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Electrospun Cartilage Glycosaminoglycans for Biomimetic Scaffolds A Thesis Submitted to the Faculty of Drexel University by Sean Michael Dowd in partial fulfillment of the requirements for the degree of Master of Science in Materials Science and Engineering May 2012

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Page 1: Electrospun cartilage glycosaminoglycans for biomimetic ... · Electrospun Cartilage Glycosaminoglycans for Biomimetic Scaffolds Sean Michael Dowd Caroline L. Schauer, Ph.D. Osteoarthritis

Electrospun Cartilage Glycosaminoglycans for Biomimetic Scaffolds

A Thesis

Submitted to the Faculty

of

Drexel University

by

Sean Michael Dowd

in partial fulfillment of the

requirements for the degree

of

Master of Science in Materials Science and Engineering

May 2012

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© Copyright 2012

Sean M. Dowd. All Rights Reserved.

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Dedications

To my family and friends; those still with us, and those who have passed on

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Acknowledgments

To begin, I must thank my advisor Professor Caroline Schauer who’s help,

support, and above all patience have been nothing short of superhuman and really helped

me to understand, and gain an appreciation of, the world of polymer science. Secondly,

I’d like to thank my mentors Keith Fahnestock and Laura Toth. These people were there

from the very beginning to aid me with my infinite amount of questions, and frequent

corrections of my errors. I would also like to thank Jennifer Atchison and Marjorie

Austero for their help in obtaining SEMs, FTIRs, and in the beginning for making sure

that what I was doing actually made sense. I must also thank Dr. Giuseppe Palmese for

the use of the equipment in his lab. I would also like to thank the rest of the Natural

Polymers & Photonics Group for their support. Last, but not least, I’d like to thank my

committee members Professor Caroline Schauer, Professor Michele Marcolongo, and

Professor Wei-Heng Shih.

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Table of Contents

List of Tables .................................................................................................................... vii

List of Figures .................................................................................................................. viii

Abstract .............................................................................................................................. xi

Chapter 1: Introduction ....................................................................................................... 1

1.1 Materials .................................................................................................................... 1

1.1.1 Overview of Glycosaminoglycans ...................................................................... 1

1.1.2 Challenges of Using Glycosaminoglycans ......................................................... 1

1.2 Applications .............................................................................................................. 2

1.2.1 Epidemiology of Knee Cartilage and Meniscus ................................................. 2

1.2.2 Cartilage Tissue Degradation ............................................................................. 3

1.3 Current Non-Tissue Engineered Treatments for OA ................................................ 4

1.4 Tissue Engineered Treatments .................................................................................. 6

1.5 Overview of Knee Cartilage and Meniscus Anatomy ............................................... 9

1.5.1 Articular Cartilage .............................................................................................. 9

1.5.2 Meniscus ........................................................................................................... 11

1.6 Overall Goal and Objectives ................................................................................... 13

1.6.1 Overall Goal ..................................................................................................... 13

1.6.2 Objectives ......................................................................................................... 13

Chapter 2: Background ..................................................................................................... 15

2.1 Materials .................................................................................................................. 15

2.1.1 Chondroitin Sulfate........................................................................................... 15

2.1.2 Hyaluronic Acid ............................................................................................... 17

2.1.3 Solvents ............................................................................................................ 18

2.1.4 Crosslinking ...................................................................................................... 19

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2.2 Electrospinning........................................................................................................ 20

2.2.1 Overview of Electrospinning ............................................................................ 21

2.2.2 Parameters for Electrospinning ........................................................................ 22

2.3 Applications of Electrospun Fibers and Membranes .............................................. 27

Chapter 3: Experimental Design and Procedure ............................................................... 29

3.1 Materials .................................................................................................................. 29

3.1.1 Dimethylformamide Solvent System ................................................................ 29

3.1.2 Acetic Acid Solvent System ............................................................................. 30

3.1.3 Divinyl Sulfone Crosslinking ........................................................................... 30

3.2 Electrospinning........................................................................................................ 31

3.2.1 Dimethylformamide Solvent System ................................................................ 32

3.2.2 Acetic Acid Solvent System ............................................................................. 33

3.3 Measuring pH .......................................................................................................... 35

3.4 Viscosity .................................................................................................................. 36

3.5 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy ............... 38

3.6 Field Emission Scanning Electron Microscopy ...................................................... 39

3.7 Energy-Dispersive X-ray Spectroscopy .................................................................. 40

3.8 Solubility ................................................................................................................. 40

Chapter 4: Results and Discussion .................................................................................... 42

4.1 Dimethylformamide Solvent System ...................................................................... 42

4.1.1 Viscosity ........................................................................................................... 42

4.1.2 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy ........ 44

4.1.3 Field Emission Scanning Electron Microscopy ................................................ 47

4.2 Acetic Acid Solvent System .................................................................................... 56

4.2.1 Viscosity ........................................................................................................... 57

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4.2.2 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy ........ 58

4.2.3 Field Emission Scanning Electron Microscopy ................................................ 61

4.2.4 Energy-Dispersive X-ray Spectroscopy ........................................................... 65

4.3 Chemical Stability ................................................................................................... 67

4.3.1 Field Emission Scanning Electron Microscopy ................................................ 68

4.3.2 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy ........ 70

4.3.3 Solubility .......................................................................................................... 72

Chapter 5: Conclusions ..................................................................................................... 73

Chapter 6: Future Work .................................................................................................... 75

6.1 Collagen .................................................................................................................. 75

6.2 Mechanical Testing ................................................................................................. 75

6.3 Cell and Clinical Studies ......................................................................................... 75

References and Works Cited ............................................................................................. 76

Appendices ........................................................................................................................ 84

Appendix A: Equations used to calculate the optimal amount of DVS to be used ....... 84

Appendix B: Previous Attempts to Electrospin the AA solvent system ....................... 86

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List of Tables

Table 1: Scaffolds that have been developed for tissue engineering applications for

human cartilage. Compiled from (19). ................................................................................ 8

Table 2: Mass and weight percent of ChS used. All other masses were held constant. .. 30

Table 3: The recorded electrospinning parameters and variables for each sample. The

accelerating voltage, distance between the syringe tip and collector plate was recorded,

pump rate, and electric field were recorded for all 13 samples. ....................................... 33

Table 4: Summary of the AA solvent system solutions that were spun and the

atmospheric conditions on that day. .................................................................................. 34

Table 5: Summary of the pH of the DMF solutions examined ......................................... 42

Table 6: Key peaks for both ChS and HA. ....................................................................... 44

Table 7: Summary table of SEMs and Histograms of fiber diameters for the DMF

optimization study. ............................................................................................................ 47

Table 8: A summary table of the fiber diameter statistical data from Figure 24, the

statistical sample size was 50 except for Sample 9 which had a sample size of 10. ........ 54

Table 9: The pH for each different wt% of ChS. .............................................................. 56

Table 10: Shared peaks between ChS and HA. ................................................................ 58

Table 11: Summary table of SEM images and histograms. .............................................. 61

Table 12: Summary data for the fiber diameters for the AA solvent system. Same data as

found in Figure 32. ............................................................................................................ 63

Table 13: The pH of the DVS crosslinker solutions. ........................................................ 67

Table 14: IR Peaks for Figure 38. ..................................................................................... 71

Table 15: Results of the solubility test. ............................................................................. 72

Table 16: Results of Eq.1. ................................................................................................. 84

Table 17: Results of the solver tool. As can be seen, the mass of each chemical correctly

correspond to the moles used for each sample. ................................................................. 85

Table 18: The same procedure used for the DMF solvent system was used. The only

solution that worked was at 15 kV, highlighted in yellow. The fiber diameter is recorded

for that sample in the far right column.............................................................................. 86

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List of Figures

Figure 1: Material components of human meniscus. Cartilage has a very similiar

composition. Image from (12). .......................................................................................... 4

Figure 2: Techniques for cartilage tissue engineering. Image taken from (19). ................ 7

Figure 3: The location of articular cartilage in the knee. The most prominent area is seen

in this image. The white area on the femur is an area of articular cartilage. Taken from

(11). ..................................................................................................................................... 9

Figure 4: Sketch of the layers of hyaline cartilage. The image is arranged such that the

superficial zone is at the top, the middle zone is in the middle, and the deep zone is on the

bottom. The image on the left containing the circular shapes indicates the orientation of

the cells. The middle image shows the alignment of the collagen fibers. The right image

is a false color image of human hyaline cartilage. Image taken from (1). ....................... 10

Figure 5: Image of the meniscus. The colored areas refer to the amount of

vascularization in the tissue. The reddest are the most vascularized, while the white areas

are avascularized. Image taken from (12). ....................................................................... 11

Figure 6: Arrangement of the fiber orientations within the meniscus, and their location.

Image taken from (12). ..................................................................................................... 12

Figure 7: Image of chondroitin-4-sulfate. ......................................................................... 15

Figure 8: Structure of aggrecan. This image contains keratin sulfate in addition to ChS.

