Bio Compatibility of Polymer Implants

7/31/2019 Bio Compatibility of Polymer Implants

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BIOCOMPATIBILITY OF POLYMER IMPLANTS

FOR MEDICAL APPLICATIONS

A Thesis

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Christopher M. Brendel

August 2009


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BIOCOMPATIBILITY OF POLYMER IMPLANTS FOR MEDICAL APPLICATIONS

Christopher M. Brendel

Thesis

Approved: Accepted:

________________________________ ________________________________

Advisor Department ChairDr. Daniel Ely Dr. Monte Turner

________________________________ ________________________________

Faculty Reader Dean of the CollegeDr. Ronald Salisbury Dr. Chand Midha

________________________________ ________________________________

Committee Member Dean of the Graduate SchoolDr. Qin Liu Dr. George Newkome

________________________________

Date


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ABSTRACT

Two separate experiments were performed to test the biocompatibility of

amphiphilic conetwork polymers for medical applications. In experiment 1, four

different types of polymers were tested in the liver and brain of 20 rats to determine

which polymer would be best suited for intervertebral disc repair or replacement. The

samples used for the brain and liver injections were Monomer: 1-cyanoacryl-2,4,4-

trimethylpentane(TMP-CA), Polymer: cyanoacrylate terminated tri-arm star PIB [(PIB-

CA)3, Initiator: N,N-diethyl telechelic [(PIB-NEt3)3] PIB, and Monomer + Polymer

Combination: cyanoacrylate-telechelic 3-arm star polyisobutylene [(PIB-CA)3] + 2,2,4-

trimethylpent-1-cyanoacrylate (pTMP-CA). The solid samples implanted into the liver

were Monomer: poly(1-cyanoacryl-2,4,4-trimethylpentane)poly(TMP-CA), Polymer:

cross linked [(PIB-CA)3] polymer, and Monomer + Polymer Combination: copolymer

of [(PIB-CA)3] + 2,2,4-trimethylpent-1-cyanoacrylate (pTMP-CA).

The rats were divided into three groups. Group number one had four different

honey-consistency gel polymers injected into the liver and a saline injection for

control. Group number two was subjected to the same injections, but this time the site of

injection was the brain. Group number three had three types of solid polymers surgically


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implanted into the liver. Tissues were then examined histologically to determine if any

damage had occurred. A polymer sample that resulted in little or no significant tissue

damage would be a good candidate for intervertebral disc replacement and/or repair.

In experiment 2, two types of polymers were used; a polyisobutylene polymer as

well as a Bionate sample for a control, (to determine the biocompatibility of the polymer

for a potential pacemaker coating). Both were implanted on the peritoneal wall of eight

rats. The rats were divided into two groups of four. Group number one had four

polyisobutylene polymer strips sutured to the peritoneal wall. Group number two had

four Bionate strips also implanted on the peritoneal wall. Tissue at the implant site was

removed and examined histologically to determine if any damage had occurred. A

polymer sample that resulted in little or no significant tissue damage was considered a

good candidate for a potential pacemaker coating.


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ACKNOWLEDGEMENTS

I would like to thank everyone who assisted me with this experimental

investigation over the course of the past two years. Without their help, my research

efforts which have culminated into this thesis would not have been possible. The

following individuals trust and support guided me throughout the laboratory work,

research, and production of this paper, of which I am most appreciative and thankful.

The first individual that I would like to thank is Dr. Kennedy. His expertise,

innovation, and enthusiasm in the field of polymer science have afforded me the

opportunity to participate in biomedical research since the final semester of my

undergraduate career. It has been through this work, that I have realized my passion

towards working in biomedical research. I am excited about the prospect of pursuing my

Ph.D. in Polymer Science in the Fall of 2009, and look forward to any future

collaboration with Dr. Kennedy. I would also like to thank Suresh Jewrajka, Dr.

Kennedys research assistant, for synthesizing the polymer samples that were used in this

study.

My gratitude is also extended to Gail Dunphy and Shannon Boehme for their

assistance in learning the proper technical and surgical protocols in the lab. Without their

insight and guidance this would have been an incredibly cumbersome task. I have gained


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a great deal of knowledge and techniques from them, and am truly grateful for all of their

efforts throughout this process.

I would also like to acknowledge Amy Kladar for her work as an undergraduate

assistant, which was invaluable to my research.

Finally, without the quality of time devoted, counseling, consideration, and

mentoring of Dr. Ely from the last semester of my undergraduate career throughout my

graduate studies, I would not be where I am today. Its hard to find the right words to

adequately express my gratitude to Dr. Ely who has played such an instrumental role in

my education and career exploration. It is thanks to Dr. Ely that I will graduate with a

clear career direction. Dr. Elys support and faith in me has been paramount to my

success and I am very fortunate to have had such a distinguished and caring advisor to

guide me through not only my research and graduate program, but also to help me realize

the full potential of the work I can accomplish in the future.

I would also like to thank my family and friends for their unwavering belief in my

ability, which means so very much to me.

As I complete my graduate coursework and embark on the new journey of earning

my Ph.D. in Polymer Science, I am thankful to have so many highly esteemed

professionals as colleagues. It is my sincere hope that I will be able to work with them in

some capacity in the future.


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TABLE OF CONTENTS

Page

LIST OF TABLES ...............................................................................................................x

LIST OF FIGURES ........................................................................................................... xi

CHAPTER

I. INTRODUCTION ............................................................................................................1

Hypotheses and Objectives ......................................................................................3

II. LITERATURE REVIEW ................................................................................................5

Medical Applications for Polymer Biomaterials .....................................................6

Biocompatibility ....................................................................................................10

Biofilms..................................................................................................................12

Biofilm Prevention .................................................................................................13

Amphiphilic Conetworks .......................................................................................14

Polymer Biodegradation ........................................................................................15

III. MATERIALS AND METHODS .................................................................................17

Experiment 1: Polymer Biocompatibility for Intervertebral Disc

Repair/Replacement ...............................................................................................17

Surgical Procedure .................................................................................................17

Polymers used for Brain and Liver Injections .......................................................18


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Solid Polymers Implanted in the Liver ..................................................................19

Tissue Removal and Histology ..............................................................................19

Experiment 2: Polymer Biocompatibility for Pacemaker Coating ........................20

Surgical Procedure .................................................................................................20

Polymer and Bionate Control Implants..................................................................21

Tissue Removal and Histology ..............................................................................21

Statistical Analysis for Experiments 1 and 2 .........................................................22

IV. RESULTS ....................................................................................................................24

Experiment 1: Polymer Biocompatibility for Intervertebral DiscRepair/Replacement ...............................................................................................24

Injection/Implant Scoring Averages for All Categories ........................................24

Wall Thickness for Liver Injections ......................................................................25

Irregular Cells for Liver Injections ........................................................................25

Lymphocytes for Liver Injections ..........................................................................25

Wall Thickness for Brain Injections ......................................................................26

Irregular Cells for Brain Injections ........................................................................26

Lymphocytes for Brain Injections .........................................................................27

Wall Thickness for Liver Implants ........................................................................27

Irregular Cells for Liver Implants ..........................................................................27

Lymphocytes for Liver Implants ...........................................................................28

Survival Rate for Injections/Implants ....................................................................28

Total Scores for Liver Injections ...........................................................................28

Total Scores for Brain Injections ...........................................................................29


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Total Scores for Liver Implants .............................................................................29

Histology for Liver Injections ................................................................................29

Histology for Brain Injections ...............................................................................30

Histology for Liver Implants .................................................................................30


Scoring Averages for Peritoneal Wall Implants and Surgical Notes .....................31

Histology for Polymer Implants.............................................................................32

Histology for Bionate control Implants .................................................................32

V. DISCUSSION ...............................................................................................................50


Liver and Brain Injections .....................................................................................50

Liver Implants ........................................................................................................53

Total Scores for Injections and Implants in Liver and Brian .................................54

Summary ................................................................................................................55


VI. CONCLUSION............................................................................................................59



REFERENCES ..................................................................................................................62

APPENDIX ........................................................................................................................67


