysis shows that Zebrafish hair cells exposed to

dissolution products have a significantly higher fluorescent intensity (*P-value = 0.032) than

those exposed to the positive Cisplatin control. Furthermore, there was no difference in

fluorescent intensity (*P-value = 0.322) between the negative (No-Treatment) control and the

dissolution products proving that the dissolution products have little to no effect on hair cell









mortality under these specific conditions. There is however, a measurable difference in

fluorescent intensity between Zebrafish hair cells exposed to both the negative and positive

control (*P-value = 0.004).

Moreover, neuromast size (area of fluoresced hair cells) is directly related to the number of

viable hair cells present within each neuromast. Figure 5-23 shows the relationship between

neuromast size after a 30 minute treatment exposure. Results show that Zebrafish hair cells

exposed to dissolution products have a significantly larger neuromast size than those exposed to

the positive Cisplatin control (*P-Value=0.004). The results were similar to the fluorescent

intensity results with no differences in neuromast size between the negative (No-Treatment)

control and the dissolution solution (*P-Value=0.235) and significant differences in neuromast

size between both the negative and positive control (*P-Value=0.000).

Furthermore, the number of viable hair cells located within neuromast P4 varied

depending on the exposure treatment. Results show that hair cell quantity is directly related to

treatment. Figure 5-24 shows that both the negative (No-Treatment) control and the dissolution

products have significantly greater numbers of viable hair cells than the positive (Cisplatin)

control (*P-Value=0.039 and *P-Value=0.000 respectively). Results are summarized in

Appendix D.

Additionally, dissolution experiments were re-done using the lower dissolution

concentration (0. 1M NaHCO3) and exposure time (30 minutes) to determine if dissolution can be

achieved. Within the 30 minute exposure time no changes were seen in dissolution of the

alginate gel. However, results show that tubes dissolved within 0. 1M NaHCO3 required -24

hours to completely dissolve (R=5.5).























Figure 5-1: Fresh Calcium alginate/PEG T-tube on glass curing rod.


Figure 5-2: Dried Calcium alginate/PEG T-tube on glass curing rod.


Figure 5-3: Dried Calcium alginate/PEG T-tube removed from glass curing rod and placed in
dissolution well.






















Figure 5-4: Fresh Reuter Bobbin Tube. Note: one flange (right side) is completely in tact while
left flange is missing part due to the manufacturing.


Figure 5-5: Dried Reuter Bobbin Tube. Note: tube overall diameter has decreased due to
shrinkage during drying.











80000.00


CaCI [0.5M]


70000.00


60000.00


50000.00


o 40000.00


2 30000.00


20000.00


10000.00


0.00


Alginate [4:1]


Alginate [2:1]


.L. Alginate [1:2]


CaCI [1.OM]






CaCI [2.OM]


Figure 5-6: Mechanical testing (Tension) average modulus results for Alginate:PEG ratio and
CaCl concentrations when compared to commercially available silicone tubes. The
calcium concentrations were made using an Alginate:PEG ratio of [4:1]. (* indicates
mechanical strength is significantly greater then silicone control, P-value <0.05)
(n= 10)











1000000.00


Day5
Day 4
T


Day 15


Day 30
T


100000.00


Silicone

S-i- --


10000.00



1000.00



100.00



10.00


1.00 -1


Day 3


Day 1
T


Day 2


Figure 5-7: Mechanical testing (Tension) results for the effect of desiccation time on the
mechanical strength of the alginate gel when compared to commercially available
silicone. (* indicates mechanical strength is significantly greater then silicone control,
P-value <0.05) (n=10)











400.00


Calcium [0.5M]


Calcium [1.OM]











Calcium [2.OM]


Figure 5-8: Compressive strength of the Alginate:PEG gels based on alginate composition and
CaCi crosslinking concentration. All results are compared to a commercially
available silicone Goode tube. The calcium concentrations are made using an
Alginate:PEG ratio of [4:1]. (* indicates mechanical strength is significantly greater
then silicone control, P-value <0.05) (n=10)


Alginate:PEG [4:1]


350.00


' 300.00
0-


' 250.00


) 200.00


S150.00

E
0 100.00


50.00


Alginate:PEG [2:1]


Alginate:PEG [1:

-----T----


Commercial


0.00











350.00 -


300.00 Day 30
3 0 0 .0 0 - - - - - - - - - -

Day 5 Day 15
a-
.250.00



0 200.00 -

) Day 2
Z 150.00 --------- signif-ca---------- Day3 3 ---- --- ----



E 1 (oo.oo -



50.00 Day 1
0. Silicone


0.00


Figure 5-9: Compressive strength of the Alginate:PEG gels based on desiccation time. All
results are compared to a commercially available silicone Goode tube. (* indicates
mechanical strength is significantly greater then silicone control, P-value <0.05)
(n= 10)










350.00 Alginate Control


Agar
Encapsulation :
15 day


Agar
Encapsulation :
30 day


Silicone Control

m-


Figure 5-10: Compressive strength of agar encapsulated alginate gel tubes after 15 and 30 days
compared to non-encapsulated alginate gel tube and silicone control. (n=5)


300.00


. 250.00


C 200.00
u,

150.00


E
o 100.00
U


50.00


0.00











Alginate
350.00 Control



300.00



a. 250.00



f 200.00

0)
Blood Serum *
150.00 *
CO Pool Water Salt Water Vinegar Ear MucusCiprodex
Soap Water
E T T H202
0 100.00
Ofloxacin

50.00 Silicone
Control


0.00 --


Figure 5-11: Compressive strength of alginate gels exposed to various ototopical solutions for 24
hours. Most detrimental treatment was Ofloxacin (73% decrease) while least
detrimental was blood serum (47% decrease). All treatments are significantly
stronger then the silicone control sample and significantly lower then the alginate
control sample (P-Value <0.05). (n=5)











Weigel, 1989
Stainless Steel


80%


70%


60%


a)
. 50%
a)

- 40%
o


0
30%


20%


10%


0%


Stainless Steel


UnCoated Alginate


HSA Coated
Alginate


Figure 5-12: Occlusion percentage of the alginate gel tubes (HSA coated and uncoated)
compared to the uncoated stainless steel tubes using the in-vitro ear chamber. Results
compared to published in-vivo stainless steel occlusion rates from Weigel et al. 1989
(n= 10).


I I














10 --------- SSTube
--- ALGINATE
0.9 ------- ----- HSA Coated

08 -

0 07-
0 --

S 0.6 L
LL

0
-0.5- -- - -


30 3

C 02 -

S01
C)
00 -

0 50 100
Time to Failure


Figure 5-13: Nonparametric Survival Plot (Kaplin-Meier Method) for stainless steel, uncoated
alginate, and HSA coated alginate tubes. Tubes were tested in the in-vitro ear
chamber. (n=10)












BAIginate:PEG [1:2] CaCI [0.5M]
OAlginate:PEG [1:2] CaCI [2M]
*Alginate:PEG [4:1] CaCI [0.5M]
OAlginate:PEG [4:1] CaCI [2M]


4.5


4.0


3.5
0)

C 3.0


o 2.5

0
-


a 2.0
5

1.5


1.0


0.5


0.0


NaCI


NaCI + NaHCO3 Vinegar


Figure 5-14: Dissolution results for additives (NaCI and Vinegar vs. NaHCO3) over 1 hour.
Tubes were completely submerged within the dissolution solution. (n=5)


Control (Water)


NaHC03 (1M)


-1 -












6.0 AIginate:PEG [1:2] CaCI [0.5M]
*Alginate:PEG [1:2] CaCI [2M]
*Alginate:PEG [4:1] CaCI [0.5M]
OAlginate:PEG [4:1] CaCI [2M]
5.0 mm


NaCI+NaHCO3 (5 hr)


a4.0
0


o 3.0




2.0




1.0




0.0


Figure 5-15: Five hour dissolution results for additives (NaCI and Vinegar vs. NaHCO3). Shows
how dissolution continues in the presence of the dissolution solution. (n=5)


Vinegar (5 hr) Vinegar+NaHCO3 (5
hr)


NaCI (5 hr)


NaHCO3 (5 hr)