The “G” numbers refer to globular domains of the protein. Taken from (6). .................. 16

Figure 9: Structure of hyaluronic acid. ............................................................................. 17

Figure 10: Structures of dimethylformamide (right) and acetic acid (left). ...................... 18

Figure 11: Mechanism of reaction for DVS. .................................................................... 20

Figure 12: Basic electrospinning set-ups. Image from (54). ............................................ 21

Figure 13: Common electrospinning parameters (58). ..................................................... 23

Figure 14: The effects of voltage on fiber morphology. Image taken from (64). ............ 26

Figure 15: Photograph of the electrospinning set-up that was used in this experiment. On

the lower left is the syringe pump. Loaded on to the syringe pump is a 10 mL syringe

with a 16 gauge needle attached. The needle is connected the high voltage supply (top

right). ................................................................................................................................. 32

Figure 16: The pH paper used in this experiment. ............................................................ 35

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Figure 17: The instruments used to measure viscosity. Top) The UGV, Bottom) the

rheometer. ......................................................................................................................... 37

Figure 18: Image of the AT-FTIR that was used in this study. ........................................ 38

Figure 19: Image of the type of SEM that was used in this experiment (75). .................. 39

Figure 20: Image of the reflectance spectrometer set-up used to characterize the solubility

of the samples. .................................................................................................................. 41

Figure 21: Viscosity of the DMF solvent systems with each polymer. ............................ 43

Figure 22: IR spectra of ChS+HA fibers for the DMF solvent system as compared to the

bulk polymers ChS and HA. ............................................................................................. 45

Figure 23: IR spectra for the DMF solvent system as compared to the bulk polymers;

focused from 2500 cm-1

to 600 cm-1

. ................................................................................ 46

Figure 24: Summary of all fiber diameters. Purple indicates no NaPO4, Yellow indicates

a constant field, Blue represents a change in voltage, Red indicates a change in pump

rate, and Green represents a change in distance. .............................................................. 51

Figure 25: Graph comparing the effect of voltage on fiber diameter. .............................. 51

Figure 26: Graph comparing the effects of distance on fiber diameter. ........................... 52

Figure 27: Graph comparing the effects of the electric field on the fiber diameters. ....... 52

Figure 28: Graph comparing the effects of pump rate on fiber diameter. ........................ 53

Figure 29: Graph showing the viscosity of the lower weight percents for the AA solvent

system. Please note the mass of HA was held constant throughout. ............................... 57

Figure 30: IR of the bulk ChS, bulk HA, and 30% mass ChS mat. .................................. 59

Figure 31: IR spectra for the range of 2500-600 showing the key peaks for the polymers.

........................................................................................................................................... 60

Figure 32: Summary of fiber diameters for the AA solvent system. ................................ 64

Figure 33: Fiber diameters for the samples demonstrate a linear trend in conjunction with

an increase of ChS. ........................................................................................................... 64

Figure 34: EDS scan of fibers found in the 30% mass ChS sample with background

elements removed. ............................................................................................................ 66

Figure 35: Image used in the EDX scan. Image is another view of the 30 wt% ChS

solution. ............................................................................................................................. 66

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Figure 36: SEM image of the fibers produced from this solution (top) and a histogram

with table containing the statistical analysis of the fiber diameters (bottom). .................. 68

Figure 37: SEM image of the fibers produced from this solution (top) and a histogram

with table containing the statistical analysis of the fiber diameters (bottom). .................. 69

Figure 38: IR spectra for the excess HA fibers. Before and after crosslinking are shown.

........................................................................................................................................... 71

Figure 39: Screen shot of the solver tool in use. ............................................................... 85

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Abstract

Electrospun Cartilage Glycosaminoglycans for Biomimetic Scaffolds

Sean Michael Dowd

Caroline L. Schauer, Ph.D.

Osteoarthritis is a condition through which cartilage is broken down by the human

body (1). Osteoarthritis of the knee affects approximately 84.5 million people over the

age of 45 in the US alone, or over 27% of the entire population (1). In order to help

combat such a debilitating condition, this study investigated electrospinning natural

polymers that are found in human cartilage: hyaluronic acid and chondroitin sulfate.

Polymer fibers were created using two different solvent systems: aqueous N,N -

dimethylformamide and acetic acid. Optimization tests were performed on each sample to

attempt to control factors such as fiber diameter and polymer concentration. In order to

understand which system would be better for the human body, crosslinking and solubility

studies were performed. When attempting to spin, it was discovered that only the

solution containing aqueous N,N – dimethylformamide and an excess of HA could be

successfully be electrospun and crosslinked.

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Chapter 1: Introduction

1.1 Materials

1.1.1 Overview of Glycosaminoglycans

Glycosaminoglycans (GAGs), linear polymers consisting of two repeating

disaccharide units, are an important part of human physiology (2). These units are

always consist of an amino-sugar and uronic acid. Occasionally, a galactose unit will be

seen in place of one of the other two (2). In mammals, virtually every cell produces at

least one GAG in order to provide support to the extra-cellular matrix (ECM) allowing

the cells to hold together (2). To use an analogy to everyday life, GAGs are the glue that

holds the body’s cells together.

Both human cartilage and meniscus have large quantities of two GAGs:

chondroitin sulfate (ChS) and hyaluronic acid, also known as hyaluronan or hyaluronate

(HA) (1, 3) . These GAGs are essential parts of the cartilage and meniscus. Specifically,

ChS and HA are responsible for most of the chemical bonding that takes place within the

tissue (1, 4–6).

1.1.2 Challenges of Using Glycosaminoglycans

As GAGs are a form of biopolymers, there are several complications that must be

dealt with in order for them to be used. For example, GAGs, and other biopolymers can

have different molecular weights (MW), purity, charge distribution, and even structure

depending on the source (7, 8). A good example of this is chondroitin sulfate.

Although more detail about chondroitin sulfate will be discussed later, it is a

perfect example of how structural differences can change the behavior of the polymer.

Chondroitin sulfate can exist in two structural forms: chondroitin-4-sulfate or

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chondroitin-6- sulfate (7, 9, 10). The only difference between the two is where the

sulfate is located (7, 9, 10). However, this change means the loss of a primary hydroxyl

group which can make chemical modification difficult.

1.2 Applications

One of the primary applications for GAGs involves the repair of the major soft

tissues of the human knee. In order to fully understand these applications, however, a

basic overview of human anatomy is required.

1.2.1 Epidemiology of Knee Cartilage and Meniscus

Around the world, both cartilage and meniscus injuries are a huge problem.

Many of these injuries are sports related, but not all. What is particularly alarming is that

a problem in either the meniscus, or the cartilage, can lead to damage of the other tissue

as well.

Meniscus injuries involve a large percentage of patients. In the United States,

approximately 200,000 people suffer from meniscus injury (11). Most of these are sports

related injuries (11). Of these, 92% require a meniscectomy (11). A meniscectomy is a

surgical procedure where the patients’ own meniscus is removed (11, 12). Furthermore,

over many years a patient that had their meniscus removed will suffer from articular

cartilage degradation as well at an average rate of 4% per year (1, 11, 12).

To compound this problem is osteoarthritis (OA). OA is a condition through

which cartilage tissue (regardless of type) is broken down by the body (1). OA of the

knee affects approximately 84.5 million people over the age of 45 in the US alone, or

over 27% of the entire population (1). What are more horrifying are the age

demographics. Of the US population, 1/5 people over the age of 45 will suffer from OA,

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as compared to 1/2 of the population over 65 that will have OA (1). With statistics like

these, it is clear that some sort of biomimetic material or regenerative template is needed.

1.2.2 Cartilage Tissue Degradation

Cartilage due to OA is due to several issues associated with the tissue and its

environment. The most important factor of cartilage loss due to OA is age (13). The age

of the tissue allows for degradation from mechanical loading and water loss in the tissue

(13). As the tissues age, the collagen and a proteoglycan named aggrecan (see Chapter

2.1.1) in the tissue does not retain as much water as before (13, 14). See Figure 1 for

the material components of human cartilage and meniscus tissue. This change in water

retention is due to changes in molecular weight, concentration, and structure that will

lead to a change in the ECM and result in lowered mechanical strength of the tissue (1,

13, 14). This decrease in mechanical strength and stability could allow for the tissue to

become permanently damaged if too much stress is placed on the joint (13). This will

lead to a cascade effect in which more water is lost and the mechanical strength is

decreased even more until physiologically normal stresses can cause the tissue to

mechanically fail (13).

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Figure 1: Material components of human meniscus. Cartilage has a very similiar

composition. Image from (12).

1.3 Current Non-Tissue Engineered Treatments for OA

As of the time of this writing, there are several clinically available or FDA

approved methods for the treatment of OA. As with all medical procedures, some

treatments are for the early stages of OA, while others are intended for late-stage OA.

Three of the major treatment options will be discussed here. They are the injection of

GAGs into the intra-articular cavity, a meniscectomy, and the use of an on the market

biomimetic material.

One of the more common treatments on the market is the direct injection of HA

into the OA site. This treatment works on the principle that since HA is a natural

component of the cartilage in this area, a direct injection should help to rejuvenate the

amount of HA that is available for the cartilage to use to heal itself (15, 16). As one

author noted:

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“HA has a variety of effects on cells in vitro that may relate to its reported

effects on joint disease. For example, it inhibits prostaglandin E2 synthesis

induced by interleukin-1 (IL-1); protects against proteoglycan depletion and

cytotoxicity induced by oxygen-derived free radicals, IL-1, and mononuclear cell–

conditioned medium; and affects leukocyte adherence, proliferation, migration,

and phagocytosis. HA has been shown to have a direct effect on control

mechanisms of cell activation in monocytes, e.g., it increases messenger RNA

expression for IL-1b, tumor necrosis factor a (TNFa), and insulin-like growth

factor 1 (IGF-1). Monoclonal antibodies against the HA receptor, CD44, block

the effect of HA on expression of IL-1b, TNFa, and IGF-1, indicating direct

interaction of HA with the cell. In cartilage, HA has been shown to suppress

cartilage matrix degradation by fibronectin fragments (15).”

Additionally, since the treatment was made clinically available, it has been discovered

that direct injections of HA may help to inhibit the progress of OA all together (15).