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LIST OF TABLES

Tables Page

1. Important Components to Consider in Material Selection ............................................11

2. Scoring Averages and Surgical Notes for Each Injection/Implant ................................33

3. Animal Survivors Associated with Polymer Type.........................................................39

4. Scoring Averages and Surgical Notes for Each Implant ...............................................45

5. Scoring Averages for Polymer and Bionate control Implants .......................................46

6. Total Mean Scores for Peritoneal Wall Implants ...........................................................46

7. Two-Tail T-Test for Comparison between Polymer and Bionate acrossAll Categories Scored ...................................................................................................47


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LIST OF FIGURES

Figures Page

1. Various Applications of Different Polymer Composite Biomaterials .............................7

2. Diagram of Intervertebral Disc ........................................................................................8

3. Schematic Representation of Stages 1-3 for Biofilm Formation ...................................13

4. Experiment 1: Wall Thickness for Liver Injections .......................................................34

5. Experiment 1: Irregular Cells for Liver Injections ........................................................34

6. Experiment 1: Lymphocytes for Liver Injections ..........................................................35

7. Experiment 1: Wall Thickness for Brain Injection ........................................................35

8. Experiment 1: Irregular Cells for Brain Injections ........................................................36

9. Experiment 1: Lymphocytes for Brain Injections ..........................................................36

10. Experiment 1: Wall Thickness for Liver Implants ......................................................37

11. Experiment 1: Irregular Cells for Liver Implants ........................................................37

12. Experiment 1: Lymphocytes for Liver Implants ..........................................................38

13. Experiment 1: Total Mean Scores for Each Group with Liver Injections. ..................40

14. Experiment 1: Total Mean Scores for Each Group with Brain Injections ...................40

15. Experiment 1: Total Mean Scores for Each Group with Liver Implants .....................41

16. Experiment 1: Representative Section for Tissue Analysis for Liver Injections ..............42


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17. Experiment 1: Representative Section for Tissue Analysis for BrainInjections ......................................................................................................................43

18. Experiment 1: Representative Section for Tissue Analysis for Liver Implants...........44

19. Experiment 2: Representative Sections for Tissue Analysis for PeritonealWall Polymer Implants ................................................................................................48

20. Experiment 2: Representative Sections for Tissue Analysis for PeritonealWall Bionate Implants .................................................................................................49


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CHAPTER I

INTRODUCTION

A large number of polymer associated implantable devices are used in medicine

today. Polymer based biomaterials such as bone plates, ligaments, intervertebral discs,

heart valves and pacemakers are used to replace or reestablish function of failing tissues

or organs. These biomaterials help to heal, increase function, repair abnormalities, and

thus improve the patients quality of life (Ramakrishna et al., 2000).

Polymers are utilized in numerous medical applications. This is mainly due to

their versatility as their composition, properties, and forms can be manipulated to readily

produce shapes and structures in the form of gels, films, fibers and solids (Deluca, 1988;

Ramakrishna et al., 2000).

A cardiac pacemaker is a small battery powered devise which produces electronic

impulses to help stimulate the heart muscle. It is used to treat heart arrythmias by

regulating an individuals heart beat. A pacemaker is similar in size to a matchbox and

weighs only between 20-27 grams. It is composed of a corresponding electronic circuit,

lithion ion battery and two tubes with electrodes to transmit the electric impulses to the

heart muscle. These components are enclosed in titanium and covered in a biocompatible


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material to isolate the devise from exposure to the bodys tissues (Lechleitner et al.,

2005).

Any and all medical implants must be properly sealed to defend against the

aggregation of surrounding body fluids, tissues and cells. Prevention of internal body

cavity substances from penetrating the coating is absolutely necessary to thwart the

destruction of the electrical components. The material(s) used to coat a surgically

implanted medical devise such as a cardiac pacemaker must be biocompatible and

biostable to reduce the possibility of tissue rejection reactions (Lechleitner et al., 2005).

Pacemaker coatings have been known to degrade prematurely due to the build up

of biofilms, which are collections/populations of bacteria, fungi and protozoa. The

microorganisms can deteriorate the protective coatings quite rapidly thus resulting in

damage and often removal and replacement of the devise itself. The function and

structure of these biomaterials can be spoiled by biofilms in a variety of ways. One way

this can occur is by masking surface properties of the protective coating and

contamination of water by microorganisms. Another way is the ability of

microorganisms to extract monomers and additives out of the polymers via microbial

degradation. Also, enzymes and/or radicals can cause brittleness and decrease of

mechanical stability to the polymer coating and its additives. There is also the possibility

of water accumulation that could potentially penetrate the polymer matrix via microbial

filaments. This could lead to swelling and an increase in conductivity. Damage and

biodeterioration can occur to the polymer coating material(s) by any one or combination

of these mechanisms (Flemming, 1998).


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that of an implanted pacemaker subject to various body tissues. It is hypothesized that

the polymer implant will elicit little or no significant tissue damage, nor will it degrade

itself, and will therefore be suitable for a cardiac pacemaker coating.


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CHAPTER II

LITERATURE REVIEW

The main objective of this research project was to provide insight to the potential

uses of polymers for medical applications by exploring their biocompatibility in vivo.

Various polymers were investigated in two separate experiments. Experiment 1

examined the possibility of discovering a polymer suitable for intervertebral disc repair

and/or replacement. Experiment 2 pursued the potential use of a polymer as an improved

pacemaker coating. This chapter will provide background information from scientific

articles to justify the reasoning for research in this interdisciplinary academic arena.

Topics relevant to this project will be discussed such as previous related work in polymer

biomaterials and their respective biocompatibility. Biofilms and cell adhesion growth, as

well as prevention, will also be reviewed concerning their roles related to polymer

implants. Mechanisms regarding biodegradation and erosion of polymers in vivo will

also be mentioned. Finally, histology of tissue sections will be discussed to better

determine the biocompatibility of the polymers tested in this project.


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Medical Applications for Polymer Biomaterials

As seen in Figure 1, polymer composite materials have been produced for many

biomedical applications (Ramakrishna et al., 2000, 2001).


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Figure 1. Various Applications of Different Polymer Composite Biomaterials. (modifiedfrom Ramakrishna et al., 2001)

Dental Implant

Vascular

Teritoneal Wall

Intramedullary

Li ament/Tendo

Cartilage

Bone plate &

Screws

External Fixation

Total Knee Replacement

Bone Cement

Total Hip

Replacement

Fin er Joint

Spine Cage, Plate, Rods,

Screws and Disc

Bone Replacement

Material


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Fibrocartilaginous intervertebral discs (IVD) separate vertebrae. They are

composed of a core, nucleus pulposus, and an outer coating known as the annulus fibrosis

which is composed of 90 concentric layers of fibers as seen in Figure 2. The disc is

coated on the lower and upper surfaces by a thin layer of cartaliginous endplates. These

endplates are perforated thus allowing the transfer of nutrients, water, and metabolism

products. The main role of the intervertebral disc is to cushion adjacent vertebral

segments that act as a shock absorber for the spine (Anderson et al., 1993; Ramakrishna

et al., 2001).

Figure 2. Diagram of Intervertebral Disc. (modified fromhttp://www.aafp.org/afp/990201ap/575.html)

Over the years many spinal disorders have been discovered. Many include disc

herniation, facet degeneration, and structural abnormalities such as scoliosis and

kyphosis. It has been reported that one disorder may have a negative cascading impact

on another (Ramakrishna et al., 2001).

Nucleus Pulposus

Transition Zone

Anulus Fibrosus

(Inner)

Anulus

Fibrosus

(Outer)


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Intervertebral disc repair is treated by the replacement of the damaged area of the

nucleus pulposus with a synthetic polymer material or by complete replacement of the

affected disc with an artificial one. Together, these methods both require the replication

of the natural substance and significant durability. Current research has proposed the

injection of silicone elastomers or hydrogels to replace the nucleus pulposus (Bao et al.,

1996; Ramakrishna et al., 2001). A polyisobutylene (PIB) based intervertebral disc

replacement has been suggested. The synthesized PIBs which carry cyanoacrylate end

groups will polymerize when exposed to moisture, proteins, and/or other weak

nucleophiles. They have the potential to be injected into the space that remains after the

nucleus pulpous of the herniated disc is excised from the spine, and will polymerize in

vivo under the influence of moisture and proteins. Adherence and space filling to the

tissue by the injectable PIB is a perfect method as leakage of the polymer into

surrounding tissues is prevented. Also, the cyanoacrylate groups of the newly formed

disc are protected from enzyme degradation by the high molecular flexible weight of the

hydrophobic PIB coils (Kennedy, 2001; Puskas, 2004).