6.0 -
---1 Hour
5 Hour

5.0 -



0 4.0 -



c 3.0



o 2.0



1.0



0.0
NaHC03 [0.1M] NaHC03 [0.5M] NaHC03 [1.OM] NaHC03 [2.OM]


Figure 5-16: Dissolution results based on NaHCO3 concentration (0.1M, 0.5M, 1.OM, 2.OM)
over 1-5 hours. All 5 hour results are dissolved significantly more then 1 hour results
(P-Value < 0.05). Tubes were completely submerged within the dissolution solution.
(n=5)














6.0



5.0 -









0
1 4.0









0.0
Day 0 (2 hour) Day 15 (2 hour) Day 30 (2 hour)


Figure 5-17: Effect of drying time (0, 15, 30 days dried) on the dissolution rate of the gel tube.
15 and 30 Day drying times dissolved significantly faster then fresh (Day 0) tubes. (*
P-Value < 0.05). Tubes were dissolved within an in-vitro ear chamber. (n=5)



















5.0
-


2 4.0
-
Cr


o 3.0
'a

O
u)
* 2.0



1.0



0.0


E HSA Coated

r Un-Coated


Flange (1 hour)


Barrel (1 hour)


Flange (5 hour)


Barrel (5 hour)


Figure 5-18: Effect of HSA coating on the dissolution rate of the alginate gel tubes. Comparison
between flange and barrel portions as well as between un-coated/coated alginate tubes
dissolved in [0.5M] NaHCO3. (No significant difference was seen between HSA
Coated and Un-Coated tubes (P-Value > 0.05)) (n=5)


* I


I I I I I




















































Figure 5-19: Dissolution within ear chamber over time (1-5 hours). (A) Front of TT (facing
flange), showing depression for dissolution solution (t=0). (B) Back of TT (facing
barrel), showing latex conforming around barrel of tube (t=0). (C) Front of TT
(flange), showing dissolution solution and deformed TT flange (t=lhr). (D) Well
"middle ear space" with excess dissolution solution, barrel dissolution (t=lhr). (E)
Front of TT (flange), completely dissolved tube and hole in elastic membrane (t=5hr).
(F) Well "middle ear space" where dissolution of barrel occurs (t=5hr).


81









Table 5-1: Dissolution Results Based on Treatment and Dissolution Time
1 Hour 2 Hour 5 Hour 24 Hour
Dissolution Dissolution Dissolution Dissolution
NaCl None None None None
NaCl + NaHCO3
al + aH 3 Partial/Complete Partial/Complete Partial/Complete
(IM)
Vinegar None None None
Vinegar +
NaHC3(aM) None None None
NaHCO3 (IM)
NaHC03 (0.1M) None None None Complete
NaHCO3 (0.5M) Partial Partial Complete
NaHCO3 (1.OM) Partial/Complete Partial/Complete Partial/Complete
NaHCO3 (2.0M) Partial Partial Partial
0 Day Drying Partial Partial
15 Day Drying Partial Complete
30 Day Drying Partial Complete
HSA Coated
lange Partial Partial Complete
(flange)
HSA Coated
HSA Coated Complete Complete Complete --
(barrel)
None = R-Value < 2; Partial = R-Value < 5; Complete = R-Value > 5

Table 5-2: Survival and Ototoxicity Results Based on Treatment and Exposure Time
24 hours 1 hour 30 minutes 15 minutes 10 minutes 5 minutes
0.5M x x x x x x
X X X X X X
NaHCO3 +
0.25M
NaHC03 +
0. iM x X SURVIVAL Survival Survival Survival
NaHCO3 +
50tM Measurable
X X X X X
Cisplatin Ototoxicity
ImM MEASURABLE Measurable
Cisplatin OTOTOXICITY Ototoxicity













Neuromast (P4)


4 0>


Head


Figure 5-20: Whole fish fluorescence after DASPEI stain. Neuromasts located along the head
and length of the body. Stomach and gut readily absorb DASPEI stain shown as dark
purple regions. Highlighted region represents neuromast P4.





Iissolution Products


at


a


Cisplatin


,i *

Negative Contrdl


*

25 um


Figure 5-21: Fluorescent intensity for all three treatments (Dissolution Products, Positive
Cisplatin Control, Negative (No-Treatment) Control). Both dissolution products and
negative (No-Treatment) control neuromasts have very high fluorescent intensity
(dark purple) whereas Cisplatin (a known carcinogen) have very low fluorescence
(light purple). (n=18)


U *


Imm


Tail


p---


Body


^^^ .No'












Negative No-Treatment
Control


120


100
u,


0
c 80






40
V,


Cisplatin-Dissolution : P-Value=0.032
Dissolutuion-No Treatment : P-Value=0.322
Cisplatin-No Treatment : P-Value=0.004


Dissolution Products



T


Positive Cisplatin
Control


Figure 5-22: Mean fluorescent intensity between the positive, negative, and dissolution product
treatments. Results normalized to the negative (No-Treatment) control. (n=18)












Negative No-Treatment
Control


Cisplatin-Dissolution :
Dissolutiuon-No Treatment:
Cisplatin-No Treatment :


500.0


450.0


400.0


C 350.0

E
3. 300.0
N
w 250.0


E 200.0


Z 150.0


100.0


50.0


0.0


P-Value=0.004
P-Value=0.235
P-Value=0.000


Positive Cisplatin
Control


Figure 5-23: Neuromast size based on exposure treatments. Both the negative (No-
Treatment) control and the dissolution products maintained a higher neuromast size
then the positive Cisplatin control. (n=18)


Dissolution Products











No-Treatment Control



T


8.0


7.0


u 6.0
C)

.0 5.0
4-
0
4.04.
E
z 3.0

2.0


1.0


0.0


Dissolution Products
Cisplatin-Dissolution : P-Value=0.039
Dissolution-No Treatment : P-Value=0.074
Cisplatin-No Treatment : P-Value=0.000

Positive Cisplatin
Control


Figure 5-24: Hair cell viability (number of hair cells per neuromast) based on treatment. (n=18)









CHAPTER 6
DISCUSSION

Specific Aim 1: Development of a Calcium Alginate Tympanostomy Tube

Developing the Alginate TT

The first step in the development of an implantable alginate TT was to create gel tubes

with the same geometry as commercial TTs. In order to achieve this, alginate tubes were

initially constructed with considerably larger dimensions. This was due, in part, to the fact that

during the drying and strengthening process alginate tubes shrink and loose up to 80% of their

initial bulk. This shrinking is caused by the removal of water from the gel matrix and was

controlled by the drying conditions. Tubes dried at 370C (rather then 250) immediately after

manufacture, shrink rapidly, rendering them useless for experimentation. These tubes lose

greater then 95% of their initial dimensions and are extremely difficult to remove from their

glass support rods, without tearing or cracking. Furthermore, manufacturing tubes from smaller

initial dimensions resulted in considerably thinner tubes, which may be incapable of

withstanding the riggers of implantation or insertion. The optimal initial conditions that produce

consistent and reliable results were those with a barrel diameter of 4.4mm, wall thickness of

1.6mm, and an overall length of 9.0mm. This resulted in a dried tube dimension of -1.1-1.2mm

inside barrel diameter (60% reduction), 0.1-0.2 mm wall thickness (80% reduction), and an

overall length of -6.0mm (33% reduction).

Similar dimensional constraints were applied to the Reuter Bobbin tubes prior to drying.

The initial barrel diameter was set to 4.0mm and the shaft length was set to 3.0mm. The final

dried dimensions were Imm inner diameter and 2mm shaft length. Furthermore, with Reuter

Bobbin tubes, it was important to maintain the separation distance between the two flanges

(Figure 5-4, 5-5). These flanges provide a location for the latex membrane to reside during









testing, and maintained the geometric constraints necessary to allow comparison between

commercial stainless steel Reuter Bobbin tubes. Results have shown that during flange repair,

additional CaCl curing solution re-crosslinks broken gel components back into a solid.

Therefore, flanges that were in contact with each other prior to CaCl application can become

permanently fused preventing the latex from sealing around the barrel. As a result, care was

taken, prior to desiccation, to separate the flanges of the tube using the back of a sharp blade

(Figure 5-4, 5-5).