This treatment has been in use for several decades, but there are still numerous

issues with this treatment. To begin, many studies that compared the injection of HA to

the injection of a placebo have shown that there is little statistical difference between the

two (15, 16). Furthermore, there is still little evidence that the treatment is effective in

humans (15, 16). Both of these issues are huge weaknesses that must be addressed.

One of the oldest, and still most common, treatments for OA is a meniscectomy.

This is the surgical removal of the meniscus tissue (11, 12). A meniscectomy is

performed when a surgeon has determined that the meniscus has taken too much damage,

and that the removal of the meniscus is the only way to help alleviate some pain (11, 12).

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This treatment has existed for nearly a century, but is beginning to die out (12). As early

as 1948, it was discovered that the complete removal of the meniscus would lead to

adhervse, and possibly worse conditions in the patient’s knee (12). As a result, most

surgeons have begun to move away from this treatment in favor only partially removed

the meniscus tissue or attempting to repair the tissue (11, 12).

Needless to say, the complete removal of a patient’s meniscus is a completely

unacceptable treatment. The meniscus is an essential component of the knee, and it must

be preserved as much as possible. Without it, the knee joint would not be able to

withstand the compressive forces that it experiences on a daily basis.

Finally, a relatively new treatment that was released on the market that may yet

alter the way meniscus and cartilage conditions are treated in the knee. The product

name is Menaflex®. Menaflex® was a collagen based implant that was either a

biomimetic that served in place of a functional meniscus, or a platform for tissue

regeneration (17, 18). This ambiguity is exactly what caused the US Food and Drug

Administration (FDA) to rescind its previous clearance for the device, and ban its use in

the US (17, 18). As a result, the company that sold the product went bankrupt and,

unfortunately, little data is available in regard to Menaflex® aside from information listed

in the legal briefs surrounding the lawsuits concerning the FDA’s actions (17, 18).

1.4 Tissue Engineered Treatments

One of the most heavily researched areas for the treatment of OA is tissue

engineering. Tissue engineering is the development of a bio-compatible scaffold that is

loaded with the appropriate cells types for the target tissue (19). The cartilage types in

the knee are ideal for tissue engineered devices because they have poor natural

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regenerative ability (19). See Figure 2 for a basic schematic for tissue engineered

cartilage.

Figure 2: Techniques for cartilage tissue engineering. Image taken from (19).

The tissue engineering approach to treating OA is a comparatively new

development. As mentioned above, tissue engineering requires the development of a

biocompatible scaffold in addition to the correct cells types for the targeted tissue. As

can be seen in Table 1, several materials have already been evaluated for use for tissue

engineered cartilage, included both HA and ChS.

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Table 1: Scaffolds that have been developed for tissue engineering applications for

human cartilage. Compiled from (19).

Scaffolds Hydrogel Available

Proteic scaffolds

Collagen Yes

Gelatin No

Fibrin Yes

Polysaccharidic scaffolds

Alginate Yes

Cellulose Yes

Chitosan Yes

Hyaluronic Acid Yes

Chondroitin Sulfate Yes

Artificial scaffolds

Carbon fiber No

Calcium phosphate No

Dacron® No

Polyethylmethacrylate Yes

Polyglycolic acid Yes

Polylactic acid No

Teflon ® No

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1.5 Overview of Knee Cartilage and Meniscus Anatomy

1.5.1 Articular Cartilage

Articular cartilage is defined as the type of hyaline cartilage that is present on the

ends of all of the bones in the knee (see Figure 3) (1). This tissue provides cushioning for

the bone from mechanical forces (1).

Figure 3: The location of articular cartilage in the knee. The most prominent area is

seen in this image. The white area on the femur is an area of articular cartilage. Taken

from (11).

Hyaline cartilage has very unique properties due to its structure. The structure is

comprised of three layers of organization: the superficial zone, the middle zone, and the

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deep zone (1). The superficial zone is comprised of collage fibers that are aligned

parallel to the surface of the cartilage (1). This zone occupies approximately 10-20% of

the total tissue (1). The middle zone occupies 40-60% of the tissue is is comprised of

collagen fibers in a random formation (1). Finally, the deep zone consists of the

remaining tissue and contains fibers that are parallel to the surface of the tissue (1). This

behavior can be seen in Figure 4. What really affects the mechanical properties of the

hyaline cartilage is the collagen. The collagen types that are present are type II (over

50% dry weight of the tissue),V, VI, IX, and XI (1). These collagen types have

extremely high mechanically strength (1).

Figure 4: Sketch of the layers of hyaline cartilage. The image is arranged such that the

superficial zone is at the top, the middle zone is in the middle, and the deep zone is on the

bottom. The image on the left containing the circular shapes indicates the orientation of

the cells. The middle image shows the alignment of the collagen fibers. The right image

is a false color image of human hyaline cartilage. Image taken from (1).

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1.5.2 Meniscus

The meniscus is a U-shaped tissue that is located between the femur and tibia (see

Figure 5) (3, 11, 12). The meniscus provides balance in the knee and transfer mechanical

loads in the joint (3, 11, 12). There are two menisci in each knee in the medial and lateral

directions (3, 11, 12). Given its mechanical function, the meniscus must be able to

withstand many mechanical forces.

Figure 5: Image of the meniscus. The colored areas refer to the amount of

vascularization in the tissue. The reddest are the most vascularized, while the white

areas are avascularized. Image taken from (12).

Not surprisingly, meniscus tissue is not very different from the articular cartilage.

In fact, the meniscus is a a type of cartilage known as fibrocartilage (3, 11).

Fibrocartilage has similar properties to hyaline, but it has a different structure. As the

name suggests, fibrocartilage consists of a fibrous structure (12). Due to its similarity to

hyaline cartilage, the meniscus can support a lot of force.

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The mechanical properties of the meniscus are also due to its structure and

materials. The meniscus is composed of three layers: superficial, lamellar, and deep (3,

11, 12). Collagen fibers in the superficial and lamellar regions are randomly orientated

(3, 12). The deep zone of the meniscus is comprised of ordered fibers that run parallel to

the surface of the meniscus (3, 12). See Figure 6 for a pictorial example of the fiber

orientation and locations. The top two layers of the meniscus are comprised of elastic like

collagen type I, while the deep zone is comprised of mostly collagen type II (12). This

mix of flexible and rigid collagen types explain how the menisci can handle both the

weight of the individual, and endure all the physical activity that the individual performs.

Figure 6: Arrangement of the fiber orientations within the meniscus, and their location.

Image taken from (12).

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1.6 Overall Goal and Objectives

1.6.1 Overall Goal

The overall goal of this study was to electrospin chondroitin sulfate and

hyaluronic acid in such a manner that the natural anatomy of cartilage tissue was

mimicked. This will lay the groundwork for a device that could be used in an

implantable device to replace either articular cartilage or meniscus.

For articular cartilage, the device will have varying dimensions. This is due to the

amount of degradation that can be variable from patient to patient (1). However, one

constant is that the thickness of the mimicked articular cartilage should be 2.26 ± 0.49

mm (20).

For the meniscus tissue, the dimensions are more quantifiable. The dimensions

do vary depending on which of the two meniscus locations is being targeted, the sex of

the patient, the age, and the activity level (12). Targeted dimensions for a device for the

medial meniscus should be 40.5 – 45.5 mm in length, and 27 mm in width (12). For the

lateral meniscus, length should be 32.4 – 35.7 mm and the width should be 26.6 – 29.3

mm (12). Furthermore, the circumferential dimension for the medial meniscus should be

approximately 90 – 110 mm, but the circumferential dimension for the lateral meniscus it

should be 80 – 100 mm (12).

1.6.2 Objectives

The objectives of this study are three-fold. First, an optimization study of an

aqueous N,N - dimethylformamide system will be performed (see Chapter 2.1.3). This

study was based on work from previous researchers (21, 22). Secondly, the use of a

solvent system that is considered to be safer to the human body, acetic acid, will be will

be used to create electrospun fiber. Finally, the chemical stability of the aforementioned

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electrospun mats will be examined through the crosslinking of the mats and solubility

testing.

In short, it is believed that the electrospun and crosslinked GAGs will form a

biomimetic or regeneration template that can be used by orthopedic surgeons.

Furthermore, this device should be simple enough that future researchers will be able to

use this device as a starting point for the attachment of collagen and growth factors to

encourage tissue regeneration.

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Chapter 2: Background

2.1 Materials

2.1.1 Chondroitin Sulfate

ChS is the most important GAG in both the cartilage and the meniscus. There are

two types of ChS found in these tissues; they are chondroitin-4-sulfate and chondroitin-6-

sulfate (see Figure 7 for structure) (1, 6, 9). Both forms play a huge role within aggrecan

in cartilage (1, 6, 23). The molecular weight of chondroitin sulfate is 50,000 (24).

Figure 7: Image of chondroitin-4-sulfate.

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ChS is part of a proteoglycan called aggrecan (1, 6, 9, 23). Aggrecan, like most

proteoglycans, is comprised of two parts: one part is a protein, and the other is a

polysaccride (see Figure 8 for structure), and its main role is to act as a structural protein

(1, 6). Over 90% of aggrecan’s mass is comprised of chondroitin sulfate and its close

relative keratin sulfate (6). Aggrecan is essentially a brittle-brush style polymer, with the

ChS acting as the brush (6). There can be up to 100 individual ChS chains on each

aggrecan molecule (20 kDa each) (6). The ChS mainly acts an attachment site. Collagen

and HA attach to the ChS, and this allows the aggrecan to perform its role as a structural

proteoglycan. More importantly, aggrecan can absorb up to 50 times its own weight in

water gives the cartilage tissue mechanical stability (6, 14).