Polymer surface coatings for implantable medical devices are of great

importance. The success of an implant is dependent on the surface chemistry developed,

which actuates interactions at the implant material-tissue interface. The human body is

selective in regard to implants as rejection of foreign material is almost inevitable thus

requiring polymer coatings that are biocompatible and biologically stable (Ramakrishna

et al., 2001).

The failure of previous pacemaker polymer coatings is notable. Polyether

polyurethane (PEU) elastomers were biocompatible and thought to be biostable. These


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attributes helped them win their campaign to replace silicone rubber as pacemaker

surface coatings. Although initial results were positive, the biostability of this polymer

did not meet long term standards (Lambda et al., 1998; Wiggins et al., 2001).

There are two mechanisms for the failure of polyther polyurethane elastomers:

metal ion oxidation (MIO) and environmental stress cracking (ESC). The MIO

mechanism explained the degradation of the PEU that was in contact with the metal ions

of the conductor coils. The ESC mechanism depicted the degradation of the PEU that

was in direct contact with tissue. The outer surface of the PEU in drastic cases has been

known to crack and completely rupture the outer insulation. (Stokes et al., 1987; Wiggins

et al., 2001).

Biocompatibility

There are many important factors that need to be taken into consideration

regarding polymer composite materials and compatibility in vivo. Table 1 lists these

components (Ramakrishna et al., 2001).


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Table 1. Important Components to Consider in Material Selection. (modified fromRamakrishna et al., 2001)


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Biofilms

Biofilms are communities of micro-organisms anchored to a surface

(substratum) and each other by EPS (exrtra-cellular polymeric substance) (Chen et al.,

1994 as cited in Cogan & Keener, 2004). Cellular adherence and formation of biofilms

to synthetic surfaces like polymer based medical materials is very well known, but may

have undesirable consequences. This colonization and bacterial adherence to medical

implants have the ability to produce infections related to the implant (Jansen et al., 1995).

In many situations, the formation of biofilms, help to protect embedded

microorganisms from antibiotics and host defense mechanisms (Evans et al., 1987; Hoyle

et al., 1990; Hoyle et al., 1991; Jansen et al., 1995). This can potentiate to their

persistence and ultimate survival on polymer devices. Biofilms may be responsible for

the complexities in treating foreign body infections and their infamous durability (Jansen

et al., 1995; Kohnen et al., 1995).

As there is a large range of applicable biomaterials performing various functions,

all can be a potential location for microbial colonization and infection. Microbial

contamination of medical devices is caused by a variety of factors. When a device is

implanted, it immediately elicits local antimicrobial immune responses that can result in a

congregation of microbials. Most patients who experience microbial infections due to

implanted medical devices are more susceptible to them as they may be more likely to be

immunocompromised (Francolini et al., 2003).

The mechanism for biofilm formation can be characterized as a three phase

process. Phase 1 consists of initial and irreversible cellular adherence to the polymer


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surface. Phase 2 is the expansion and maturation of the biofilm scaffold. Lastly, phase 3

is the adhesion of individual cells or cellular colonies from the biofilm. These stages are

depicted in Figure 3 (Francolini et al., 2003).

Figure 3. Schematic Representation of Stages 1-3 for Biofilm Formation. (modified fromFrancolini et al., 2003)

Biofilm Prevention

Upon implantation of a medical device, the human body responds to the device by

coating it with a protein layer. This layer consists mainly of albumin, laminin, fibrin, and

fibronectin (Francolini et al., 2004; Jeng et al., 2003). There are several approaches to

counteract the formation of this protein layer, or biofilm growth, on medical devices.

One in particular refers to polymers which are chemically modified to prevent primary

microbial adherence and are able to release antimicrobials to prevent surface growth

(Francolini et al., 2004).

Phase 1 Phase 2 Phase 3


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A decrease in bacterial adherence occurs by changing the surface charge of the

polymer. Since most bacteria are negatively charged, a polymer which has a negative

charge repels bacteria better than an uncharged polymer (Jansen et al., 1995).

Another way to deter antimicrobial growth and adhesion is by the addition of a

silver coating. By binding to microbial DNA and sulphydryl groups, silver ions act to

restrict bacterial replication and deactivate metabolic enzymes (Francolini et al., 2003;

Petering et al., 1976).

Amphiphilic Conetworks

The polymers used in this study for experiments 1 and 2 for the potential

applications of intervertebral disc repair and/or replacement and pacemaker coatings are

amphiphilic conetworks (ACPN). Amphiphilic conetworks are two-component

networks of covalently interconnected hydrophilic/hydrophobic (HI/HO) phases of

cocontinuous morphology; as such they swell both in water and hydrocarbons, and

respond to changes in the medium by morphological isomerization (smart networks)

(Erdodi et al., 2006). ACPNs are responsive to stimuli as they have the ability to

rearrange their morphology very rapidly. This allows the conetwork to produce favorable

conformations in reaction to a medium of any polarity as well as amphiphilic protein

contact (Erdodi et al., 2006).

Amphiphilic conetworks have the potential to be used in a variety of ways for

biomedical applications. They are currently used as controlled implantable drug delivery

devices. In previous work, they exhibit delayed drug delivery profiles, and in some cases


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yield desirable zero-order drug delivery kinetics (Chen et al., 1989; Erdodi et al., 2006;

Ivan et al., 1989, 1990; Kennedy et al., 1990; Keszler, 1993). Most recently, ACPNs

have been used to support cell proliferation (Erdodi et al., 2006; Haigh et al., 2000,

2002). Another application was for a thin film, or antimicrobial coating, which showed

that the biocide was released into the environment over a long time period (Erdodi et al.,

2006; Tiller, 2005a; Tiller, 2005b). Peroxides that become trapped in the hydrophilic

domains of the ACPNs have displayed increased activity and stability. The increased

activity has been attributed to the hydrophilic/hydrophobic interphase of the ACPN

(Bruns, 2005a; Bruns, 2005b; Erdodi et al., 2006).

Polymer Biodegradation

Investigations of polymeric applications in medicine have elucidated serious

questions about the suitability and degradability of polymers in certain circumstances.

The stability of sensitive compounds such as protein and peptide drugs as well as the

ability of living cells to survive in close proximity to an eroding polymer are of concern.

Research regarding the loss of mechanical stability of polymers during erosion can occur

too quickly and cause high concentrations of toxicity due to the degradated products. An

understanding of polymer erosion and degradation is key to the solution of these

undesirable effects (Gopferich, 1996).

In this study for both experiments 1 and 2, polymer toxicity was measured by

lymphocyte infiltration (used as a marker in this thesis). Lymphocytes are inflammatory

cells which migrate to the wound site to assist in the healing process and rid the body of


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any foreign invaders. So the level of polymer toxicity can be directly correlated to the

amount of lymphocytes found at the implant site. An increased number of lymphocytes

discovered at the site is indicative of an undesirable chronic inflammatory response

(Jewrajka et al., 2007).

The process of degradation refers to the chain scission process in which polymer

chains are cleaved resulting in the formation of oligomers, then monomers. Erosion is

described as the loss of material owing to monomers and oligomers leaving the polymer

(Gopferich, 1996; Tamada et al., 1993).

There are two principal ways by which polymer bonds can be cleaved: passively

by hydrolysis or actively by enzymatic reaction (Lenz et al., 1993; Gopferich, 1996).

The most important criterion for monitoring polymer degradation is an observed decrease

in molecular weight. Other parameters include loss of mechanical strength and absolute

chemical degradation into monomers, or monomer release by scission of the polymer

backbone (Gopferich, 1996).