Furthermore, the addition of CaCl to the gel prior to drying served two purposes. One

purpose was access to additional CaCl, which helped crosslink the remaining alginate within the

gel. The second was that the extra moisture allowed the gel to dry at a slower rate producing a

more flexible (elastic) gel tube. Preliminary experiments were conducted without the addition of

any solutions prior to drying. The resulting tubes were very rigid and difficult to remove from

their glass support rod. It was then discovered that perhaps the tubes were "too dry" resulting in

very fragile brittle tubes. Therefore, additional liquid (in the form of water) was added to the

tube and it was discovered that the resulting tubes were more elastic and could more easily be

removed from the support rod. Further investigation yielded gels with higher concentrations of

Ca that were often softer and had a more elastic consistency, making them easier to manipulate

and insert. Therefore, CaCl solution was later substituted for water to help maintain mechanical

strength, flexibility, and crosslinking.

Maximizing Mechanical Strength

Alginate:PEG Ratio: The four most important parameters associated with developing a

reliable tympanostomy tube are mechanical strength, smooth geometry, dissolution, and

biocompatibility. In designing our alginate tube, the primary focus was on maximizing

mechanical strength. We achieved this by first drying the tubes over extended time periods and









by adjusting the alginate concentration. The results show (Figure 5-6 (tension experiments)) that

as the alginate ratio doubles from 1 to 2 to 4 parts per sample the mechanical strength increased

by -40% and again by ~ 50%.

A similar trend was seen in Figure 5-8 for tube compression testing. However, the rate

was not as consistent. Compression tests show a considerable increase in strength (-70%

increase) as the alginate concentration increases (1 part to 2 parts) with a more gradual increase

(~ 25% increase) in strength from 2 parts to 4 parts. Increased alginate concentration results in

an increased crosslink density and higher solid volume fraction which increases gel stiffness in a

fairly predictable manner 53. PEG was added to the gels based on preliminary experiments using

100% alginate. Gel tubes manufactured using just alginate were very difficult to remove from

the glass support rods, and were not very elastic. PEG provided a way to predictability

decreasing the mechanical strength of the gel tubes using biocompatible components.

Ca Concentration: The calcium concentration used to crosslink the gel also plays an

important role in mechanical strength with increased concentrations resulting in decreased

strength. This goes against our initial assumption of a mechanical increase as Ca concentration

increases 49. Initially it was seen (at time = 0) that the higher Ca gels appeared stronger resulting

from rapid crosslinking (high crosslink density) occurring at high Ca concentrations. Rapid

crosslinking traps larger quantities of water within "fresh" gel tubes increasing strength initially.

This has been observed in other studies showing increased tendencies of water molecules to

diffuse into gels with high crosslinking density 53. However, as the tubes are dried the water

content within the gels dictates their final mechanical strength (Figure 5-7 and 5-9). Therefore,

as the Ca concentration doubles, the mechanical strength decreased by -55% from [0.5M] to

[1.OM] and then -80% from [1M] to [2M]. Water content within the gel may reduce the solid









volume fraction which may contribute to the decrease in mechanical strength. Compression

results follow a similar, although less substantial trend with a -25% decrease from [0.5M] to

[1M] and -50% decrease from [1M] to [2M].

Tube Drying Time: The compressive strength of the alginate gel tubes were all

significantly greater then the silicone control, independent of drying time (Figure 5-9). This is

due to the initial (non-dried) wall thickness of the alginate gel tubes. After crosslinking and

before complete drying the wall thickness was more then 6 times greater then the silicone control

which contributed to the increase in the compressive strength seen within the first few days (3

days).

Material Properties (Physical Characteristics): Previous studies have labeled alginate

gels as viscoelastic resulting from crosslinks and entrapped entanglements contributing to its

elastic behavior and other physical characteristics contributing to viscous behaviors (such as

slippage) 39. However, due to the aforementioned drying process, it is reasonable to believe that

the alginate gels have a very low or no viscous component and are considerably less elastic than

fresh crosslinked gels used in the above study. Therefore, numerical solutions for tension and

compression calculations should assume the alginate gels to be elastic, isotropic, and symmetric

materials.

Furthermore, the calcium alginate gels' physical appearance changes as a result of

ingredient concentration, with lower CaCl concentrations producing "brittle-dry" white gels and

high CaCl concentrations producing "clear-elastic" gels. This clarity could be the result of the

additional water entrapped within the gels produced with high CaCl concentrations. Also, as the

alginate ratio decreased, the gels became more fragile and difficult to handle due to less solid

volume fraction and crosslink density.









In addition, the magnitude of the tension and compression results highlight the various

testing methods. Tension experiments load the gel samples to failure and are therefore

considerably higher (several orders of magnitude) than the compression tests which are a non-

destructive experiment measuring functional compression. Therefore, it is important that

mechanical comparisons be made only among experiment types.

Moreover, disparities between experiments of similar ingredient components and drying

times (Alginate:PEG/CaCl concentration) can be attributed to drying variability. As shown in

Figure 5-6 the average modulus for [0.5M] CaCl gel (Alginate:PEG [4:1]) dried for 7 days was

~50,000kPa. However, during the extended drying experiments (Figure 5-7) the modulus

increased to -100,000kPa after drying for ~7 days. Similar although less substantial differences

were seen among tube compression experiments. Figure 5-8 shows [0.5M] CaCl gel

(Alginate:PEG [4:1]) dried for 7 days with a compressive strength of -300kPa whereas extended

drying experiments (4-30 days) (Figure 5-9) show a compressive strength of -250kPa. One

explanation for this is attributed to large standard deviations due to fluctuations in the drying rate

of the gels within the incubator. Furthermore, the testing environment (temperature/humidity)

changes from day to day, which may have some affect on the mechanical properties of the gel.

Although, considerations were made to normalize experimental conditions, it was not possible to

completely control all aspects of testing due to equipment limitations. One solution, in the

future, would be to perform all experiments within a temperature controlled environment such as

an incubator.

Mechanical Strength Limitations: There are, however, tradeoffs associated with

maximizing gel tube strength, including a possible reduction in dissolvability. Higher alginate

ratios within the gel decrease the dissolution rate (Figure 5-14) and can result in incomplete tube









removal. Furthermore, tube strength is directly related to geometry, with thick walled gel

samples having superior strength to thin walled ones. Once again there is a tradeoff in that thick

walled tubes can create larger tympanic perforations within the eardrum, allowing pathogens to

aspirate into the middle ear space. This can lead to secondary infection and further

complications. Therefore, these parameters must be optimized to promote healing and in some

cases can be specifically developed based on the patients needs.

Physiological Mechanics

Alginate tube compressive strength after agar encapsulation

Tubes encapsulated within an agar substrate, simulated conditions similar to in-vivo tissue

encapsulation environments. Due to the high water content of the agar during the first 20-25

days, a reduction in compressive strength of the alginate gel was seen. As shown before, (Figure

5-7, 5-9) water within the matrix of the gel reduces compressive strength. After about 20-25

days, the moisture within the agar had decreased (via evaporation), allowing the 30 day

compression results to increase. However, due to the lack of moisture it was impossible to

remove the gel tube from the agar (after 30 days). Therefore, water was added to the agar gel,

after 30 days, in order to soften the gel prior to tube extraction. However, after a 1 hour

submersion, the resulting compressive strength was still statistically similar to the non-

encapsulated control. Furthermore, all treatment results were more then 5 times stronger then the

commercial silicone control tubes indicating agar encapsulation (wet or dry) has little effect on

gel compressive strength.

Based on the drying of the agar gel over the length of the experiment, alterations were

considered to amend the agar drying experimental protocol. However, the application of water

to the agar every few days (preventing drying) was rejected because increased moisture would

artificially reduce compressive strength. Therefore, it could not be determined if moisture









application or agar encapsulation was contributing to compressive loss. As a result, the agar

encapsulation protocol was not altered over the 30 days and the gel was allowed to naturally dry

over time. On the other hand, studies have shown that mammalian cells may not readily interact

with alginates due to the absence of cell-surface receptors and lack of protein absorption 54

limiting the development of complete tissue encapsulation in-vivo.

Alginate tube compressive strength after exposure to ototopical solutions

Over the lifetime of tube implantation it is reasonable to believe that various solutions will

come in contact with the tube (other then the dissolution solution) resulting in premature

dissolution or weakening. The mechanical strength of commercial silicone tubes were used as a

minimum threshold for mechanical stability. Alginate gel tubes with a compressive strength

below silicone were considered non-functional. The results show that all solutions caused a

significant decrease in strength, compared to the no treatment alginate control (Figure 5-11).