Figure 8: Structure of aggrecan. This image contains keratin sulfate in addition to ChS.

The “G” numbers refer to globular domains of the protein. Taken from (6).

As briefly described earlier, aggrecan plays a key role in OA. As previously

mentioned, water loss is one of the key factors of OA (13). As the body ages, aggrecan

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will undergo changes in its molecular weight due to the native cells not being able to

produce the same quality and quantity aggrecan as before (1, 13, 14). Since the aggrecan

changes, the amount of water that aggrecan can hold decreases (6, 13, 14). This in turn

leads to the progression of OA (13).

2.1.2 Hyaluronic Acid

Figure 9: Structure of hyaluronic acid.

Hyaluronic acid (HA) is another essential GAG (Figure 9). Unlike ChS, HA acts

as an individual molecule, and not part of a proteoglycan. Its main role and structure are

very similar to ChS. In hyaline cartilage, HA acts as a structural component connecting

the aggrecan and collagen together (4, 6, 25). Additionally, HA is responsible for cellular

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signaling and wound healing in this tissue (4, 21, 25). Needless to say, it is not a part of

an essential proteoglycan, but it is essential for cartilage tissue.

2.1.3 Solvents

Figure 10: Structures of dimethylformamide (right) and acetic acid (left).

GAGs such as HA and ChS are fairly easy to solubilize. Both ChS and HA are

extremely hydrophilic and will dissolve in water rapidly (5, 21, 25). However, water is

not the only solvent that can dissolve these GAGs. Many aqueous organic solvents can

also dissolve ChS and HA. Several studies have shown that both HA and ChS can be

dissolved using a combination of distilled water and N,N - dimethylformamide (DMF)

(21, 22, 26–30). DMF can be toxic to the environment, but a study has shown that the

DMF can evaporate out of electrospun fibers (see Chapter 2.2) (31). Furthermore, both

ChS and HA have been shown to be able to dissolve in acetic acid (AA) (32, 33). AA

can also be toxic, but it has a high vapor pressure and will evaporate at room temperature

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and low pressure (34–36). The chemical structures for both DMF and AA can be seen in

Figure 10.

2.1.4 Crosslinking

Crosslinking is the process of chemically bonding at least two polymer chains

together (37). For GAGs and similar biopolymers, there are numerous crosslinking

agents that could be used. These include, but are not limited to genipin, epichlorohydrin,

glutaraldehyde, and hexamethylene-1,6-diaminocarboxysulphonate (38–44). Ideally, a

crosslinker should be water soluble, nontoxic, and cost-effective (45). One crosslinking

agent that comes comparatively close to this description is divinyl sulfone (DVS). DVS

has been used many times in the past to crosslink polymers that contain primary hydroxyl

groups (45–47). In addition to the above qualities, one upside to using DVS is that its

unique sulfone group can be detected using sulfur microanalysis (45). In short, compared

to other crosslinkers, DVS is very easy to identify.

The mechanism of reaction for DVS is simple. DVS crosslinks via a modified

form of a 1,4 addition, also known as a Michael addition (45). In the most general form,

a Michael addition is a reaction in which a carbanion undergoes a conjugate addition to a

carbonyl compound (48). The general form, however, is not what describes the DVS

crosslinking reaction. As can be seen in Figure 11, the vinyl groups on the DVS

molecule prefer to attack primary hydroxyl groups in alkaline environments (45).

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Figure 11: Mechanism of reaction for DVS.

2.2 Electrospinning

Electrospinning was chosen as the primary processing technique for the proposed

device. This was chosen for several reasons. The first is that electrospinning can

produce fibrous structures similar to natural cartilage tissue, and produce the fibers in a

low-cost way (21, 49, 50). Secondly, electrospinning can produce fibers that are an ideal

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diameter for a biomimetic device. Specifically, a fiber diameter of 0.5 – 2 μm would be

ideal for such an application (51–53).

2.2.1 Overview of Electrospinning

Electrospinning was first developed in the early part of the twentieth century, with

the first US patent being awarded in 1934 to Anton Formhals (54, 55). Electrospinning is

a process where electrostatic forces are applied to a polymeric solution or melt for the

fabrication of nanofibers (fibers with diameters less than 100 nm) (54).

This simple process is flexible, scalable and cost-effective (54). The basic

components of an electrospinning set-up are a high-voltage electrical supply, an infusion

pump, a collector, and of course a container for the polymeric solution with a thin

capillary to serve as a spinneret (8, 54, 56). An example of how a typical electrospinning

set-up appears can be seen in Figure 12.

Figure 12: Basic electrospinning set-ups. Image from (54).

The process is fairly simple. First, the polymer solution or melt is loaded into a

syringe pump with a small capillary at one end (54, 56, 57). Next, an electrical charge is

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applied to the solution via an electrode that is connected to the voltage supply (54, 56,

57). At the same time, the collector (usually a flat plate) is charged, but with the opposite

charge of the solution (54, 56, 57). This collector is placed at a pre-determined distance

from the edge of the capillary (54, 56, 57). As both the polymer and collection plate are

oppositely charged, and since only the polymer is capable of movement, it is the natural

tendency of the now electrically charged polymer to attempt to complete the created

electric circuit (54, 56, 57). The charged polymer will attempt to travel toward the plate,

and as a result, the polymer cone at the end of the capillary form a Taylor cone from

which jets of polymer will begin to eject from tip of the capillary (54, 56, 57).

The jets will spin in the air until they have reached the plate (54). All the while,

the polymer jet will continue to become thinner and thinner due to the increased strain on

the jet, and the solvents that were in solution with the polymer will evaporate off (54).

When the polymer jets finally land on the plate they are extremely small in diameter (54).

2.2.2 Parameters for Electrospinning

As with most manufactured items, there are several parameters that must be met

in order to use the process effectively. As can be seen in Figure 13, these parameters can

be broadly broken up into three different categories. For the purposes of this study,

however, the parameters will be re-categorized. Specifically, the parameters of the

solution, the electrospinning machinery, and environmental conditions will be examined.

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Figure 13: Common electrospinning parameters (58).

2.2.2.1 Solution Parameters

There are several properties of the solution that can affect the quality and quantity

of fibers produced. Several of these properties can be adjusted, but most should be taken

into consideration before attempting a spin. They are viscosity, conductivity, and

concentration.

Viscosity, and closely related elasticity, is one of the most important

electrospinning parameters. In terms of solution properties, it is the viscosity of the

solution that determines whether or not a jet will form off of the droplet at the end of the

capillary (8, 56, 59). Specifically, the viscosity parameter affects the critical

entanglement, or concentration, that will determine if a solution will electrospin

successfully (60, 61). Critical entanglement is the concentration where the polymer

chains in solution begin to overlap each other and become more entwined (60–62). As a

result, this entanglement will cause a bending instability in the fibers, and this is what

causes the polymer jet to whip around during the electrospinning process (60, 61).

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Needless to say, the viscosity is an essential property to understand. The type of

viscosity that must be known is the relative viscosity. This is because the relative

viscosity that determines the fiber diameter and the morphology of electrospun fibers

(63). If the viscosity is too high then the solution will not be able to spin (56).

Interestingly enough, if the viscosity is too low then it still will not spin (56). Rather, a

balance must be reached in order to optimize fiber production for a solution to spin.

Unfortunately, given that every polymer solution is different, there are no set guidelines

or recommendations in terms of desired viscosity for electrospinning

Another parameter of note is the electrical conductivity of the solution. As the

name “electrospinning” implies, the solution must be able to carry an electric current if it

is to spin successfully (54–57). However, much like viscosity, there is no real guideline

for what is to be considered a “good” conductivity reading for electrospinning a solution

(56). What is certain is that the solution must be conductive, or else electrospinning will

not occur.

Finally, the concentration of a solution can affect how well a solution will spin.

Concentration generally ties in closely with viscosity, in that an increased concentration

usually means an increased viscosity (56, 57, 59). This is not always the case however.

Several studies have shown that the concentration of the individual components of a

solution can be modified and have various effects on the viscosity (56, 57). More

specifically, there appears to be an inverse relationship between concetration and fiber

diameter size. One study showed that a decrease in concentration led to an increase in

fiber diameters (57). This study was performed while examining the effects of viscosity,

but instead the researchers stumbled across an interesting behavior concerning the

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concentration. This further implies the underlying importance of taking concentration

into consideration when attempting to electrospin a solution.

2.2.2.2 Electrospinning Parameters

It comes as no surprise that the electrospinning equipment can affect how well a

solution will spin, or if it will spin at all. As alluded to previously, there are three

parameters that can be changed easily by just adjusting the various components of the

electrospinning set-up. Those parameters are target distance, accelerating voltage, and

pump rate.

The distance between the capillary and collector is an important part of

electrospinning. Distances in the centimeter to millimeter range usually yield more

oriented fibers (56). However, once again there are no set guidelines for how close a

sample should be (56). Furthermore, the distance can actually be determined by the

accelerating voltage because a higher voltage at a close distance could result in electrical

damage to the sample (56).

As it has been made clear, the accelerating voltage is one of the most important

parts that can be controlled via the electrospinning set-up. The accelerating voltage

directly effects the fluid flow from the capillary (56, 64). As one researcher noted, “the

changes in the applied voltage will be reflected on the shape of the suspending droplet at

the nozzle of the spinneret, its surface charge, dripping rate, velocity of the flowing fluid,

and hence on the structural morphology of electrospun fibers (64).” The same

researchers, however, did notice that the accelerating voltage can affect the morphology

of the fibers that were formed. This can be seen in Figure 14.

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Figure 14: The effects of voltage on fiber morphology. Image taken from (64).