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CHAPTER III

MATERIALS AND METHODS

Experiment 1: Polymer Biocompatibility for Intervertebral Disc Repair/Replacement

It is the goal of experiment 1 to determine if any of the four polymers (polymer,

monomer, initiator, polymer + monomer combination) could be used for intervertebral

disc replacement, or to repair any degenerated discs or disc space that has occurred in the

spine.

Surgical Procedure

A group (n=27) of adult male, SHR/y rats were injected with the anesthesia,

sodium pentathol (50mg/kg,ip) before surgery. For the rats designated for liver

injections, an incision approximately 8 cm long was made on the ventral side of the

animal. The liver was exposed and injected with the respective polymer, or saline for

control.

For the rats designated for brain injections, an incision approximately 5 cm in

length was made on dorsal side of the cranium. With the skull exposed, a dremel with


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drill bit attachment was used to manufacture a hole adequate for a 10 gauge needle to be

inserted. The brain was then injected with the respective polymer, or saline for control.

Polymers used for Brain and Liver Injections

Monomer: 1-cyanoacryl-2,4,4-trimethylpentane(TMP-CA)

Polymer: cyanoacrylate terminated tri-arm star PIB [(PIB-CA)3

Initiator: N,N-diethyl telechelic [(PIB-NEt3)3] PIB

Monomer + Polymer Combination: cyanoacrylate-telechelic 3-arm star polyisobutylene

[(PIB-CA)3] + 2,2,4-trimethylpent-1-cyanoacrylate (pTMP-CA)

The incision made on the ventral side of the animal for the liver injections was

closed with sutures.

The incision made on the dorsal side of the cranium for this procedure was closed

with staples.

For the group of rats designated for solid polymer liver implants, an incision was

generated approximately 8 cm long on the ventral side of the animal. A 2 cm incision

was made on a lobe of the exposed liver, and the respective solid polymer was implanted.

The incision site was then sutured to prevent the solid implant from leaving the

appropriated tissue site.


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Solid Polymers Implanted in the Liver

Monomer: poly(1-cyanoacryl-2,4,4-trimethylpentane)poly(TMP-CA)

Polymer: cross linked [(PIB-CA)3] polymer

Monomer + Polymer Combination: copolymer of [(PIB-CA)3] + 2,2,4-trimethylpent-1-

cyanoacrylate (pTMP-CA)

The incision made on the ventral side of the animal for the liver implants was

closed with sutures.

Upon completion of each surgical procedure, suturing and/or stapling, the post-

operative antibioticadministered to the animals was penicillin (5,000 units, im). Theanimals were housed one to a cage with Sani-Chips bedding (Murphy Forest Products,

Montville, NJ) changed once per week and food (PMI Nutrition Intl., Brentwood, MO)

and water were provided ad libitum. The animals were regularly examined for signs of

pain, infection, or implant/injection rejection.

Tissue Removal and Histology

After two weeks the rats were euthanized via a sodium pentothal overdose, and

the tissues containing the polymer injections and/or implants were removed. The tissue

samples were then placed in a 10% buffered formalin phosphate solution for

preservation.

Tissues were dehydrated and embedded with paraffin in a tissue processor

(Tissue-Tek, Miles Scientific). The tissue samples were then embedded into paraffin wax


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blocks. The tissue blocks were sectioned at 7 microns thick by a Reichert-Jung 820-II

microtome. Sections were taken at the site of the polymer implantation or injection and

placed in a 40 degree Celsius water bath. These sections were transferred to gelatin

coated slides where they were de-waxed, rehydrated, and stained with hematoxylin and

eosin stain (H&E) for microscopic analysis. The slides were observed through a light

microscope and micrographs were taken using an Olympus BX 60 microscope, an MTI

digital camera and the SCION image program. To attain an accurate quantitative

evaluation of the tissue site where the polymer had been implanted or injected, wall

thickness, irregular cells, and lymphocyte infiltration were scored to measure tissue

response. These catergories were blindly scored on a scheme of 0-3 (0 = none, 1 =

negligible, 2 = moderate, 3 = excessive) (Jewrajka et al. 2007).

Experiment 2: Polymer Biocompatibility for Pacemaker Coating

It is the goal of experiment 2 to determine if the polymer tested here, can be used

as an improved coating for cardiac pacemakers.

Surgical Procedure

During experiment 2, each rat was injected with the anesthesia, sodium pentathol

(50mg/kg,ip) before surgery. For the first group of animals (n=4) designated for polymer

implants, an incision approximately 8 cm long was made on the ventral side of the

animal. The peritoneal wall was exposed and the 3 cm long polymer strip was sutured to


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the wall at both ends. For the second group of animals (n=4) designated for the control

Bionate implants, an incision approximately 8 cm long was made on the ventral side of

the animal. The peritoneal wall was exposed and the 3 cm long polymer or Bionate strip

was sutured to the wall at both ends.

Polymer and Bionate Control Implants

Polymer: Polyisobutylene polytetramethylene oxide

Bionate: Thermoplastic polycarbonate urethane

The incision made on the abdominal muscle wall of the animal for the polymer

and Bionate implants were closed with sutures, and the skin was sealed with staples.

Upon completion of each surgical procedure, the post-operative antibioticadministered tothe animals was penicillin (5,000 units, im). The animals were housed one to a cage with

Sani-Chips bedding (Murphy Forest Products, Montville, NJ) changed once per week and

food (PMI Nutrition Intl., Brentwood, MO) and water were provided ad libitum. The

animals were regularly examined for signs of pain, infection, or implant rejection.

Tissue Removal and Histology

After an interval of four weeks, the rats were euthanized via a sodium pentothal

overdose, and the tissue areas containing the polymer and Bionate implants were

removed. The tissue samples were then placed in a 10% buffered formalin phosphate

solution for preservation.


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Tissues were dehydrated and embedded with paraffin in a tissue processor

(Tissue-Tek, Miles Scientific). The tissue samples were then embedded into paraffin wax

blocks. The tissue blocks were sectioned at 7 microns thick by a Reichert-Jung 820-II

microtome. Sections were taken at the site of polymer or Bionate implantation and

situated in a 40 degree Celsius water bath. These sections were transferred to gelatin

coated microscope slides and were allowed 24 hours to dry. The slides were then de-

waxed, rehydrated, and stained with hematoxylin and eosin stain (H&E). The slides were

observed through a light microscope and micrographs were taken using an Olympus BX

60 microscope, an MTI digital camera and the SCION image program. To attain an

accurate quantitative evaluation of the tissue site where the polymer/Bionate had been

implanted, lymphocyte infiltration, irregular tissue, irregular cells and fat infiltration were

scored to measure tissue response. These catergories were blindly scored on a scheme of

0-3 (0 = none, 1 = negligible, 2 = moderate, 3 = excessive) (Jewrajka et al., 2007).

Statistical Analysis for Experiments 1 and 2

Statistical tests performed in this study were 1 way ANOVA and t-test. One way

ANOVA tests were used in each graph to test for significance between injections and

implants. A two-sample t-test was used by way of SPSS (Statsistal Package for the

Social Sciences) to compare the four categories scored between the polymer and Bionate

implants to explore the possibility of any statistically significant differences. Total scores

and averages for each category respective to experiments 1 and 2 are presented. A


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survival table is also shown to focus on the toxicity of the polymers tested. Graphs were

created using Microsoft Excel. Tables were created using Microsoft Word.


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CHAPTER IV

RESULTS


In experiment 1, three categories were blindly scored on a scale of 0-3, by four

individuals, to determine wall thickness around the injection/implant site, number of

irregular cells, and number of lymphocytes present. A score of zero represents a thin

wall thickness and little or no cells present. A score of three is indicative of a thick wall

and a considerably high number of cells present at the site.

Injection/Implant Scoring Averages for All Categories

Table 2 depicts the scoring averages for each injection/implant to compare each

category (wall thickness, irregular cells, lymphocytes) scoring average. Injection/implant

site is shown, as well as surgical notes during removal from each tissue.


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Wall Thickness for Liver Injections

Figure 4 is a graph showing the comparison of wall thickness between polymer

injections and saline control delivered to the liver (mean, s.e.m., 1 way ANOVA). The

scoring means for each group were respectively: Monomer (1.44), Initiator (.57),

Polymer (1.88), Monomer + Polymer (2.25), Saline (.38). There was statistical

significance between Monomer and Saline; p=.016. There was statistical significance

between Polymer and Saline; p=.004. There was statistical significance between

Monomer + Polymer and Saline; p=.003.