However, none of the solutions resulted in a decreased strength below the commercial silicone

tube. In fact, all treatments were more then 50% stronger then the commercial silicone tube

(after 24 hours). In addition, unlike the majority of the tested solutions, ear mucus and blood

serum are unique, given that they may have prolonged tube exposure resulting from the healing

process. Therefore, significant loss in compressive strength from either solution can be used as

an indicator of long-term performance. However, both blood serum and ear mucus showed a

loss in compressive strength of 47-58% respectively (well above that of the silicone control).

This is despite the need to submerge the ear mucus tube prior to compression testing within a

PBS solution for 1 hour. Ear mucus over the 24 hour exposure period (at 370C) dried around the

tube and created a crust, preventing removal. PBS was added (1 hour soak) to remove the tube

from the mucus without damage. Even with the re-introduction of "water", the ear mucus tube









lost only 58% of its initial strength and was still more then twice the strength of the commercial

silicone tube.

Furthermore, upon contact with the tube, it was observed that soap water created a milky

white solution. However, it proved to have very little effect on the overall strength after 24

hours. Antibiotic solutions, specifically Ofloxacin, showed a 73% decrease in mechanical

strength after 24 hours, making it the most detrimental of the solutions. However, the resulting

gel was still -50% stronger than the commercial tube. It is important to note that for each

experiment, the tubes were completely submerged in their respective solutions, simulating a

worst case scenario. In-vivo conditions will most likely not provide complete 3600 submersion

and may potentially limit mechanical loss. Furthermore, although significantly lower than the

alginate control, all the alginate gel tubes have the potential to re-dry after ototopical application,

regaining strength.

Minimizing plugging

Tube plugging is one of the most prevalent complications associated with TT implantation.

Tubes manufactured with very smooth inner lumens have decreased occlusion prevalence. As a

result, alginate tubes were developed on glass rods providing a smooth inner diameter. Also,

previous studies have shown that higher concentrations of alginate within the gel matrix

produces a smoother surface 55, further validating the use of high alginate concentrations when

developing tubes. This smooth lumen prevents mucus (egg white) from adhering to the inner

surface of the tube and, therefore, allows fluid to extrude into the external ear canal rather then

building up on the inner surface of the tube.

HSA Application: Further tube enhancements included the addition of HSA to reduce

mucous adhesion. HSA coatings indicate that it may be possible for microscopic imperfections

in the surface of the tube to increase resistance and facilitate plugging (Figure 5-12). HSA









coating provides a very thin barrier to adhesion that can be applied prior to tube insertion 7,56,57

However, HSA application involves the submersion of the tube, without its glass support rod, in

a liquid solution (Phosphate Buffered Saline (PBS)) containing HSA for 12 hours. This

submersion re-hydrates the gel tube which, in tern, decreases its mechanical strength.

Nevertheless, this loss in mechanical strength appears minimal, having little to no effect on the

manipulation and insertion of the alginate tube into the latex membrane for testing. Furthermore,

removal of the glass rod permits coating of the inner barrel surface with albumin. However

without its glass support, it was noted that the tube lumen shrank due to swelling of the gel. This

swelling can artificially increase the occlusion rate by decreasing the tube diameter, while

limiting comparisons with commercial tubes. As a result, after submersion each tube was re-

inserted onto its glass rod to re-form the lumen of the tube and maintain its uniform circular

state. During insertion, care was taken to not tear the gel or to artificially enlarge the tube

opening using the glass rod.

Occlusion results were compared between both un-coated tubes and to albumin coated

alginate tubes in order to determine if albumin coatings reduce the occlusion rate. It was shown

that un-coated alginate tubes have a reduced occlusion rate when compared to un-coated

commercial stainless steel tubes. This can be partially attributed to the possible decreased

adhesion properties of the mucus simulant to the alginate gel. Based on these results it was not

surprising to see a further reduction in occlusion rate with the application of albumin. Albumin

coated alginate tubes showed a 50% reduction in occlusion due to the decreased resistance

provided by the albumin.

Moreover, HSA coated/un-coated tubes were tested immediately after PBS submersion and

were not dried prior to experimentation. It is unknown what affect moisture will have on









occlusion rates. However, previous experimental protocols 7,56,57 did not include drying after

albumin application. Despite this, throughout the 2 hour occlusion experiment, the alginate gels

did dry somewhat (visually) and after experimentation appeared to have regained most of their

original strength.

Specific Aim 2: Optimize the Gel Dissolution Components for Complete Dissolution

The ability to dissolve the gel tube after implantation is one of the novel features of our TT

design. Prior research studies have focused on the use of absorbable tubes that dissolve at a set

rate independent of physiological conditions. Therefore, once inserted there is no control over

absorption. Our tube design provides the ability to dissolve the tube using a dissolution solution

which affords extra control to the physician and alleviates the need to "guess" the absorption

time upon insertion. Dissolution is critical to promoting long term healing, since prolonged tube

implantation has been shown to be detrimental 2. However, it is not necessary to sacrifice

mechanical strength for rapid dissolution. Dissolution time frames of around one week may be

acceptable in order to limit ototoxicity and maintain mechanical strength. Furthermore, the use of

a minimally invasive dissolution technique (solution injection vs. surgical removal) further

reduces trauma to the patient and promotes natural healing.

Effect of Various Dissolution Solutions on Dissolution Rate

Complete tube dissolution was limited to contact with dissolution solutions and not

exposure to ototopical solutions. Dissolution is thought to occur when calcium within the

calcium alginate gel is replaced by Na ions available from the dissolution solution, remaking

sodium alginate solution. Three dissolution solutions were tried with the intention of optimizing

the dissolution rate of the gel. Optimal tube dissolution consists of complete removal of the tube

with little physical remains. However, as shown in the results (Figure 5-14 5-15), material

properties (gel tube composition) greatly affect the dissolution rate and complete dissolution









cannot be achieved in every case. The three solutions used for our experiments were NaC1,

Vinegar and NaHCO3. The only two dissolution combinations that resulted in any noticeable

changes in the tube removal were NaCl + NaHCO3 (50:50 mixture) and NaHCO3 alone. Both

solutions showed considerable affinity for dissolving [1:2] Alginate:PEG gels crosslinked with

[0.5M] CaCl. Dissolution was achieved by the re-introduction of Na into the matrix of the gel at

a higher concentration than Ca present in the gel, which increased swelling and promoted

dissolution 39

Furthermore, prior to the use of NaHC03, NaC1, and vinegar, several other dissolution

solutions were tried with minimal success including sodium phosphate dihydrate (NaH2PO4

-2H20) (result: soft, denatured tube), potassium chloride (result: soft, denatured tube), sodium

hydroxide (result: soft, denatured tube), magnesium sulfate (result: soft, denatured tube),

potassium hydroxide (result: almost complete tube dissolution) and sodium sulfate (result:

almost complete dissolution). In each case, the tubes remained in tact after several hours with

the exception of potassium hydroxide and sodium sulfate. These two solutions showed

promising dissolution but could pose ototoxicity concerns. In the end, there are limitless

possibilities for additional dissolution solutions however, very few if any have been previously

tested for ototoxicity and therefore should be used with caution.

In addition, the dissolution solutions used to remove the solid gels (NaCl + NaHCO3 and

NaHCO3 alone) play an important role in tube removal. However, the rate of dissolution across

all tube types using NaCl + NaHCO3 was not significantly greater than NaHCO3 alone and did

not justify its continued use. Initially (within one hour), NaHCO3 appears to dissolve the gels

faster than those solutions containing NaCl + NaHCO3. Furthermore, vinegar, along with

NaHCO3, did produce a vigorous reaction (C02 gas). However, the reaction was not sufficient









enough to last longer than a few seconds and did not facilitate degradation of the tube. It is

possible, though, that after degradation has begun, a vinegar solution can be applied to break up

larger gel pieces and achieve compete dissolution.

NaCl alone showed very slow dissolution. Furthermore, over 24 hours there appears to be

no change in the tube submerged in NaCl. This phenomenon has been confirmed by others 39

dissolving alginate gels with NaCl. Immediately after submersion (NaCl several hours),

mechanical strength of the dried gel tube is decreased due to gel hydration and swelling.