Lastly, the pump rate can also affect the electrospinning process. Generally, the

pump rate can determine the quality of the fibers that are formed (56, 57, 65). The pump

rate, more commonly referred to as the feed rate, affects the quality by altering the

droplet that forms at the end of the capillary. This is respect, it is similar to viscosity, but

the pump rate does not usually affect if fibers are formed or not. It usually affects only

the morphology, but there are several examples where this is not the case (56, 57, 65).

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2.2.2.3 Environmental Parameters

Not surprisingly, the environment in which a solution is spun can affect the entire

electrospinning process. Of all the parameters discussed, these can be some of the

hardest to control or alleviate the effects of. The primary environmental factors that

affect electrospinning are temperature and humidity.

Temperature affects several other parameters in the electrospining process. The

temperature mostly affects if a reaction will take place within the spinning solution, the

vapor pressure of the solvents in the solution, and also the degradation of any temperature

sensitive polymers (56, 57). Obviously, temperature plays a huge role in determining

which polymers and solvents must be used, and how they should be used. Without it, the

solution could evaporate before it makes contact with the collector.

Finally, the humidity of the environment plays a dramatic role in the

electrospining process. The humidity can affect anything from the fiber morphology, the

pore size of the fibers, or if the fibers (56). The exact mechanisms behind how humidity

generates such varying effects are still unknown, and are still being investigated.

2.3 Applications of Electrospun Fibers and Membranes

Needless to say, electrospun fibers have a large amount of possible applications.

Electrospun fibers and membranes can be used in filtration, clothing, drug delivery,

biomaterials, tissue engineering, nanotubes, and nanoelectronics (66–72). Within the

scope of this study, focus was placed on biomaterials and tissue engineering.

Electrospinning has been used in the field of biomaterials for many years. One of

the natural advtanges of this process is that the fibrous membrane naturally resembles the

ECM of most tissues in both size and mechanical properties (67). As a result,

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electrospun fibers and mats have become fairly popular, but the most common

application is in the overlapping field of tissue engineering.

Tissue engineering has used electrospinning for almost as long as biomaterials,

and for the same reason (67). Electrospun mats and membranes have seen use in

attempts to tissue engineer bone, nerve cells, and many other tissues (67, 69, 73). The

reason why electrospinning was used was because the mats offered a way for the cells to

attach to a mimicked environment and behave physiologically normal (67, 69, 73).

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Chapter 3: Experimental Design and Procedure

3.1 Materials

The basic materials that were used in this study were:

Solids:

Chondroitin sulfate sodium salt, 90+% purity (Indofine Chemical Company,

Hillsborough, NJ, 5 gram container)

Hyaluronic acid sodium salt (Dali Hyaluronic Acid Company, Liuzhou City,

Guangxi, China, 1000 gram package)

Disodium phosphate, Anhydrous, ≥99.999% (metals basis), (Fluka, St. Louis,

MO, 100 gram container)

Liquids:

N,N – Dimethylformamide, Anhydrous, 99.8% (Sigma-Aldrich, St. Louis, MO, 2

Liter bottle)

Acetic acid 99.7% (Sigma-Aldrich, St. Louis, MO, 2.5 Liter bottle)

Deionized water (Milli-pore Filtration System, Ultra-pure water filter, water

resistivity: 18.2Ω at 25°C)

Divinyl sulfone 97% (Sigma-Aldrich, St. Louis, MO, 25 gram container)

3.1.1 Dimethylformamide Solvent System

The first solvent system to be used during this study contained N,N - dimethyl

formaldehyde (DMF) as one of the primary solvents for ChS and HA. The procedure for

creating this solution was taken and modified from a previous study (21, 22). First 0.6 g

of HA sodium, 0.2 g ChS sodium salt, and 0.6 g of disodium phosphate were mixed

together in a glass beaker with a glass stirring rod. Next, the solids were mixed in a

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20mL volume of 1:1 DMF and distilled water. This solution was stirred with a glass

stirring rod until the polymer completely dissolved into solution.

3.1.2 Acetic Acid Solvent System

The acetic acid (AA) solvent system was made in a similar fashion to the DMF

system. For each sample, 0.3 g of HA was used and a varying amount of ChS was used.

Unlike the DMF samples, the materials in the spun solution were altered depending on

what was to be examined. Specifically, the mass of the ChS was varied for each

experiment. This data can be seen in Table 2. Once the polymers were thoroughly mixed

with a glass stirring rod, the polymers were then added to a 6 mL, 1:5 solution of AA:

distilled water. Once again, the solution was stirred with a glass stirring rod until the

polymer completely dissolved into solution.

Table 2: Mass and weight percent of ChS used. All other masses were held constant.

Mass of ChS (g) Weight Percent (M/M) of ChS

0.33 5

0.69 10

1.10 15

1.56 20

2.68 30

3.1.3 Divinyl Sulfone Crosslinking

DVS crosslinking was attempted on both the DMF and AA solvent systems in

order to compare them on a one-for-one basis. Such a comparison would determine

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which solution would be the best for a biomimetic type device. A low concentration for

both polymers was chosen so as to be more economical. Calculations were performed to

determine the ideal amounts of polymer and DVS that would be needed using the number

of moles of each substance. It was shown, however, that given the different molecular

weights of the polymers, mathematically there would always be an excess of one

polymer. For solutions with an excess of ChS the amount of moles used was 4.30x10-4

moles, and for solutions with an excess of HA the amount of moles used was 5.30x10-4

moles. These calculations can be seen in Appendix A. Both solvent systems were mixed

in the same ways as previously, but DVS was added simultaneously with the aqueous

solvents attempting to electrospin. Once again, the solution was stirred with a glass

stirring rod until the polymer completely dissolved into solution.

3.2 Electrospinning

The electrospinning apparatus consisted of a syringe pump (Harvard Apparatus,

70-2209, Holliston, MA), a high voltage power supply (Gamma High Voltage Research,

ES60P-10W, Ormond Beach, FL), a 10mL syringe (Becton, Dickinson and Company ,

REF309604, Franklin Lakes, NJ), and a 21 or 16 guage Luer-lock needle (both Becton,

Dickinson and Company , 305165 and 305198 respectively, Franklin Lakes, NJ). An

image of the apparatus can be seen in Figure 15.

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Figure 15: Photograph of the electrospinning set-up that was used in this experiment.

On the lower left is the syringe pump. Loaded on to the syringe pump is a 10 mL syringe

with a 16 gauge needle attached. The needle is connected the high voltage supply (top

right).

3.2.1 Dimethylformamide Solvent System

The polymer solution was electrospun with several parameters being studied for

each trial. The control for this study was based on the previous work from (21, 22).

Specifically, the control solution was electrospun at a distance of 7 cm, with an

accelerating voltage of 17 kV, and a pump rate of 5 μL/min. All samples were allowed to

spin for 15 minutes. Table 3 shows the variables that were changed for each sample in

each trial. Please note that both the temperature, and relative humidity in the

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environment during electrospinning were recorded using a digital thermohygrometer

(Cole-Palmer, RH520A, Vernon Hills, IL).

Table 3: The recorded electrospinning parameters and variables for each sample. The

accelerating voltage, distance between the syringe tip and collector plate was recorded,

pump rate, and electric field were recorded for all 13 samples.

Sample Conditions Voltage

(kV)

Distance

(cm)

Rate

(µL/min)

Electric

Field

(kV/cm)

Temp

(°C)

RH

(%)

0 No NaPO4 17 7 5 2.4 23 22

1

Control

Constant

Field 17 7 5 2.4 22 22

2 Constant

Field 21 9 5 2.4 22 22

3 Constant

Field 27 11 5 2.4 22 22

4 Varied

Voltage 15 7 5 2.1 22 22

5 Varied

Voltage 12 7 5 1.7 22 22

6 Varied

Voltage 9 7 5 1.3 22 22

7 Varied Pump

Rate 17 7 7 2.4 22 22

8 Varied Pump

Rate 17 7 3 2.4 22 22

9 Varied Pump

Rate 17 7 1 2.4 22 22

10 Varied

Distance 17 5 5 3.4 22 22

11 Varied

Distance 17 9 5 1.9 22 22

12 Varied

Distance 17 11 5 1.5 22 22

3.2.2 Acetic Acid Solvent System

Similar to the DMF system, all AA systems were electrospun at a distance of 7

cm, with an accelerating voltage of 17 kV, and a pump rate of 5 μL/min. All samples

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were allowed to spin for 30 minutes in order to allow for a large quantity of fibers. See

Table 4 for a breakdown of the spin conditions at the time of experiment.

Table 4: Summary of the AA solvent system solutions that were spun and the atmospheric

conditions on that day. Weight Percent of ChS

(wt% [m/m])

Temp

(°C) RH (%)

1 20.0 24

5 22.2 22

10 22.2 74*

15 22.2 69*

20 22.2 22

30 22.8 26

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3.3 Measuring pH

The pH was taken using a pH paper (VWR North American, BDH35309.606).

Images of the equipment can be seen in Figure 16.

Figure 16: The pH paper used in this experiment.

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3.4 Viscosity

The viscosity of all samples was determined using either an Ubbelohde glass

viscometer (UGV) (Fisher Scientific, 13-614A, Pittsburgh, PA), or a rheometer (TA

Instruments AR Rheometer) as can be seen in Figure 17. The UGV measures the

kinematic viscosity of the polymers. The kinematic viscosity was calculated using

ASTM D 445 standards. The kinematic viscosity was then converted to relative

viscosity. The capillary diameter of the UGV was 1 mm. Each solution was measured

three times in order to get an average kinematic viscosity at 22 – 23 °C (the operating

limit for the UGV was 22 ± 2°C). The UGV could only measure kinematic viscosity

from 2 cSt to 10 cSt.