Irregular Cells for Liver Injections

Figure 5 is a graph which depicts the comparison of irregular cells for injections

across all treatments administered to the liver (mean, s.e.m., 1 way ANOVA). The

scoring means for each group were: Monomer (2.38), Initiator (.63), Polymer (.25),

Monomer + Polymer (1.0), Saline (0). There was statistical significance between

Monomer and Saline; p=9.73E-5. There was statistical significance between Monomer +

Polymer and Saline; p=.003.

Lymphocytes for Liver Injections

Figure 6 is a graph showing the comparison of lymphocytes between liver

injections for all treatments (mean, s.e.m., 1 way ANOVA). The scoring means for each


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treatment and control group were: Monomer (2.63), Initiator (.82), Polymer (1.13),

Monomer + Polymer (2.25), Saline (.13). There was statistical significance between

Monomer and Saline; p=.001. There was statistical significance between Initiator and

Saline; p=.007. There was statistical significance between Polymer and Saline; p=.001.

There was statistical significance between Monomer + Polymer and Saline; p=0009.

Wall Thickness for Brain Injections

Figure 7 is a graph which shows the comparison of wall thickness between

polymer injections and saline control delivered to the brain (mean, s.e.m., 1 way

ANOVA). The scoring means for each treatment and control group were respectively:

Monomer (.75), Initiator (.63), Monomer + Polymer (1.0), Saline (.82). There was no

statistical significance between these average scores.

Irregular Cells for Brain Injections

Figure 8 is a graph depicting the comparison of irregular cells between brain

injections for all treatments used (mean, s.e.m., 1 way ANOVA). The scoring averages

for each brain injection were respectively: Monomer (.50), Initiator (.38), Monomer +

Polymer (.38), Saline (0). There was statistical significance between Monomer and

Saline; p=.049. There was statistical significance between Monomer + Polymer and

Saline; p=.002.


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Lymphocytes for Brain Injections

Figure 9 is a graph showing the comparison of lymphocytes between all

treatments administered to the brain (mean, s.e.m., 1 way ANOVA). The scoring

averages for each brain injection were: Monomer (1.25), Initiator (1.75), Monomer +

Polymer (.63), Saline (.57). There was statistical significance between Initiator and

Saline; p=.001.

Wall Thickness for Liver Implants

Figure 10 is a graph comparing wall thickness between all treatments implanted in

the liver. Means and s.e.m. were shown in this graph. The scoring averages for each

liver implant were: Monomer (.69), Polymer (2.38), Monomer + Polymer (.88). There

was statistical significance between Polymer and Monomer; p=.0002. There was

statistical significance between Polymer and Monomer + Polymer; p=.0002.

Irregular Cells for Liver Implants

Figure 11 is a graph which compares irregular cells between all types of liver

implants used. Means and s.e.m. were shown in this graph. The scoring averages for

each implant delivered to the liver were: Monomer (.69), Polymer (1.5), Monomer +

Polymer (.44). There was statistical significance between Polymer and Monomer +

Polymer; p=.009.


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Lymphocytes for Liver Implants

Figure 12 is a graph comparing lymphocytes between all treatment implants in the

liver. Means and s.e.m. were shown in this graph. The scoring averages for each implant

in the liver were: Monomer (1.25), Polymer (2.0), Monomer + Polymer (1.82). There

was no statistical significance between treatment groups.

Survival Rate for Injections/Implants

Table 3 shows the number of rats injected or implanted with each polymer or

saline control, site at which the injection or implant was placed, and the number that

survived the procedure. All injections/implants experienced 100% survival rates except:

Monomer + Polymer injection delivered to the liver (50%), Monomer + Polymer

delivered to the brain (50%), Polymer implanted in the liver (50%). Polymer injection

delivered to the brain (0%).

Total Scores for Liver Injections

Figure 13 compares the total score averages across all treatment injections in the

liver (mean, s.e.m., 1 way ANOVA). Total score represents the sum of the averages for

all three categories (wall thickness, irregular cells, lymphocytes). Total score averages

for each treatment was: Monomer (6.45), Initiator (2.02), Polymer (3.26), Monomer +

Polymer (5.50), Saline (.51). There was statistical significance between Monomer and


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Saline; p=.006. There was statistical significance between Initiator and Saline; p=.02.

There was statistical significance between Monomer + Polymer and Saline; p=.018.

Total Scores for Brain Injections

Figure 14 compares the total score averages for brain injections between all

treatments used (mean, s.e.m., 1 way ANOVA). Total score averages for each treatment

was: Monomer (2.50), Initiator (2.76), Monomer + Polymer (2.01), Saline (1.39). There

was no statistical significance between treatment injections and Saline.

Total Scores for Liver Implants

Figure 15 compares the total score averages for treatment implants delivered to

the liver. Means and s.e.m. were shown in this graph. Total score averages for each

treatment was: Monomer (2.63), Polymer (5.88), Monomer + Polymer (3.14). There was

significance between Polymer and Monomer; p=.027.

Histology for Liver Injections

Figure 16 is a representative section of liver tissue used for the analysis and

scoring of tissue response to treatment injections. Digital pictures of the slides were

taken at 40x magnification. Notice the area between the pointers for the combo

(monomer + polymer) slide. The monomer + polymer injection caused an increased wall


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thickness formation around the injection site. Also, notice the increased amount of

irregular cells between the pointers for the monomer slide.

Histology for Brain Injections

Figure 17 is a representative section of brain tissue used for the analysis and

scoring of tissue responses to treatment injections. Digital pictures of the slides were

taken at 40x magnification. On the initiator injection slide, there is an increased

lymphocyte infiltration depicted between the pointers. The combo (monomer + polymer)

slide shows a significant amount of irregular cells, a result from the injection.

Histology for Liver Implants

Figure 18 is a representative section of liver tissue used for the analysis and

scoring of tissue response to treatment implants. Digital pictures of the slides were taken

at 40x magnification. The set of pointers labeled A on the polymer slide specifies wall

thickness caused by the implant. The area between the B labeled pointers represents the

increased amount of irregular cells present from the polymer implant.


In experiment 2, four categories were blindly scored on a scale of 0-3, by three

individuals, to determine lymphocyte infiltration, fat infiltration, irregular cells, and


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irregular tissue. A score of zero represented little or no cells/tissue present. A score of

three was indicative of a considerably high number of cells/tissue present at the site. The

following table depicts the scoring averages for each category listed as well as notes

taken during the surgical removal of each sample.

Scoring Averages Peritoneal Wall Implants and Surgical Notes

Table 4 depicts the scoring averages for each implant delivered to the peritoneal

wall to compare each category (lymphocyte infiltration, fat infiltration, irregular cells,

irregular tissue) scoring average. Surgical notes regarding tissue adherence and polymer

condition during the removal of each respective tissue is provided.

Table 5 shows the scoring averages for Polymer and Bionate control implants to

compare each category respectively. Lymphocyte infiltration average scores for Polymer

implant (1.9) is compared to Bionate (2.3). Fat infiltration average scores for Polymer

implant (.8) is in comparison to Bionate (1.4). Irregular cells average scores for Polymer

(1.1) is compared to Bionate (1.6). Irregular tissue average score for Polymer (1.2) is

compared to Bionate (1.7).

Table 6 compares the total score averages for Polymer and Bionate implants

delivered to the peritoneal wall. Total score represents the sum of the averages for all

four categories (lymphocyte infiltration, fat infiltration, irregular cells, irregular tissue).

The total scores were: Polymer (4.99) and Bionate (6.99). There was no statisitcal

significance.


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Table 7 shows the results of a two-tailed t-test to measure any significance

between categories scored in comparison of Polymer and Bionate implants. There was

statistical significance for fat infiltration at 95% confidence; p=.032.