However, after that initial time period no further degradation was achieved (up to 1 week 39). In

addition, the NaCl solution used was of relatively low concentration (0.85% w/v or 0.303M).

Based on a weight per volume ratio there was more NaCl (1.515 x 10-4 mol) than calcium

alginate (9.09 x 10-5 mol) present during dissolution (Appendix E). Therefore, NaCl should be

capable of dissolving alginate gels. However, a difference of 6 x 10-5 mol (or 1.6x) appears to

not be sufficient enough to result in rapid dissolution. Literature has also confirmed that

degradation of low a-L-guluronic acid alginates (M/G ratio > 1.0, our alginate M/G ratio = 1.6)

can be prevented by submersion in solutions with a Na :Ca2+ ratio of 3:1. The amount of NaCl

relative to calcium alginate is less than 3:145 (1.515 x 10-4 mol vs. 9.09 x 10-5 mol) preventing

dissolution.

An additional factor that may contribute to dissolution includes byproduct solubility. The

dissolution of the alginate gel using NaCl may result in the production of CaCl as a byproduct.

The solubility of CaCl (74.5g/100ml, 200C) is greater than that of CaHCO3 (calcium

bicarbonate) (16.6g/100ml, 200C). Comparing the solubility of the possible dissolution products

CaHCO3 vs. CaCl there is support for higher dissolution rates with NaHCO3. The production of

CaHCO3 precipitate is less soluble in aqueous solution, thus driving the reaction in favor of









NaHCO3 dissolution over that of NaC1. CaCi is soluble in H20 therefore, Ca ions are available

in solution to re-crosslink the alginate gel limiting disintegration. On the other hand CaHCO3

precipitates out of solution at relatively low concentrations removing Ca from solution and

promoting disintegration. This supports results that the CaHCO3 shows faster, more complete

dissolution than the NaCl.

Furthermore, other factors can come into play, including the relative pH of the solution,

and porosity of the polymer surface. Alginate gel porosity can be reduced via desiccation.

Furthermore, pore size can be further reduced in the presence of low pH solutions. NaCl has a

pH of -7.0, whereas NaHCO3 has a pH of 8.6. This small change in pH may potentially limit

rapid dissolution when using NaCl. As a result, the reduction in pH, along with solubility

constraints and a Na:Ca ratio below 3:1, may combine to limit degradation via NaCl submersion.

Based on these results it appears that it is not just the Na present in the solution that drives

dissolution, but rather a combination of factors leading to tube removal.

Gel Tube Composition: Results show that gel composition plays a vital role in the rate of

dissolution. Gel compositions tested for dissolution represent the max and min (extreme) values

of gel composition and were used to obtain values of the max and min dissolution rates for

various tube compositions. Gels such as those composed of [1:2] Alginate:PEG crosslinked with

[0.5M] CaCl showed an affinity for dissolution (R=4.6). However, gels composed of similar

Alginate:PEG ratios [1:2] but with higher [2.OM] CaCl curing solutions has slower dissolution

rates (Figure 5-14 5-15). This can be explained by the excess CaCl remaining within the [2.OM]

crosslinked gel, which is capable of maintaining the high crosslinking density necessary for gel

formation. As expected, it appears that over time (1-5 hours), tubes composed of low alginate

concentrations and low CaCl concentrations had a higher dissolution affinity than all other









combinations. Although slower in terms of dissolution, [4:1] Alginate:PEG crosslinked with

[0.5M] CaCl has superior mechanical strength when compared to all other gel combinations.

Therefore, the extra increase in dissolution time (generally >5 hours using 0.5M NaHCO3) was

justified over the long implantation time. All other tube combinations (after 5 hours in NaHCO3)

achieved an average dissolution value of R=4 which is quite respectable considering their

varying composition and mechanical strength.

Effect of NaHC03 Concentration on Dissolution Rate

Results show that the concentration of NaHCO3 in solution does affect the rate of

dissolution. However, the results were not linear; rather dissolution peaks at 0.5M followed by

decreases in dissolution at l.OM and 2.0M (Figure 5-16). At a concentration of O.1M, the

amount of NaHCO3 (5 x 10- mol) present in solution was less than the amount of alginate (9.09

x 10-5 mol) in solution. Therefore, dissolution should be limited. However, degradation

progressed slowly (requiring -24 hours to complete). According to the literature 39,45 NaHCO3

dissolution should be limited due to low Na availability along with a Na :Ca2+ ratio of < 1

(described above). Therefore, other factors may come into play, such as pH (8.6 vs. NaCl 7.0)

along with the possible development of secondary byproducts such as CaHCO3. The increase in

pH may facilitate an increase in gel porosity, which may promote dissolution using 0.1M

NaHCO3. Furthermore, the development of CaHCO3 precipitate, with its low solubility, may

provide additional bonding sites for Ca during dissolution. However, due to the relativity short

longevity ofNaHCO3 solution, it may be more efficient to re-apply the 0.1M solution several

times to enhance its effect.

On the other hand, dissolution using 0.5M NaHCO3 contained approximately twice as

much NaHCO3 (2.5 x 10-4 mol) as alginate (9.09 x 10-5 mol) and appears to dissolve alginate gels

rapidly. The ratio of Na:Ca present in solution was ~3x which is necessary for alginate









dissolution. Moreover, very high concentrations of NaHCO3 (1.OM, 2.OM) (5 x 10-4 mol and 1 x

10-3 mol vs. Alginate at 9.09 x 10-5 mol) approach the saturation point of NaHCO3 in solution

(10g/100ml at 200C). Therefore, Na is precipitated out of solution and is not available to replace

Ca within the alginate gel (Appendix E). However, since one of the goals of this project was to

minimize ototoxicity, it is preferable to use lower NaHCO3 concentrations for experimentation.

Physiological Dissolution

Two methods were used to dissolve the tube: complete submersion (within a 24 well plate)

and physiologic dissolution (within an in-vitro apparatus). Complete submersion (0.5-1.0ml

solution) provided total envelopment of the gel tube and promoted uniform dissolution. This

represents the "best case" for dissolution because the entire tube is dissolved at one time.

However, this method was not possible after in-vitro tube implantation. Therefore, a

physiological method was developed that simulates conditions the clinician will see during

dissolution (access to only one side of the tube via external ear canal). Using this method, tube

dissolution was achieved first with consumption of the external "flange". Over time, the barrel

portion of the tube was degraded to the point where it released from the flange and was dissolved

within the holding well. Dissolution results using both methods were similar, with both

techniques achieving complete dissolution within parallel time frames (Figure 5-16 and Figure 5-

17).

However, physiological dissolution appears to have a slight edge due to separation of the

thinner barrel portion from the thicker flange portion. During the first hour, dissolution rates

were slower (R-3.2) for the flange portion of the tube due to increased gel thickness. However,

the barrel portion of the tube dissolved rapidly, sometimes within one hour due to its small size

and minimal thickness. This is a consequence of the manufacturing process, which produces gel









tube flanges more than two times thicker than the barrel portion, resulting in longer dissolution

times. After 5 hours both the barrel and flange portions of the tube had dissolved to R=5.5.

Although the flange portion takes longer to achieve complete dissolution, its location

during testing was designed to maximize dissolution time. Knowing that the flange dissolves

slower than the barrel its placement within the in-vitro ear chamber was inverted (from in-vivo)

in order to present the thicker portion for dissolution. In this way the maximum dissolution time

can be determined for tubes with thick external flanges.

Although all in-vitro experiments were performed with the flange portion of the tube

facing towards the external ear canal, in-vivo implantation requires that the flange face medially

preventing premature extrusion. Therefore, based on our results, in-vivo dissolution times may

be enhanced within the middle ear space. On the other hand, the thinner barrel portion can

dissolve rapidly within the external ear canal (in-vivo), allowing the thicker flange to fall into the

middle ear space to begin dissolution. As a result, after in-vivo implantation and subsequent

dissolution, it is the thick flange portion of the tube that will contribute to any ototoxic effects.

However, dissolution and perhaps ototoxicity can be reduced with the development and

subsequent dissolution of smaller more uniform TTs. In the present experiments, all dissolution

experiments were performed using T-shaped tubes which can have barrel lengths of up to 6 mm

and thick flanges. Larger tubes were used to determine the maximum time (worst case) for

dissolution.