Additionally, a rheometer (TA Instruments AR Rheometer) was used for solutions

that were either too viscous, or not viscous enough, to be measured in the UGV. The

conditions used were a temperature of 25 °C and a 40 mm circular parallel plate, with a

shear rates ranging from 0 – 300 s-1

. The rheometer recorded the dynamic viscosity in

Pa*s. This was converted to kinematic viscosity (cSt) so as to allow for a comparison

with the DMF solutions, and then to relative viscosity for the same reason. Unlike the

kinematic viscosity, the dynamic viscosity does not take the density of the solution into

consideration, thus a conversion was needed in order to perform a comparison. The

viscosity that was recorded was averaged from several points according to when the shear

stress was near zero in order to obtain a near-zero viscosity, which in theory remains

constant (8). This procedure was adapted from a previous study (8).

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Figure 17: The instruments used to measure viscosity. Top) The UGV, Bottom) the

rheometer.

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3.5 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)

was performed to determine if any mats contained both HA and ChS. Specifically, ATR-

FTIR (Thermo Scientific, Nicolet Nexus 870 FT-IR Spectrometer, Waltham, MA.) had a

temperature controller that ranged from 22-110°C (22°C was used), could measure near

and mid infrared, and also contained the single bounce ATR accessory. The ATR-FTIR

was used to examine the percent absorption peaks from 3500 cm-1

to 500 cm-1

. The

device can be seen in Figure 18.

Figure 18: Image of the AT-FTIR that was used in this study.

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3.6 Field Emission Scanning Electron Microscopy

Field emission scanning electron microscopy (FESEM, Zeiss Supra 50VP) was

used to determine if fibers were successfully electrospun, and to determine the diameters

of the spun fibers. All samples were mounted on a stub using a double-sided carbon tape

then coated with platinum/palladium for 35 s, 40 mA using Denton vacuum desk II

sputtering machine. This was done to improve the charging on the samples. See Chapter

4 for a detailed breakdown for each SEM images. See Figure 19 for an image of the

SEM.

The FESEM images were analyzed using ImageJ software from the US National

Institutes of Health. This program uses Java programming in order to perform image

enhancement and measurement (74). This software is open source and available to the

public. ImageJ was used to measure the diameter of the fibers. For each sample, 50

fibers were measured. This was done to ensure a large statistical population.

Figure 19: Image of the type of SEM that was used in this experiment (75).

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3.7 Energy-Dispersive X-ray Spectroscopy

Energy-dispersive x-ray spectroscopy (EDX) was used to determine if both ChS

and HA were in specific electrospun fibers. Specifically, since ChS contained a sulfate

group, the EDX would confirm that ChS was in a fiber because it is the only component

that would contain such a group. EDX was done in conjunction with, and in support of,

FTIR. The EDX operates as an attachment to the SEM. The EDX device was from

Oxford Energy-Dispersive X-ray Microanalysis.

3.8 Solubility

Solubility testing was performed to determine how well an electrospun mat would

survive in an aqueous environment. This was performed using three different aqueous

baths: one bath contained 20 mL of 1.0M of acetic acid, another contained 20 mL of

1.0M sodium hydroxide, and the last bath contained 20 mL of distilled water (38). Six

samples (each sample was 1 mm2 in area) were prepared, such that each bath would

contain two samples. One of each sample was immersed in the baths for 15 minutes and

72 hours respectively (38). After the appropriate amount of time, the solution was

examined for any change in transmittance. Transmittance at 600 nm as taken using the

reflectance spectrometer seen in Figure 20 (Ocean Optics, Inc., USB2000 Miniature Fiber

Optic Spectrometer, and DH-2000 Deuterium Tungsten Halogen Light Sources, Dunedin,

FL) (38).

The solubility was determined by the transmittance of the bath liquids. Cuvettes

filled with 1.5 mL of sample from each bath were used. The transmittance of the samples

was compared to that of control baths. If the sample had a transmittance from 80 - 90%,

the sample was dissolving (38). If the sample had a transmittance greater than 90%, and

there was no visual confirmation, the sample has dissolved (38). If the sample’s

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transmittance was above 90%, and there was visual confirmation of the sample, then the

sample was still dissolving (38). If any samples were below 80%, they were considered

to be not dissolving (38).

Figure 20: Image of the reflectance spectrometer set-up used to characterize the

solubility of the samples.

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Chapter 4: Results and Discussion

4.1 Dimethylformamide Solvent System

The DMF systems had multiple successful electrospin mats. As mentioned

previously, two different solutions were tested. The general parameters are seen below in

Table 5.

Table 5: Summary of the pH of the DMF solutions examined Solution pH

NaPO4 7

No NaPO4 7

4.1.1 Viscosity

The viscosity profiles of the solutions were unsurprising. On average, there was

an increase in viscosity with increasing amounts of polymer solution. This is consistent

with literature (76, 77). The 1 wt% ChS with 2 wt% HA in DMF co-polymer system was

not tested because it was not observed to flow once placed in the UGV. Furthermore,

time constraints prevented a rheological exam.

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Figure 21: Viscosity of the DMF solvent systems with each polymer.

What is interesting, however, is the difference between the addition of ChS and

HA individually to the solvent system. As can be seen in Figure 21, the relative viscosity

of ChS was only 1.04 ± 0.01. The average kinematic viscosity was 0.10 ± 0.01 cSt. This

viscosity for the ChS is to be as expected. A previous study determined that for the

weight percent of ChS that was used, the average kinematic viscosity was approximately

0.09 cSt (78). This value is very close to what was expected.

Another trend was observed with the HA sample. Although data were recorded, it

was beyond the viscometer’s accuracy. The UGV used could only measure up to 10 cSt,

while the lowest HA value was 55.38 cSt. It is safe to say that the solution with only HA

is more viscous than the ChS solution. The kinematic viscosity recorded for the HA

sample is close to the lower end of the reported range of 50 – 1000 cSt that was been

recorded in other studies (79, 80). What data that was able to be obtained from this

0

10

20

30

40

50

60

70

DMF + Water

1 wt% ChS

2 wt% HA

Rel

ativ

e V

isco

sity

Polymer Concentration

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experiment suggests that the HA being used is of lower purity due to ultrapure HA

having a kinematic viscosity of approximately 1000 cSt (79, 80).

4.1.2 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

The IR spectroscopy data of the DMF solvent system fibers reveal that ChS is

present in the fibers. As can be seen in both Figure 22 and Figure 23, the IR spectrum for

the ChS and HA mat contain the same key peaks that are found in the bulk HA and ChS

peaks. The key characteristic peak for ChS, specifically Ch4S, is found at 848 cm-1

(9,

81). These can be seen below in Table 6.

All IR spectra for this solution were taken using the Na2PO4 solution. This was

done because it had the largest fibers overall, and as a result would most yield a more

defined spectrum. Furthermore, it was used because the majority of the mats used during

this part of the study were those containing Na2PO4.

Table 6: Key peaks for both ChS and HA.

Peak Wavenumber (cm-1

) Species

848 ChS Characteristic Peak: C–O–S (9, 81)

1396 C-O (21)

1633 C=O/C-N (21)

2800-2900 C-H (21)

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Figure 22: IR spectra of ChS+HA fibers for the DMF solvent system as compared to the

bulk polymers ChS and HA.

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Figure 23: IR spectra for the DMF solvent system as compared to the bulk polymers;

focused from 2500 cm-1

to 600 cm-1

.

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4.1.3 Field Emission Scanning Electron Microscopy

As can be seen in both Table 7 and Table 8, fibers were produced using the DMF

solvent system. The diameters of the fibers varied greatly depending on the spin

conditions. In both Figure 24 and Figure 25, there appears to be a general trend with the

data. For example, all samples that had a constant field showed an increasing linear trend

despite both the voltage and the distance were increased.

Table 7: Summary table of SEMs and Histograms of fiber diameters for the DMF

optimization study. Sample SEM Image Histogram of Fiber Diameters

0

1

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2

3

4

5

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6

7

8

9

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10

11

12

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Figure 24: Summary of all fiber diameters. Purple indicates no NaPO4, Yellow indicates

a constant field, Blue represents a change in voltage, Red indicates a change in pump

rate, and Green represents a change in distance.

Figure 25: Graph comparing the effect of voltage on fiber diameter.

y = -0.0343x + 0.1996 R² = 0.7931

0

0.05

0.1

0.15

0.2

0.25

0.3

9 12 15 17

Fib

er

Dia

me

ter

(µm

)

Voltage (kV)

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Figure 26: Graph comparing the effects of distance on fiber diameter.

Figure 27: Graph comparing the effects of the electric field on the fiber diameters.

y = 0.0367x + 0.0243 R² = 0.8385

0

0.05

0.1

0.15

0.2

0.25

0.3

7 5 9 11

Fib

er

Dia

me

ter

(µm

)

Distance (cm)

y = 0.0237x + 0.0454 R² = 0.9967

0

0.05

0.1

0.15

0.2

1 2 3

Fib

er

Dia

me

ter

(µm

)

Sample Number

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Figure 28: Graph comparing the effects of pump rate on fiber diameter.

y = -0.028x + 0.2015 R² = 0.4311

0

0.05

0.1

0.15

0.2

0.25

0.3

1 3 5 7

Fib

er

Dia

me

ter

(µm

)

Pump Rate (µL/min)

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Table 8: A summary table of the fiber diameter statistical data from Figure 24, the

statistical sample size was 50 except for Sample 9 which had a sample size of 10.