Histology for Polymer Implants

Figure 19 is a representative section of peritoneal wall tissue used for the analysis

and scoring of tissue response to polymer implants. Digital pictures of the slides are

shown. Polymer 1 is depicted at 40x magnification. Polymer 2 and Polymer 3 are shown

at 100x magnification. The set of pointers on the polymer 1 slide represents an

aggregation of lymphocytes. The area between the pointers on the polymer 2 slide

depicts healthy tissue.

Histology for Bionate control Implants

Figure 20 is a representative section of peritoneal wall tissue used for the analysis

and scoring of tissue response to Bionate implants. Digital pictures of the slides were

taken at 100x magnification. The set of pointers on the Bionate 1 slide represent an area

of irregular cells. The set of pointers labeled A on the Bionate 2 slide specifies

irregular tissue. The set of pointers on the Bionate 2 slide labeled B shows fat

infiltration. The set of pointers on the Bionate 3 slide is representative of lymphocyte

infiltration in response to the implant.


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Table 2. Scoring Averages and Surgical Notes for Each Injection/Implant

Injection/Implant

TissueSite

WallThickness

IrregularCells

Lymphocytes Surgical Notes

Monomer Liver 1.44 2.38 2.63 Abdominal adhesion

to liverInitiator Liver .57 .63 .82 Abdominal adhesionto liver

Polymer Liver 1.88 .25 1.13 Minimal adhesionCombo Liver 2.25 1.0 2.25 Minimal adhesionSaline Liver .38 0 .13 Minimal adhesionMonomer Brain .75 .50 1.25 Injection pushed outInitiator Brain .63 .38 1.75 Injection pushed outCombo Brain 1.0 .38 .63 Injection site goodSaline Brain .82 0 .57 Injection site goodSolid

Monomer

Liver .69 .69 1.25 Significant adhesion

SolidPolymer

Liver 2.38 1.5 2.0 Mesentery adhesion

SolidCombo

Liver .88 .44 1.82 Intestinal adhesion

Table 2. Experiment 1: Scoring Averages and Surgical Notes for Each Injection/Implant.Wall thickness, irregular cells and lymphocytes represent average tissue response scoresfor each implant/injection. A low score denotes a low tissue response vs. a high scorewhich denotes a high tissue response.


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Figure 4. Experiment 1: Wall Thickness for Liver Injections. Comparison of wallthickness across treatment injections (means, s.e.m., 1 way ANOVA) in the liver. There

was statistical significance between Monomer and Saline; p=.016. There was statisticalsignificance between Polymer and Saline; p=.004. There was statistical significancebetween Monomer + Polymer and Saline; p=.003.

Figure 5. Experiment 1: Irregular Cells for Liver Injections. Comparison of irregular cells

across treatment injections (means, s.e.m., 1 way ANOVA) in the liver. There wasstatistical significance between Monomer and Saline; p=9.73E-5. There was statisticalsignificance between Monomer + Polymer and Saline; p=.003.

*

**

**


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Figure 6. Experiment 1: Lymphocytes for Liver Injections. Comparison of lymphocytesacross treatment injections (means, s.e.m., 1 way ANOVA) in the liver. There was

statistical significance between Monomer and Saline; p=.001. There was statisticalsignificance between Initiator and Saline; p=.007. There was statistical significancebetween Polymer and Saline; p=.001. There was statistical significance betweenMonomer + Polymer and Saline; p=0009.

Figure 7. Experiment 1: Wall Thickness for Brain Injection. Comparison of wallthickness across treatment injections (means, s.e.m., 1 way ANOVA) in the brain. Therewas no statistical significance between treatment injections and Saline.

***

***

***

**


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Figure 8. Experiment 1: Irregular Cells for Brain Injections. Comparison of irregular cellsacross treatment injections (means, s.e.m., 1 way ANOVA) in the brain. There was

statistical significance between Monomer and Saline; p=.049. There was statisticalsignificance between Monomer + Polymer and Saline; p=.002.

Figure 9. Experiment 1: Lymphocytes for Brain Injections. Comparison of lymphocytesacross treatment injections (means, s.e.m., 1 way ANOVA) in the brain. There wasstatistical significance between Initiator and Saline; p=.001.

**

*

***


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Figure 10. Experiment 1: Wall Thickness for Liver Implants. Comparison of wallthickness across treatment implants (means, s.e.m., 1 way ANOVA) in the liver. There

was statistical significance between Polymer and Monomer + Polymer; p=.0002. Therewas statistical significance between Monomer and Polymer; p=.0002.

Figure 11. Experiment 1: Irregular Cells for Liver Implants. Comparison of irregular cellsacross treatment implants (means, s.e.m., 1 way ANOVA) in the liver. There wasstatistical significance between Polymer and Monomer + Polymer; p=.009.

******

**


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Figure 12. Experiment 1: Lymphocytes for Liver Implants. Comparison of lymphocytesacross treatment implants (means, s.e.m., 1 way ANOVA) in the liver. There was no

statistical significance between implant groups.


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Table 3. Animal Survivors Associated with Polymer Type

Type Site Number Injected Number Survived Survival Rate

Monomer Liver 2 2 100%

Initiator Liver 2 2 100%Polymer Liver 2 2 100%Combo Liver 2 1 50%Saline Liver 2 2 100%

Monomer Brain 3 1 33%

Initiator Brain 2 2 100%Polymer Brain 2 0 0%Combo Brain 2 1 50%Saline Brain 2 2 100%Solid

MonomerLiver 2 2 100%

SolidPolymer

Liver 2 1 50%

SolidCombo

Liver 2 2 100%

Table 3. Experiment 1: Animal Survivors Associated with Polymer Type. Number ofanimals which received the respective implant and/or injection and the number of thoseanimals that survived the duration of the experiment.


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Figure 13. Experiment 1: Total Mean Scores for Each Group with Liver Injections.Comparison of total scores across all treatment injections (means, s.e.m., 1 way

ANOVA) in the liver. Total score represents the sum of the averages for all threecategories (wall thickness, irregular cells, lymphocytes). There was statisticalsignificance between Monomer and Saline; p=.006. There was statistical significancebetween Initiator and Saline; p=.02. There was statistical significance between Monomer+ Polymer and Saline; p=.018.

Figure 14. Experiment 1: Total Mean Scores for Each Group with Brain Injections.Comparison of total scores across all treatment injections (means, s.e.m., 1 wayANOVA) in the brain. Total score represents the sum of the averages for all threecategories (wall thickness, irregular cells, lymphocytes). There was no statisticalsignificance between treatment injections and Saline.

**

*

*


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Figure 15. Experiment 1: Total Mean Scores for Each Group with Liver Implants.Comparison of total scores across all treatment implants (means, s.e.m., 1 way ANOVA)

in the liver. Total score represents the sum of the averages for all three categories (wallthickness, irregular cells, lymphocytes). There was statistical significance betweenMonomer and Polymer; p=.027.

*


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Figure 16. Experiment 1: Representative Section for Tissue Analysis for Liver Injections.Slides were used for tissue response scoring and analysis. All five pictures here are shown at40x magnification. Combo refers to Monomer + Polymer injection. Notice the area betweenthe pointers for the combo slide. The combo injection caused an increased wall thicknessformation around the injection site. Also, notice the increased amount of irregular cellsbetween the pointers for the monomer slide.


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Figure 17. Experiment 1: Representative Section for Tissue Analysis for Brain Injections.Slides were used for tissue response scoring and analysis. All five pictures here areshown at 40x magnification. Combo refers to monomer + polymer injection. On theinitiator injection slide, there is an increased lymphocyte infiltration depicted between thepointers. The combo slide shows a significant amount of irregular cells, a result from theinjection.


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Figure 18. Experiment 1: Representative Section for Tissue Analysis for Liver Implants.Slides were used for tissue response scoring and analysis. All five pictures here areshown at 40x magnification. Combo refers to monomer + polymer injection. The set ofpointers labeled A on the polymer slide specifies wall thickness caused by the implant.The area between the B labeled pointers represents the increased amount of irregularcells present from the polymer implant.