Effect of desiccation on dissolution

Dehydration of the gel tube removes moisture and creates a strong rigid tube. Alginate

TTs were designed to be implanted for extended time periods (months/years) and moisture loss

will continue as implantation time progresses. The results show that throughout drying, the rate

of dissolution increases, achieving complete dissolution. Tubes dried for only 7 days achieved









complete dissolution after only 5-24 hours (depending on tube/dissolution solution ingredients)

(Table 5-1). However, tubes dried for extended time frames (15-30 days) showed a marked

decrease in dissolution time (1-2 hours using 0.5M NaHCO3) (Figure 5-17). This may be the

result of the reduction in surface area (80% smaller dried tubes) compared to fresh tubes.

Furthermore, the porosity of fresh tubes is most likely larger than that of dried tubes (drying

decreases porosity 45), but fresh tubes are significantly thicker than dried tubes. The increased

size of "fresh" tubes may prevent Na from rapidly penetrating the gel and replacing Ca during

the dissolution process. Therefore, fresh tubes dissolve slower.

Effect of HSA coatings on dissolution

Tubes coated with HSA limit occlusion formation and have been extensively studied in

literature 7,56-58. In addition, tubes coated with albumin appear to dissolve within 5 hours posing

no negative affect on dissolution (Figure 5-18). These results can be linked to the albumin

coating process, which involves submerging the tube within a PBS solution containing 0.01%

HSA. This application process allows sodium along with potassium ions, located within the PBS

solution, to begin to break down the tubes prior to formal dissolution. However, uncoated

alginate tubes submerged in PBS show a slight decrease in dissolution, implying albumin

application may aid dissolution as well. One limitation of this technique was that, prior to

experimentation, the albumin coated tubes were not dried, which may artificially increase the

performance (dissolution/occlusion) of the tube.

Removal of retained tube components

After application of the dissolution solution it is possible for part of the tube to remain un-

dissolved. Un-dissolved components (along with broken tube parts) within the ear canal can be

retrieved by the clinician via the external ear canal. However, components of the tube within the

middle ear space can not be physically removed and will become absolved over time. The









biocompatible nature of the gel inhibits the immune response and over time, via cellular

encapsulation and degradation, the warm moist environment of the middle ear will absolve the

remaining alginate gel components. The timeframe for complete tube dissolution should be

limited to within one week. Prolonged dissolution times (> one week) may lead to the

development of secondary complications.

Specific Aim 3: Quantify Ototoxicity from Exposure to Dissolution Products

Biocompatibility associated with TT dissolution can be related to Zebrafish ototoxicity.

Ototoxicity was minimized by manufacturing TTs from ingredients previously used in medial

implants and with the use of dissolution solutions chosen from clinical ototopical treatments.

Across all parameters (fluorescent intensity, neuromast area, and number of viable hair

cells) dissolution products outperformed known ototoxic treatments (Cisplatin). There were no

significant differences between the no-treatment (negative control) and those fish exposed to the

dissolution products. However, a decreasing trend was seen between the two groups (Figure 5-

22, 5-23, 5-24). This declining trend could be the result of the increased salinity of the

dissolution products. However, effects can not be isolated between dissolution products and

salinity based on the Zebrafish model limitations. Mammalian experiments with a higher

threshold for salinity will need to be conducted in order to isolate individual effects.

Zebrafish were used to confirm the viability of hair cells after exposure to dissolution

products. However, due to tonicity limitations (fresh water fish in a salt water environment),

Zebrafish embryos could only be maintained within a 0. 1M NaHCO3 solution for 30 minutes.

Mortality associated with longer exposure times (>30 minutes) was accelerated due to high

concentrations of NaHCO3 within the dissolution product. This concentration (0.1M) was more

than 20 times greater than the NaHCO3 concentration found in embryo media (extreme testing









condition)(Appendix E). Therefore, any ototoxicity associated with the dissolution solution can

be partially attributed to the high salinity of the water (Zebrafish model limitation) and not

necessarily the dissolution products themselves. Along with the increased sensitivity of

Zebrafish and extreme testing conditions ototoxicity was still minimized after exposure to

dissolution products. This indicates the potential viability of using dissolvable alginate gels as

an alternative to commercial tubes.

While a direct correlation could not be made between Zebrafish ototoxicity and

dissolution (30 minute exposure vs. 24 hour exposure at 0. 1M NaHCO3), previous clinical

studies have provided a link. It has been shown that (Appendix E) NaHCO3 is the primary

component within the dissolution solution making up more then 20 times the volume of the

dissolution products. Therefore, it may be possible to assume that NaHCO3 is the primary

ingredient driving potential ototoxicity. As a result, previous studies 22,23,25 have shown NaHCO3

use (to relieve obstructed tubes) at concentrations up to 5% or -0.59M. Although minimal pain

was associated with in-vivo application 23 hearing loss was not attributed, indicating NaHCO3

efficacy in humans up to 0.59M. At this concentration, ototoxicity is not anticipated and

dissolution was achieved within a short time frame (1-2 hours). Further testing using mammals

(ex. guinea pigs) will have to be conducted to measure ototoxicity at higher NaHCO3

concentrations.

pH may also be an issue associated with Zebrafish ototoxicity. However, results show

that pH remains constant (Table 6) as NaHCO3 concentration (in embryo media) is increased

from 0.001M up to 2M. Furthermore, previous studies have also shown that Zebrafish can

survive within solutions as high as (pH 8.9) for up to 15 days 59









Furthermore, osmotic affects associated with NaHCO3 injection may lead to hearing loss

in mammals. Increasing the sodium bicarbonate concentration can lead to fluxuations in the

osmotic conditions within the lymphatic system of the cochlea. These alterations, between the

perilymph and endolymph fluids, can affect the pressure within each system. Increased pressure

results from an osmotic imbalance of salts (potassium (K) and/or sodium (Na)) which may cause

fluid to be drawn into the perilymph extracellularr fluid) pushing the basilar membrane

(separates scala tympani and scala media within inner ear) towards the scala tympani (perilymph

cavity within the cochlea). This movement of the basilar membrane can affect the

electrophysiology of the cochlea resulting in decreased hearing 60. However, the method of entry

into the cochlea after tube dissolution is thought to occur through the round window (middle ear)

which is selectively permeable. It has been shown that round window permeability can be

decreased in the presence of overlying residual effusion, the presence of granulation tissue, or

thickening of the round window membrane which can all be present in infected ears.

Permeability also appears to be a function of concentration as seen with HSA levels, which are

impermeable below a minimum concentration. Therefore, if the concentration is below the

threshold value then the toxic chemicals may not be able to enter into the inner ear to cause

damage. Furthermore, upon entering the round window the concentration of the chemical must

be high enough to stimulate biochemical or immunochemical reactions. Therefore, ototoxicity

relies on several factors combining to cause hearing loss and as a result may be minimized 61












Table 6-1: Embryo media solution pH as NaHCO3 concentration is increased from O.O1M-1.OM
Solution Concentration pH

NaHCO3 0.01M 8.6

NaHCO3 0.05M 8.6

NaHCO3 0.1M 8.6

NaHCO3 0.5M 8.5

NaHCO3 1.OM 8.3

Embryo Media 7.2









CHAPTER 7
FUTURE WORK

Based on the results obtained from this report, additional experiments will need to be

performed in order to implant alginate tubes into human patients.

Tube Manufacturing: Limitations including variability in geometry and dissolution

times will need to be addressed prior to in-vivo application. The use of an enclosed injection

mold for fabricating geometrically uniform tubes is necessary for the production of implantable

tubes. Dissolution variability associated with differences in flange/barrel thickness can be

eliminated using more sophisticated molds. Furthermore, uniform drying can be achieved via

lyophilization (freeze drying) techniques, further increasing uniformity. Also, mechanical

strength can be enhanced with the use of high G-block alginate gels (long length G-blocks),

which result in more molecular entanglement and higher mechanical strength, although it may

limit dissolution.

Experimental Recovery: Based on our results, it is unknown what permanent

mechanical losses, if any, are associated with repetitive exposure to various solutions. Therefore,

experiments will need to be conducted to determine if there are any lasting mechanical effects of

repetitive exposure to ototopical solutions. Moreover, additional experiments will need to be

conducted using other ototopical agents with potential exposure to the tube. These treatments

can include (Sofradex 22, lipase, hyaluronidase 24, locorten vioform, and olive oil, 25 among

others.)