Sample Conditions Mean (µm) St. Dev.

(±µm) Max (µm) Min (µm)

0 No NaPO4 0.046 0.015 0.083 0.013

1 Constant

Field 0.068 0.033 0.187 0.024

2 Constant

Field 0.094 0.039 0.184 0.030

3 Constant

Field 0.116 0.040 0.218 0.043

4 Varied

Voltage 0.104 0.039 0.227 0.048

5 Varied

Voltage 0.098 0.034 0.182 0.039

6 Varied

Voltage 0.185 0.077 0.377 0.074

7 Varied

Pump Rate 0.129 0.044 0.236 0.048

8 Varied

Pump Rate 0.126 0.040 0.218 0.068

9 Varied

Pump Rate 0.203 0.069 0.307 0.103

10 Varied

Distance 0.102 0.044 0.240 0.044

11 Varied

Distance 0.105 0.042 0.220 0.031

12 Varied

Distance 0.190 0.086 0.373 0.056

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The reasons for this are because the field was maintained constant. To put it

simply, the electric field aids in creating the initial instability in the polymer solution that

eventually results in the jets that create the polymer fibers (50, 82, 83). In the case of this

experiment, since the field was kept constant, fibers would always be produced.

The increasing fiber diameter is the result of the increasing distance (65). When

the voltage of a system is kept constant, but when the distance is not, it is possible that

the polymer solution will not be stretched as thinly (65). Although this previous study

was not examining the effect of the electric field on the fiber diameter, the same principle

applies. To summarize, the further away from a target the solution is, the more voltage is

required to spin successfully. This also helps to explain some of the linear behavior seen

in the samples where the distance was increased, but with no change in voltage. These

conclusions are supported by the data shown in both Figure 26 and Figure 27.

As the name would suggest, the behavior seen in the sample set where the voltage

was changed was due to the change in voltage. As noted earlier, a change in voltage can

severely affect the size and morphology of the fibers (56, 64, 65). For Sample 6

specifically, the drastic change in fiber diameters suggests that the voltage change created

more instability in the fibers than was necessary, and as a result led to the fibers being

larger than the others. This conclusion is supported by data shown in Figure 25.

Furthermore, the change in pump rate was the main reason for the behavior in the

sample set where the pump rate was varied. Although pump rate usually does not

drastically affect fiber morphology, this was not the case in this experiment (59, 65, 84).

Under certain circumstances, the pump rate can affect fiber diameter and morphology

(56, 64, 65). As the rate was lowered, less polymer solution was available on the needle

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tip to spin which explains the overall low yield of this series. As can be seen in Figure

28, there does not appear to be a trend associated with altering the pump rate.

4.2 Acetic Acid Solvent System

The pH data for each sample can be seen in Table 9. The data showed that on

average the solutions were acidic in nature. However, it appeared that ChS did make the

solution more basic in the higher concentrations. This solvent system was spun at a

voltage of 15 kV, 7 cm distance, and with an infusion rate of 5 uL/min. Other distances

and voltages were attempted, but these yielded no fibers. Data for those attempts can be

seen in Appendix B.

Table 9: The pH for each different wt% of ChS.

ChS (wt %) pH

5 3

10 3

15 4

20 4

30 4

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4.2.1 Viscosity

The viscosity measurements of the AA solutions follow the expected trend for

such a system. As can be seen in Figure 29, the viscosity of the solution decreased with

increasing amounts of ChS. This is to be expected as it is known that at room

temperature in a non-alkaline solution, the viscosity number of ChS is 41 (85). This

indicates that beyond a certain mass, the system will become oversaturated and the

viscosity will drop (85). This is due to less entanglement from the polymers (78, 85).

The data supports this assertion. The relative viscosities of the solutions were 253,029 ±

8.08 for the HA sample, 198,478 ± 5.94 for the 1wt% ChS sample, and 32,129 ± 0.36 for

the 5wt% ChS sample. Compared to the DMF solutions, these solutions were generally

more viscous due to the larger relative viscosity that is exhibited in these solutions.

Figure 29: Graph showing the viscosity of the lower weight percents for the AA solvent

system. Please note the mass of HA was held constant throughout.

0

50000

100000

150000

200000

250000

300000

Control 0 wt% ChS (0.25wt% HA)

1 wt% ChS 5 wt% ChS

Re

lati

ve V

isco

sity

Concentration of the polymer systems

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4.2.2 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

FTIR of the electrospun fibers confirmed that both HA and ChS were found in the

mat. As can be seen in both Figure 30 and Figure 31, the IR spectrum for the ChS and

HA mat contain the same key peaks that are found in the bulk HA and ChS peaks. The

key characteristic peak for ChS, specifically Ch4S, is found at 854 cm-1

(9). Given the

similarities in functional groups, both HA and ChS have similar peaks. These can be

seen below in Table 10.

All IR spectra for this solution were taken using the 30 wt% ChS solution. This

was done because it had the largest fibers overall, and would most yield a more defined

spectrum.

Table 10: Shared peaks between ChS and HA. Peak Wavenumber (cm

-1) Species

848 ChS Characteristic Peak: C–O–S (9, 81)

1396 C-O (21)

1633 C=O/C-N (21)

2800-2900 C-H (21)

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Figure 30: IR of the bulk ChS, bulk HA, and 30% mass ChS mat.

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Figure 31: IR spectra for the range of 2500-600 showing the key peaks for the polymers.

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4.2.3 Field Emission Scanning Electron Microscopy

The SEM images of the electrospun fibers yielded fascinating results. Only the

samples that were subjected to the concentration study were examined using the SEM.

To begin, all polymer solutions that were tested successfully spun fibers. The images and

statistics of each sample can be seen in Tables 11 & 12.

Table 11: Summary table of SEM images and histograms.

Sample SEM Image Histogram of Fiber Diameters

5

10

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15

20

30

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Table 12: Summary data for the fiber diameters for the AA solvent system. Same data as

found in Figure 32.

Percent % of

ChS

Average

(μm)

St. Dev.

(±μm)

Max

(μm)

Min

(μm)

5 0.08 0.03 0.16 0.012

10 2.04 0.89 4.63 0.61

15 3.75 2.74 17.47 0.75

20 6.31 2.75 14.42 2.00

30 11.97 14.89 82.96 3.86

The SEM images for the AA solution systems revealed a very surprising trend.

As can be seen in both Figure 32 and Figure 33, there was a general increase of fiber

diameter of nearly 2-3 times for each increase in the weight percent of ChS. Although,

this would seem to be in defiance of the general rule of, “increased concentration,

decreased fiber diameters,” this is not the case. As noted earlier, there is evidence to

suggest that the increase in concentration will lead to an increase in fiber diameter (56,

64, 65). As the only variation between these samples was the concentration of ChS, it is

very likely that the concentration of ChS caused an increase in the fiber diameters.

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Figure 32: Summary of fiber diameters for the AA solvent system.

Figure 33: Fiber diameters for the samples demonstrate a linear trend in conjunction

with an increase of ChS.

y = 0.4657x - 2.5546 R² = 0.9831

p = 0.01, α = 0.05

0

5

10

15

20

25

30

0 5 10 15 20 25 30

Ave

rage

Fib

er

Dia

me

ter

(mic

ron

s)

wt% ChS

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Additionally, as can be seen in Figure 33, the relationship between polymer

concentration and fiber diameter is very linear. A linear regression was performed on the

data that confirmed this suspicion. This behavior lends further support to the theory that

an increase in concentration will lead to increased fiber diameters.

Finally, the viscosity and concentration of these solutions could explain the

increased fiber diameters. Previous studies noticed that there was a correlation between

fiber diameters, concentration, and viscosity (86, 87). One researcher did notice that

viscosity had a small effect on fiber size due to a longer polymer jet (87). The

researchers speculated that because the polymer jet was more elongated in higher

viscosity solutions which would lead to smaller diameter polymers (87). The researchers

concluded that concentration had a greater effect on electrospin fibers (87).

Furthermore, as mentioned earlier, an increase in the concentration of ChS can

lead to less entanglement of the polymer chains due to oversaturation (78, 85). This

could also explain the larger fiber diameters since the polymers were not as entangled as

others, but were beyond the critical entanglement point.

4.2.4 Energy-Dispersive X-ray Spectroscopy

EDX scans of the 30wt% ChS sample further confirmed that ChS was in the

sample fiber in addition to HA. As HA has been known to spin very well, it was assumed

that HA was already present in the fibers, or was the only compound to spin (21, 22).

FTIR did confirm that ChS was present in mat, but it could not determine if ChS was

actually in the fiber. As can be seen in Figure 34, EDX did show that sulfur was in the

fibers. The image used to acquire these scans can be seen in Figure 35.

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Figure 34: EDS scan of fibers found in the 30% mass ChS sample with background

elements removed.

Figure 35: Image used in the EDX scan. Image is another view of the 30 wt% ChS

solution.

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4.3 Chemical Stability

The DVS crosslinked solutions yielded some surprising results. As the DMF

solution without Na2PO4 was found to be among the best of the DMF solutions due to the

fewer materials required, and the smallest fibers, this was used as basis for the testing

solution for the DMF species. The 5 wt% ChS AA solution was used as the starting point

for the AA solutions with DVS. As noted previously, four solutions were used in this

part of the study. The pH of these solutions can be seen below in Table 13.

Table 13: The pH of the DVS crosslinker solutions.

Solution pH

DMF Excess HA 6

DMF with Excess ChS 6

AA with Excess HA 4

AA with Excess ChS 4

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4.3.1 Field Emission Scanning Electron Microscopy

The DMF-DVS systems contained one of the best SEM images. The best image

was from the sample that contained excess HA. The solution containing excess ChS

failed to spin properly and as a result no SEM was performed.