A

B


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Table 5. Scoring Averages for Polymer and Bionate control Implants

Implant Lymphocyte

Infiltration

Fat

Infiltration

Irregular

Cells

Irregular

Tissue

Polymer 1.9.11 0.8.22 1.1.26 1.2.22

Bionate

Control

2.3.33 1.4.18 1.6.18 1.7.17

Table 5. Experiment 2: Scoring Averages for Polymer and Bionate Control Implants.Lymphocyte infiltration, fat infiltration, irregular cells and irregular tissue scoresrepresent the average for each respective implant in the peritoneal wall. A high score isindicative of a high tissue response vs. a low score which denotes a low tissue response.

Table 6. Total Mean Scores for Peritoneal Wall Implants

Implant Total Score

Polymer 4.99.23

Bionate Control 6.991.19

Table 6. Experiment 2: Total Mean Scores for Peritoneal Wall Implants. Comparison oftotal scores for both treatment implants (means, s.e.m., 1 way ANOVA) in the peritoneal

wall . Total score represents the sum of the averages for all four categories (lymphocyteinfiltration, fat infiltration, irregular cells, irregular tissue). There was no statisticalsignificance.


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Table 7. Two-Tail T-Test for Comparison between Polymer and Bionate across AllCategories Scored

Levenes Test

for Equality of

Variances t-test for Equality of Means

95% Confidence

Interval of the

Difference

F Df

Sig. (2-

tailed)

Std. Error

Difference Lower Upper

Lymphocyte

Infiltration

Equal variances assumed 24.123 16 .224 .35136 -1.18930 .30041

Equal variances not assumed 9.756 .235 .35136 -1.22999 .34110

Fat

Infiltration

Equal variances assumed .038 16 .032 * .28328 -1.26719 -.06614

Equal variances not assumed 15.191 .032 .28328 -1.26980 -.06353

Irregular

Cells

Equal variances assumed .400 16 .176 .31427 -1.11067 .22178


Irregular

Tissue

Equal variances assumed .291 16 .129 .27778 -1.03331 .14442


Note. Comparison of polymer and Bionate control for all four categories (lymphocyteinfiltration, fat infiltration, irregular cells, irregular tissue). F=F variance, df=degrees offreedom. * denotes significance.


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Figure 19. Experiment 2: Representative Sections for Tissue Analysis for Peritoneal WallPolymer Implants. Polymer 1 is shown at 40x magnification. Polymer 2 and Polymer 3are shown at 100x magnification. The set of pointers on the polymer 1 slide represents anaggregation of lymphocytes. The area between the pointers on the polymer 2 slide depictshealthy tissue.


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Figure 20. Experiment 2: Representative Sections for Tissue Analysis for Peritoneal WallBionate Implants. Slides were used for tissue response scoring and analysis. All fivepictures here are shown at 100x magnification. The set of pointers on the Bionate 1 sliderepresent an area of irregular cells. The set of pointers labeled A on the Bionate 2 slidespecifies irregular tissue. The set of pointers on the Bionate 2 slide labeled B shows fatinfiltration. The set of pointers on the Bionate 3 slide is representative of lymphocyteinfiltration in response to the implant.

A

B


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CHAPTER V

DISCUSSION


The goal for Experiment 1 was to determine if any of the four polymers (polymer,

monomer, initiator, polymer + monomer combination) tested in the brain and liver could

be used for intervertebral disc replacement, or to repair any degenerated discs or disc

space that occurred in the spine. It was hypothesized that the polymer sample will elicit

little or no significant tissue damage, and will therefore be best suited for intervertebral

disc replacement and/or repair.

Liver and Brain Injections

Prior experimentation regarding the polymer (cyanoacrylate terminated tri-arm

star PIB [(PIB-CA)3) and monomer + polymer combination (cyanoacrylate-telechelic

3-arm star polyisobutylene [(PIB-CA)3] + 2,2,4-trimethylpent-1-cyanoacrylate (pTMP-

CA)) tested for swelling, extractables, and oxidative resistance among others.


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Under well defined laboratory conditions, these materials exhibited mechanical

properties promising for medical applications, specifically intervertebral discs (Jewrajka

et al., 2008).

The liver was chosen as an alternative tissue site to the brain to evaluate potential

biocompatibility issues with the polymers. Tissue damage analysis for liver injections

showed three treatments: monomer, polymer, and monomer + polymer all having a

higher wall thickness in comparison to the saline control. The initiator may have

recorded the lowest wall thickness for the liver injections because of its lack of

cyanoacrylate, found in the above three mentioned treatment injections. The

cyanoacrylate has induced an increased immune response, in particular wall thickness

around the injection site. Literature supports this result as the polymerization of CA is

exothermic, and the heat released may be responsible for the cell damage in cell culture

(Leggat et al., 2004). Another possible mechanism for CA toxicity refers to the by-

products of degradation: cyanoacetate and formaldehyde, responsible for evoking an

intense inflammatory response (Schwade et al., 2008).

When analyzing irregular cells in the liver it was determined that the monomer

and monomer + polymer injections elucidated an increased response in comparison to the

saline control. This result may be due to the 2, 2, 4-trimethylpentane-1-cyanoacrylate

(TMP-CA) groups that are found in these samples. The polymer and initiator groups did

not contain TMP-CA, and recorded scores that were not statistically significant from the

saline control.

Tissue analysis showed three treatments as having higher scores for lymphocytes

in the liver when compared to saline: initiator, polymer, and monomer + polymer.


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Although the liver responded to the monomer with an increase in wall thickness around

the injection site, lymphocytes were not statistically significant when compared to the

saline injection. This result may be due to the absence of polyisobutylene (PIB), which is

found in the other three treatment injections. The initiator, polymer, and monomer +

polymer may have activated an increase in lymphocyte infiltration in response to an

infection caused by the PIB.

In support of the hypothesis, the irregular cell score of the polymer injection to

the liver was very similar to that of the saline injection. So although an initial immune

response was evoked in the form of lymphocyte infiltration and increased wall thickness,

no tissue damage in the form of irregular cells occurred in comparison to the saline

control. These results suggest negligible polymer toxicity, and little or no cellular

damage.

It is difficult for the chemicals to leak out from the polymer injections and enter

the central nervous system (CNS) via a systemic route, due to the presence of the blood

brain barrier (Kou et al., 1997). This makes it difficult to determine biocompatibility

when referring to a potential polymer application that will be located in the CNS having

direct contact with cerebral spinal fluid (CSF). Therefore, treatment injections into the

brain were performed as suggested by Dr. Kennedy. The brain is among the softest of

biological tissues, but was chosen as a target tissue site to help create an environment

similar to that of the spinal column, in which the polymer implanted there would be

bathed in CSF.

It was found that monomer and monomer + polymer injections into the brain

resulted in an increase in irregular cells compared to the saline control. This finding is


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exactly the same as the results for irregular cell scores recorded for the liver injections in

this study. This finding in the brain once again suggests that cellular damage in the form

of irregular cells was due to the TMP-CA in the monomer and monomer + polymer

combination. Different percentage compositions should be examined in the future for

better biocompatibility results.

Tissue damage analysis showed an elevated lymphocyte infiltration in the brain

for the initiator injection compared to the saline control. To explain this result, the

initiator was the only sample that contained PIB without TMP-CA. The absence of this

chemical interaction may have caused this increased immune response in the brain. The

monomer contained TMP-CA, but not PIB, and resulted in the second highest average

score for this category. Perhaps the PIB with TMP-CA is more biocompatible at this

tissue site in regards to lymphocyte infiltration. This suggestion can be supported, as the

monomer + polymer combination recorded a score (.63) very similar to the saline

injection (.57).

Liver Implants

The polymer implant recorded a higher statistically significant score for wall

thickness when compared separately to the monomer and monomer + polymer implants.

This may be the result of the polymer implant not having a TMP-CA group attached.

The monomer and monomer + polymer implants both contained TMP-CA, and had a

lesser tissue response in the liver.


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The polymer implant also recorded a higher statistically significance score for

irregular cells when compared to the monomer + polymer combination. The lesser score

of the monomer + polymer supports the idea that an implant, which is composed of PIB

and TMP-CA, may be the most biocompatible. So a polymer composed of PIB alone

may not be as biologically compatible as compared to PIB-TMP-CA.