Dissolution: Premature dissolution, resulting from in-vivo cellular/tissue encapsulation

associated with alginate gel implantation can be limited with the application of poly-L-lysine-

MPEG and poly-L-lysine. These coatings have been shown to limit protein absorption while

decreasing cellular interactions and immunogenicity 45. Furthermore, dissolution limitations,









including injection techniques, can be modified to facilitate long term dissolution. Dissolution

application can be adjusted to include dissolution gels, which can maintain their position within

the ear canal after implantation. Currently, dissolution solutions have a low viscosity and can

become expelled during standing and walking. Dissolution is limited if contact is not made

between the dissolution solution and alginate tube. Therefore, thick dissolution gels, such as

those composed of Dextran can be infused with NaHCO3 and applied to the tube. These gel

solutions have the potential to maintain their position after insertion, decreasing treatment time.

Furthermore, dissolution can be localized with the application of a gelfoam pledgette that can be

soaked in NaHCO3 solution and applied to the lateral surface of the gel tube. The delivery

mechanics of gelfoam may allow the barrel/flange portion of the tube to dissolve leaving the

remaining portion to act as a scaffold for tissue growth. This dissolution method may also limit

NaHCO3 from entering the middle ear space (reducing ototoxicity), and therefore, higher

concentrations of NaHCO3 can be used to dissolve the gel. After dissolution, the gel foam can

be removed by the clinician. In addition, dissolution time may be reduced with the use of

Reuter-Bobbin tubes with their 2mm shaft length and significantly smaller (weight and volume)

dimensions. Further work can focus on optimizing the dissolution concentration to minimize

dissolution time, while simultaneously minimizing ototoxicity in mammals and eventually

humans.

Ototoxicity: Furthermore, additional ototoxicity experiments will have to be conducted

first on small mammals followed by human clinical trials. Successful Zebrafish experiments

indicate that the dissolution products have a minimal affect on hair cell function after 30 minute

exposures. However, the limitations of Zebrafish as an ototoxicity model preclude their use in

further experimental analysis. The high salinity produced by the sodium bicarbonate solution









provides a habitat that is inhospitable for Zebrafish testing over extended time periods.

Although, good for rapid, inexpensive, qualitative results, Zebrafish ototoxicity experiments are

deficient for optimizing dissolution time. Future experiments must be conducted using mammals

(ex. guinea pigs) to determine if hearing loss is associated with implantation and removal of

alginate gel TTs using higher concentrations and longer exposure times, approaching 0.5M and

up to one week. Following this, clinical patient studies will have to be conducted to validate

experimental results and to optimize components.









CHAPTER 8
CONCLUSION

Based on the results presented in this paper it can be concluded that it is possible to

develop a calcium alginate TT. We have shown that the mechanical strength is superior to that

of commercial tubes, providing reliable ventilation. Furthermore, these tubes can be removed

from a simulated tympanic membrane with the use of a dissolution solution reducing the need for

more invasive procedures. Finally, the resulting tube dissolution has been shown to have a

minimal affect on hair cell function using a Zebrafish model. As a result, it may be possible to

develop a biocompatible alginate TT capable of providing and maintaining reliable ventilation

while dissolving on demand. Individual conclusions concerning the development of a

biocompatible and dissolvable TT are summarized below.

1. Experiments have proven that the mechanical properties of calcium alginate gels exceed
those of commercial silicone tubes.

2. In the presence of an agar substrate, mechanical superiority was maintained after in-vitro
encapsulation for 30 days.

3. Exposure to ototopical solutions including ear mucus and blood serum proved to be
detrimental. However, after 24 hours (complete submersion) the resulting compressive
strength was still more then 50% greater then that of commercial silicone tubes.

4. When compared to commercial stainless steel Reuter Bobbin tubes, tube plugging was
minimized with the use of alginate tubes (in-vitro). Un-coated alginate gel TTs showed a
20% decrease in plugging using an in-vitro ear chamber.

5. Mechanically superior alginate tubes can be completely dissolved with the use of solutions
containing NaHCO3, potentially reducing the need for more invasive surgical procedures.

6. Dissolution efficacy may be enhanced with increased drying time and remains unaffected by
the application of albumin coatings.

7. Using a Zebrafish ototoxicity model, biocompatibility (hair cell viability) associated with
alginate gel dissolution was maintained.









APPENDIX A
TENSION AND COMPRESSION TESTING FOR ALGINATE GEL SPECIMEN

Mechanical testing apparatus (tension) (Figure A-1, A-2, A-3) showing the "dog-bone"

specimen as it is pulled to failure. Figure A-4 describes the compression of the alginate gel tube

between two flat platens.


Figure A-1: "Dog-Bone" specimen in clamps (time=0)


Figure A-2: Specimen pulled and elongated. Sample becomes thinner.

























Figure A-3: Sample fractured with a clean even break.


I D .,, ^ '-',
0 tBi-** a
I ^ 'S.^ ...' c .
*~ *.. I '; *- ,, O.


i Aluminum
Inmn


Figure A-4: Alginate gel tubes deformed between aluminum and PDMS platen. (A) Shows the
un-deformed state of the gel tube placed between two flat platens. The top platen is
composed of PDMS and the bottom platen is composed of aluminum. (B) Shows the
deformed state (25% of initial diameter) after compression. (NOTE : Initial diameter
measured prior to addition of the aluminum platen (A) -120 pixels, final diameter (B)
~90pixels. Pictures are inverted due to camera mounting, bottom section represents
the aluminum funnel).









APPENDIX B
NUMERICAL SOLUTION FOR TUBE COMPRESSION

Compression Deflection:


CD =
A


(B1)


CD = compression deflection compressivee strength) force per unit of specimen area, (kPa) 46. F


= force required to compress the specimen 25% of the outer diameter, (N). A

compression contact surface area, (m2).


A


r2


V


specimen


Figure B-1: Compression of alginate gel tubes between two flat platens. (A) Undeformed
silicone and alginate gel tubes. d = distance between the two platens. ri = inner
radius. r2 = outer radius. F = force (load) applied to the barrel of the tube. (B)
Deformed tube (25%) showing contact width of 2b. Two half cylinders with radius rib
= inner radius deformed tube, r2b =outer radius deformed tube, h = thickness of tube
barrel.

Assumptions:
Material is isotropic
Geometry is uniform across all tube types
Deformation is uniform/symmetric
Wall thickness is constant
Contact area is constant across all tube types


I I


I IV









Based on the conservation of volume and the constraint that h = constant, the front surface area

of the undeformed tube (Figure B-i-A) and the deformed tube (Figure B-i-B) should be equal,

assuming the tube deforms symmetrically into two half circles separated by two rectangular

contact sections. Since the geometry of the tubes are equal (independent of gel composition) and

the tubes are all equally deformed by 25%. It is reasonable to assume that the contact area for all

tubes is equal. The only change being the load (F) required to compress the tubes 25%.

Therefore, the contact surface area (A) from the above equation was calculated using Equations

B2-B5 at the time the load compressed the tube 25% (Figure B-1). L = length of contact the tube

has with the bottom platen. 2b = the width of contact with the surface.

Contact Area (rec tan gle) = 2b x L (B2)

Area of undeformed tube (A ) = A2 A, = i2 m^Z (B3)

2 2 >
Area of deformed tube (ADb) 22 m -b
2 2 (B4)



^b =r2b-h

r2b = (0.75)(d)
Where: 2
h = wall thickness
d = tube outer diameter

Setting (B3) equal to (B4) and solving for b gives:

^A ;Tr(r22 rl )
b U
4h (B5)
0.637mm2 .r(0.5625mm2 0.4125mm2)
b = 0.295mm
4(0.15mm)

Force (F)

F = force required to compress the Alginate:PEG specimen 25%.










Force = ma (m = mass, a = acceleration of gravity (constant)) (B6)

m = 35.5g (average mass for calcium alginate, changes based on composition), g = 9.81 m/s2


F = ma = (35.5g)(9.81m/s2)= 348.255 g = 0.348255kg = 0.348255N
2 2
s s

Contact Length (L)

L = length of contact the tube has with the bottom platen.