Figure 36: SEM image of the fibers produced from this solution (top) and a histogram

with table containing the statistical analysis of the fiber diameters (bottom).

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The fibers that were spun prior to the cross-linking step were very good. As can

be seen in Figure 36 above, the fibers have excellent morphology and have an average

fiber diameter of 88.89 ± 32.32 nm. The largest fiber diameter was 187.92 nm, while the

smallest fiber diameter was 25.00 nm.

Figure 37: SEM image of the fibers produced from this solution (top) and a histogram

with table containing the statistical analysis of the fiber diameters (bottom).

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The fibers that were spun after the cross-linking step were drastically altered. As

can be seen in Figure 37 above, the fibers appear to have either bonded together, or have

become swelled due to the water bath that was used to remove excess salt. Given that

the average diameter of the remaining fibrous structures was 6.78 ± 2.61 µm, and the

largest fiber diameter was 11.97 µm, while the smallest fiber diameter was 2.22 µm, the

swelling choice appears to be correct. The fibers grew approximately 7000% from the

start of the cross-linking process to the end.

Despite this large jump, this is not a bad thing. The crosslinked “fibers” very

closely match the natural structure of human cartilage. In fact, this accidentally

discovered structure is ideal for a biomimetic device.

4.3.2 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

The IR spectra of the excess HA fibers, as seen in Figure 38, show a successful

crosslinking. There is a clear change in the peaks at 2912 and 2848 which indicates a

change in the C-H bonds (88). Furthermore, the change in C=C appears at 1643 could

indicate that the vinyl groups on DVS have become less prominent, and suggests that the

crosslinking mechanism may have worked (88). Although it does not show definitive

proof of a successful crosslinking step, the IR does offer some support to the theory that

the polymers were successfully crosslinked. Table 14 contains the peak wavenumbers.

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Table 14: IR Peaks for Figure 38. Peak Wavenumber (cm

-1) Species

848 ChS Characteristic Peak: C–O–S (9, 81)

1396 C-O (21)

1633 C=O/C-N (21)

2800-2900 C-H (21, 88)

Figure 38: IR spectra for the excess HA fibers. Before and after crosslinking are shown.

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4.3.3 Solubility

The solubility test performed on the DMF-HA sample yielded some surprising

results. After 15 minutes, the mats were almost completely dissolved in the acidic and

water baths. The transmittance values at the 15 minute mark confirm that the mats were

dissolving at that time (Table 15). At the 72 hour mark, there was no visible trace of any

mat. All transmittance values indicated that the mats had dissolved into solution.

The results of this experiment concur with findings in literature. Although, it

should be pointed that there is little data available that relates to the solubility of

crosslinked ChS and HA in the same material; there is a plethora of information,

however, for each polymer crosslinked individually. The data that most closely matches

the behavior observed in this experiment is seen in several studies that examine the

solubility of crosslinked HA. The researchers found that crosslinked HA will dissolve

rapidly when exposed to hydroxyl radicals (89, 90). In one study, the crosslinked HA

was dissolved within minutes of being exposed to hydroxyl radicals (90).

Table 15: Results of the solubility test.

15 minutes 72 hours

Solution Visual

Observation T600 Comments

Visual

Observation T600 Comments

1.0 M AA

Reference N/A 99.17 ± 0.39 Control N/A 98.46 ± 1.97 Control

1.0 M AA No 99.33 ± 1.15 Dissolved No 95.56 ± 0.72 Dissolved

1.0 M NaOH

Reference N/A 98.53 ± 1.63 Control N/A 98.63 ± 1.43 Control

1.0 M NaOH Yes 88.38 ± 0.65 Partially

Dissolved No 92.11 ± 0.05 Dissolved

DI Water Reference N/A 98.44 ± 0.68 Control N/A 100.13 ± 0.09 Control

DI Water Yes 101.47 ± 1.20 Partially

Dissolved No 101.30 ± 0.47 Dissolved

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Chapter 5: Conclusions

First, ChS-HA nanofibers were successfully electrospun using the previously

known method to electrospin HA. Secondly, a predominantly ChS mat was successfully

electrospun using an AA solvent system which may be better for the body than a DMF

based system. Finally, it was shown that despite its promise, the AA system did not spin

with the added crosslinking agent, but the DMF system was spun and crosslinked

successfully.

The DMF based solvent system operated far better than anticipated. The solution

spun extremely well in the ideal conditions of room temperature and low humidity.

Additionally, it was discovered that the DMF solvent system could be spun without

disodium phosphate, an additive that is required in the spinning of HA. In an industrial

application, this would be an excellent advantage as it would drive down the cost of

manufacture. Once this solution was identified, the viscosity of the solution, and its pH,

were determined in order to better understand the solution parameters.

The ability to spin the AA system in a lower volume solution was another result

that was unexpected. As a result, an attempt was made to discover the most ChS that

could be spun successfully and it was discovered that larger concentrations of ChS

produces larger fiber diameters.

After some calculations, it was decided to test each system against each other

using DVS to crosslink the fibers. This would create a common ground for both solvent

systems and allow for one on one comparison. The optimal amounts of polymer were

added to their respective solvents, and electrospun. The only solvent system that

successfully electrospun was the DMF based solution that contained an excess mass of

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HA which was subsequently tested using solubility tests. In conclusion, this study

successfully laid the groundwork for the development of an electrospun biomimetic

scaffold for both human cartilage and knee meniscus. There is much more work to be

done in regard to these systems, but this study has shown that with some refinement, two

different systems can work can be used to accomplish this goal.

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Chapter 6: Future Work

6.1 Collagen

As noted previously, collagen is the primary polymeric material by mass in both

the knee and cartilage. Future direction will be to include collagen into the CS-HA

crosslinked fibrous mats. The reason that collagen was not spun in this study was because

it is known to cause scar tissue formation, and possibly cause an immune system reaction

(21). Collagen should provide mechanical strength that is needed for this biomimetic

scaffold to function properly in the human body.

6.2 Mechanical Testing

The mechanical testing of this biomimetic scaffold should be attempted in the

future. One of the primary reasons why mechanical testing was not performed during

this study was that collagen was missing from the scaffold. Without collagen, it is

unlikely that the electrospun fibers would have matched the natural tissue in any form of

mechanical testing. Given the discovered structural properties of the electrospun and

crosslinked fibers, however, this should not be a challenge to future researchers.

6.3 Cell and Clinical Studies

As with all devices that will be inserted into the human body, cellular studies

must be performed on the device. Such a study will be able to definitively prove which

solvent system will be the best for a biomimetic scaffold. Hopefully, such tests will lead

to a new medical device on the market for the treatment of OA.

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Appendices

Appendix A: Equations used to calculate the optimal amount of DVS to be used

The moles for each polymer were calculated using the following equation:

Eq.1

The results of this calculation are seen below in Table 16. The mass of all components

was 0.2 g to start off. Any mass could have been used, but 0.2 g was used because it

would translate to a low weight percent.

Table 16: Results of Eq.1.

Chemical Mass (g) Density (g/mL) Mw (g/mol) Moles

ChS 0.2 - 463 0.00043

HA 0.2 - 379 0.00053

DVS 0.2 1.17 118.1 0.00169

Once the number of moles had been determined, it was realized that there would always

be an excess of one polymer compared to the other. With this in mind, the Solver tool in

Microsoft Excel 2010 was used to back calculate the ideal mass for a polymer and DVS,

using the other polymer as the reference. The Solver tool used a GRG nonlinear

mathematical modeling to return the correct mass that would be needed to crosslink all

moles of polymer with DVS. A screenshot of Solver can be seen in Figure 39. The

results of Solver can be seen in Table 17.

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Table 17: Results of the solver tool. As can be seen, the mass of each chemical correctly

correspond to the moles used for each sample.

Moles ChS as the Starting Point

Chemical Mass (g) Density (g/mL) Mw (g/mol) Moles

ChS 0.20 - 463 0.00043

HA 0.16 - 379 0.00043

DVS 0.03 1.17 118.1 0.00043

Moles HA as the Starting Point

Chemical Mass (g) Density (g/mL) Mw (g/mol) Moles

ChS 0.24 - 463 0.00053

HA 0.20 - 379 0.00053

DVS 0.03 1.17 118.1 0.00053

Figure 39: Screen shot of the solver tool in use.

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Appendix B: Previous Attempts to Electrospin the AA solvent system

Table 18: The same procedure used for the DMF solvent system was used. The only

solution that worked was at 15 kV, highlighted in yellow. The fiber diameter is recorded

for that sample in the far right column. Conditions Voltage

(kV)

Distance

(cm)

Rate

(µL/min)

Electric

Field

(kV/cm)

Temp

(°C)

RH

(%)

Fiber Diameter

(µm)

Constant

Field

17 7 5 2.4 24 22 - -

Constant

Field

21 9 5 2.4 24 22 - -

Constant

Field

27 11 5 2.4 24 22 - -

Varied

Voltage

15 7 5 2.1 24 22 0.398 ± 0.147

Varied

Voltage

12 7 5 1.7 24 22

Varied

Voltage

9 7 5 1.3 24 22 - -

Varied

Pump Rate

17 7 7 2.4 24 22 - -

Varied

Pump Rate

17 7 3 2.4 24 22 - -

Varied

Pump Rate

17 7 1 2.4 24 22 - -

Varied

Distance

17 5 5 3.4 24 22 - -

Varied

Distance

17 9 5 1.9 24 22 - -

Varied

Distance

17 11 5 1.5 24 22 - -