Total Scores for Injections and Implants in Liver and Brain

The monomer injected in the liver recorded the highest total tissue response score

(6.45) and statistical significance was noted compared to the saline control. The initiator

and monomer + polymer treatments also recorded a higher statistically significant score

when compared to the control. The polymer injection recorded a total score that was

found not to be statistically significant compared to saline.

The solid polymer implant in the liver registered the highest total score (5.88).

This finding contradicts the result for total score regarding the polymer hydrogel injected

in the liver. The difference seen in the study may be explained by the morphology of the

polymer implant vs. polymer injection. Biomaterial interactions with tissues are directed

by surface molecules, specific receptors, atomic geometry and the electronic state of the

biomaterial surface (Gadkari et al., 1989). It appears that both of these polymer forms are

not biocompatible.


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Summary

It was very difficult to find literature pertaining to toxic polymer effects in the

liver and brain for the application of intervertebral disc replacement. Searches were

carried out on various websites, electronic journal databases, and search engines

including: Google.com, Googlescholar.com, Dogpile.com, and OhioLink. Experiments

involving polymer injections and/or implants in the brain were inclusive of successful

biodegradable polymers associated with controllable drug carriers.

The brain may be the most important site to focus our attention to when

examining these results, because as mentioned above, this area has a similar environment

to that of the spinal column with the presence of cerebral spinal fluid (CSF). There are

some drawbacks with brain injections which include the risk of infection due to very

frequent reservoir replenishment and peripheral toxicity due to rapid CSF turnover (Kou

et al., 1997). The chemical makeup of the monomer, polymer, and monomer + polymer

combination is the most logical explanation for this increased rate of mortality (~ 55%)

with these injections. The samples are responsible for delivering toxic effects to the

CNS.

The initiator generated the highest total score (2.76) for the brain injections, but

there was not any significant difference between it and the saline control. The polymer

injection failed to record a score here, because both animals injected failed to survive.

Any type of injection into the brain has to be done very carefully as room for error is

small, but I do not believe technique was to blame for this particular result. Both the

initiator and saline injections for this group achieved a 100% survival rate. Two of the


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three animals injected with the monomer at this site died, as well as one of the two

animals injected with the monomer + polymer combination. It was discovered that the

animal with the monomer injection into the brain that did survive had actually pushed the

injection out of the site. Had the injection stayed in the brain, this animals chance of

surviving the two week time interval may have significantly decreased. Based on this

data, it seems as if any injection into the brain other than that of the initiator or saline

control may illicit negative effects on the animal model, most likely leading to death.

Only one of the two animals receiving the solid polymer implant survived. So the

polymer hydrogel and solid form elicited a high degree of mortality (75%) for animals

receiving these treatments, deeming this particular sample extremely toxic.

In 16 of 20 animal models, tissue involvement/adherence was noted. This would

undoubtedly be problematic when pertaining to a prosthetic polymer disc or polymer

injection into intervertebral disc space. Of the four animals that did not show tissue

adhesion, two of the injections (initiator and monomer) were displaced from the target

site (brain), one was a Saline injection into the brain, and the other was a monomer +

polymer combination injection into the brain.


The goal for Experiment 2 was to determine if the polymer tested, in the

peritoneal wall, could be used as an improved coating for cardiac pacemakers. It was

hypothesized that the polymer implant will elicit little or no significant tissue damage,


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nor will it degrade itself, and will therefore be a suitable candidate for a cardiac

pacemaker coating.

It should be noted that tissue sections were not obtained from two of eight animals

in this study. The two tissue samples were extremely brittle, dry, and flaky during the

sectioning process. I met with Dr. Ely and Dr. Salisbury to discuss the matter and it first

was suggested to me to soak the wax block samples in 10% ammonia hydroxide for ten

minutes and then try to section them. After doing this the sections still remained brittle

and dry, so I soaked the wax blocks for three hours. Again, I tried to section each sample

to no avail. I then spoke with Dr. Salisbury once more and we decided to reprocess the

tissue samples. The tissues were removed from their wax blocks and placed in a cassette

to soak in Hemo-De for five days to help remove the paraffin wax still infiltrating the

tissue. After this, the tissue samples were removed from the cassettes and placed in a

cartridge (approximately 2.5x 1 in.) containing Hemo-De, and placed on a rotator for six

days to remove any wax that may have still been present in the tissue. The tissues were

then reprocessed and re-embedded in paraffin wax. Sectioning resumed with the

reprocessed tissue samples and the same result was found as before; the samples

remained too dry to section properly.

Lymphocyte infiltration, irregular cells, and irregular tissue are all signs of the

bodys immune response to a foreign material, in this case, polymer implants onto the

peritoneal wall. A high score in any of these areas is indicative of tissue damage and

overall poor biocompatibility.

The Bionate control implant achieved the highest total score (6.99) compared to

the polymer implant (4.99). This result suggests that although no statistical significance


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between the polymer and Bionate groups was noted, the polymer implant did elicit less of

a tissue response versus the Bionate control. In fact, the polymer induced less of a tissue

response in every category scored, including a statistically significant difference in the fat

infiltration category. The hydrophilic and hydrophobic cross-linked domains that

compose this amphiphilic polymer allows for the counter-current diffusion of water and

oxygen and better overall performance compared to the control (Jewrajka et al., 2007).

In 8 of 8 animals, fibrous encapsulation of the polymer and fat adherence to it

was noted. There was no evidence of fluid accumulation or a visual infectious response.

None of the animals demonstrated any signs of implant rejection (implants protruding

from the skin) (Jewrajka et al., 2007). In support of the fat adherence finding, a similar

result was discovered in a study conducted for a polymer (polypropylene) implanted in

the abdominal wall. The tissue site where the polymer was implanted was removed after

four and eight week time periods. Fat infiltration was found after both durations, but was

greater after twelve weeks (Kaleya, 2005).

The adipose tissue adherence is an interesting phenomenon. The reason for the

fat adhesion to the implants may be a line of defense associated with these animal

models. The fat may have migrated from the epididymis to the implant site on abdominal

wall to protect any potential injury to vital organs

(http://massage.largeheartedboy.com/archive/2009/01/role_of_fat.html).


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CHAPTER VI

CONCLUSION


As the progression of intervertebral disc degeneration fails to cease, research

involving candidate biomaterials for the spinal column increases. Improving implant

design and delivery to the target site is of importance, but biocompatibility and durability

of the polymer is of chief concern.

It is extremely difficult to support my hypothesis from the data presented in this

experiment. The solid polymer implant into the liver recorded the highest total score

compared to the other implants and one out of two animals implanted with the solid

polymer failed to survive. Also, there were no survivors with the polymer injection into

the brain.

The in vivo findings in this study contradict the in vitro results discovered by

Jewrajka et al. Indeed physiological settings are difficult to reproduce in the laboratory,


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but the polymer tested here was extremely toxic and not well tolerated in the brain tissue,

thus the hypothesis was rejected.

To understand the mechanisms involved in the immunological response for these

implants and injections requires a close relationship between multiple disciplines such as

polymer science, biomedical engineering, and biology.


As a medical device coating becomes susceptible to chemical degradation it

becomes brittle and cracking ensues. Biological enzymes attack these weakened areas

and can promote further damage via electrical malfunction of the pacemaker itself.

Improved polymer coatings for pacemakers should prevent cracking and biodegradation

and serve as a successful protective barrier for pacemaker.

Both implants in this experiment failed to repel tissue adherence completely, but

lower inflammatory values in each category scored were recorded for the polymer

compared to the Bionate control. Fat adherence, which was noted in each implant, was

significantly less in the polymer implant compared to the control. The low tissue

inflammatory response suggested by the data supports the hypothesis that the polymer

implant will elicit little or no significant tissue damage.

All four polymers recovered were visually in tact, meaning they looked the same

post-surgery as they did pre-surgery without tears or physical damage. It is my belief

that this sample is both biocompatible and biostable, thus making it suitable as a synthetic

polymer pacemaker coating.


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The polymer coating tested in experiment two may have other roles in future

medical devices. Applications for this polymer may include catheter and cardiac stent

coatings, but future research is needed in the area of biological durability. This study

requires further research to test the long-term biostability of this material in physiological

conditions as in vivo testing involves much more complex biological effects on polymers

and does not always support in vitro testing.


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