Assumption: Length of contact with the PDMS is equal to the length of contact of the

aluminum funnel. L = 2mm = 0.002m

Therefore, the compressive strength (CD) can be calculated below.

kgm kgm
0.348255
CD = =0.2951 = 0.2951 = 295.13kPa
A 2(0.295mm)(2mm) mm mm









APPENDIX C
ZEBRAFISH SOLUTION CONCENTRATION

All solution concentrations (Cisplatin, DASPEI, MS-222, Dissolution products) were

created using Embryo Media as the base.

Cisplatin: (50pM 1mM)


Cisplatin (MW) =00.05g
mol
SImol 300.05g Cisplatin Imol 0.30005g Cisplatin
1000ml mol 1000ml Iml media
For 50pM = 0.000050M

0.000050M x 0.30005g Cisplatin 0.000015g Cisplatin
Iml media ml media
1 i/h 10 ml of soltuion
0.000015g Cisplatin
10mlx = 0.00015g Cisplatin in 10ml of media
ml media
For ImM
0.30005g Cisplatin
O.OOM x x 10nml solution -> 0.003g Cisplatin in 10ml media
Iml media

DASPEI: (1mM)

380.27g
DASPEI (MW) = 3827
mol

ImM = 0.001 mol
1000ml
380.27g DASPEI 0.001 mol 0.00038027g DASPEI
mol 1000ml ml media
Want 10 ml of solution
0.00038027g DASPEI
1Omlx 00038027g 0.0038027g DASPEI in 10ml media
ml


MS-222: (10pg/ml-250mg/L)









To make 10 jg/ ml solution :

1 Oml solution : 1 Omlx 10i g = 100jg in l0ml media (0.0001g)
ml
For Euthanisa:
250mg MS- 222 L 0.25mg MS 222
x = x 10nml media -> 2.5mg MS 222 in 10ml media
L 1000ml ml
Buffered i/ i/h
~ 200mg NaHCO3 L 0.2mg NaHCO media 2mg NaHC in m media
-x = x 10ml media = 2mg NaHCO, in 10ml media
L 1000ml ml


Dissolution Products: (0.5M-0.1M)

Embryo Media contains 0.035g NaHCO3 /ml. In a 100ml of embryo medial there is

0.00035g NaHCO3 per ml of embryo media (or a total of 0.035g). A 0.5M NaHCO3 solution

contains 4.2g of NaHCO3 per 100ml of solution.

0.5mol NaHCO, 84.01g NaHCO, L 0.042g NaHCO,
x x = x 100ml -> 4.2g NaHCO3
L mol 1000ml ml

Therefore, to make 0.5M solution using embryo media as the base instead of DI water

subtract 4.2g-0.035g to get 4.1659g NaHC03 in 100ml. We only need 10 ml of solution so we

divided by 10 resulting in 0.4165g NaHCO3 in 10 ml of solution. 0.25M and 0.1M solutions

were created by dividing 0.4165 by 2 (0.208g) and 5 (0.083g in 10ml of embryo media)

respectively.

The molar concentration of NaHCO3 in embryo media was determined using the

molecular weight of NaHCO3 (84.01g/mol) and the following formula (given the amount of

NaHCO3 in embryo media is 0.00035g/ml). Therefore, the molar concentration of NaHCO3 used

to dissolve the tube (0. 1M) was more then 20 times greater then the molar concentration in

embryo media.

0.00035g 0.35g Imol mol
-- x 1000ml = x = 0.00416 (M)
ml L 84.01g L










APPENDIX D


NEUROMAST FLUROESCENT INTENSITY

Neuromast P4 was isolated from each Zebrafish treatment and the image colors were

inverted in order to clearly see the individual hair cells. The results (Table D-l) show that the

fluorescent intensity (dark purple area) (Figure D-l) was significantly higher for the hair cells

exposed to the dissolution products and negative no-treatment control when compared to the

positive Cisplatin control. It was also shown that there are more viable hair cells per neuromast

and a larger effective neuromast area in those Zebrafish exposed to the dissolution products and

negative no-treatment control than the positive Cisplatin control.

Table D-l: Fluorescent intensity, neuromast area, and hair cell number based on Zebrafish
treatment.
No Treatment Negative Positive Control Dissolution Products
Dissolution Products
Control (Cisplatin)
Fluorescence Area # Fluorescence Area # Fluorescence Area #
1 218.19 299.4 7 106.25 185.0 6 190.12 214.2 6
2 205.74 252.6 8 160.37 244.0 6 161.79 111.4 4
3 197.37 288.3 8 81.75 131.1 3 135.49 110.9 4
4 149.02 219.3 7 151.14 313.0 6 194.39 214.2 3
5 199.3 215.8 6 199.52 117.5 4 73.92 224.8 8
6 180.48 225.8 8 171.83 173.9 5 183.32 174.9 5
7 141.89 376.6 6 145.69 134.6 4 105.82 441.1 6
8 150.61 302.0 6 157.88 112.9 4 134 265.7 6
9 195.03 328.7 7 128.3 128.0 4 229.79 240.5 5
10 88.14 705.7 4 109.96 184.5 3 134 501.6 7
11 182.47 382.6 9 108.08 178.5 4 127.27 200.6 4
12 224.8 218.8 8 166.89 250.5 7 174.41 455.7 9
13 166.09 499.6 9 127.79 156.8 5 189.14 335.2 7
14 139.13 320.1 5 81.35 226.8 5 161.51 373.5 7
15 124.26 321.1 6 112.04 159.3 4 171.93 337.7 7
16 -- -- -- 125.15 179.0 6 155.05 317.1 7
17 -- -- -- 128.11 163.8 4 152.65 113.9 2
18 -- -- -- 124.41 217.8 5 165.86 368.0 8
Average 170.8 330.4 6.9 132.6 180.9 4.7 157.8 277.8 5.8
St. Dev. 38.2 129.0 1.4 31.4 52.7 1.1 36.1 120.2 1.9












Dissolution Solution


am


S


t a


I
Si r


V g


a.


&


Cisplatin






Negative Control


Figure D-1: Zebrafish neuromast P4 hair cells for each experimental treatment.


0


4 q* A










APPENDIX E
DISSOLUTION SOLUTION QUANTITY

The amount of each dissolution component in solution was calculated for calcium alginate,

NaCl, and NaHC03.

Calcium Alginate:


Molecular Weight


121g Ca Alginate
mol


Molar Con. (Tube weight = 0.01 Ig Ca Alginate)

mol
0.01 g Ca Alginate x
121g Ca Alginate


9.09x 10 mol


Molecular Weight


28g NaCl
mol


Molar Con. (at 0.85%) (0.5ml solution)


8.5g NaCl mol
x 0.
L 28g NaCl

03:


Molar Con. [0.1M] (0.5ml solution)



Molar Con. [0.5M] (0.5ml solution)



Molar Con. [l.OM] (0.5ml solution)



Molar Con. [2.0M] (0.5ml solution)


303 mo x 0.5ml
1000ml




0.1 mol
= 0.5ml x =
1000ml

0.5 mol
= 0.5mlx =
1000ml

1.0 mol
= 0.5ml x =
1000ml

2.0 mol
= 0.5ml x =
1000ml


= 1.515 x10- 4ol





5 x 10 5mol



2.5 x 10-4 ol



5x104 mol


1 x 10-3 mol


NaCl:


NaHC









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BIOGRAPHICAL SKETCH

The author was born in 1978, in Manhattan, New York to an elementary school teacher

and a secretary. He has one younger sibling, a very talented actress and artist, Nicole. In 1980,

the author's parents moved to West Palm Beach, Florida. It was here that the author grew up and

discovered the wonderful world of engineering. As a child, he constantly took apart everything

he could get his hands on and used the parts to create his own inventions. It was here that he fell

in love with science, a lifelong passion that is with him constantly. In May 2002, he received his

Bachelor of Science in engineering science with a minor in biomechanics from the University of

Florida and entered graduate school in the Mechanical Engineering Department. Shortly before

finishing his bachelor's degree, he started working for Dr. Roger Tran-Son-Tay in the

Biorheology Lab and he continues pursuing his research there today. In August 2005, he

received his Masters in Engineering in mechanical engineering from the University of Florida.

In the summer of 2001, he met the love of his life Amy and from then on knew that he had found

his soul mate. On February 1, 2004, he asked her to marry him, and on June 6, 2006, they were

married. He and his wife currently live in Gainesville, Florida.





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