PREPARATION OF SOFT MACROPOROUS HYALURONAN...
Transcript of PREPARATION OF SOFT MACROPOROUS HYALURONAN...
Academic year 2012-2013
PREPARATION OF SOFT MACROPOROUS
HYALURONAN GELS
Tine JANSEN
First Master of Pharmaceutical Care
Promoter Prof. Dr. K. Braeckmans
Co-promoter Prof. Dr. A. Larsson
Commissioners Dr. K. Remaut
Prof. Dr. T. De Beer
GHENT UNIVERSITY
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and
Physical Pharmacy
Master thesis performed at:
CHALMERS UNIVERSITY OF
TECHNOLOGY
Department of Chemical and Biochemical
Engineering
Laboratory of Pharmaceutical Technology
COPYRIGHT
"The author and the promoters give the authorization to consult and to copy parts of this thesis
for personal use only. Any other use is limited by the laws of copyright, especially concerning the
obligation to refer to the source whenever results from this thesis are cited."
June 3, 2013
Promoter Author
Prof. Dr. K. Braeckmans Tine Jansen
SUMMARY
Tissue engineering (TE) is a technique that has gained a lot of attention in recent years since
it provides an alternative for the painful and costly transplantation surgeries. A three-
dimensional scaffold is used that meets some requirements such as biocompatibility and
biodegradability. Cryogels are a new kind of material that can be used for this purpose
because they are macroporous structures with sufficient mechanical strength. Hyaluronic
acid (HA) is a suitable polymer to prepare this scaffold as it meets the criteria for a good
scaffold. HA is a negatively charged linear polysaccharide that is found in for example the
skin, cartilage and synovial fluid.
In this study, the reproducibility of previously obtained HA cryogels with ethylene glycol
diglycidyl ether (EGDE) as chemical crosslinker is verified. HA was dissolved in 1% NaOH
before adding the crosslinker. The cryogels were prepared by freezing the gel mixture at -
18°C using liquid cooling. Two series of gels were studied: a series with a fixed HA
concentration and varying EGDE ratios and a series where the EGDE ratio is fixed but the HA
concentration varied. The same series were prepared using a different method in order to
lower the HA concentration required to form a proper gel. The HA hydrogel was prepared in
Milli-Q and then mixed in a 50:50 ratio with a 2% NaOH solution 30 minutes before EGDE
was added. The gels of both preparation methods were analysed using an Instron for the
mechanical properties. Swelling/de-swelling tests were performed too. Water and PBS were
used to swell the gels, aceton for de-swelling the gels.
After being in aceton, the gels absorbed the water within a minute and went back to their
initial weight, indicating excellent swelling behaviour. In PBS the swelling occurred slower.
The gels could be compressed up to 100% without crack development and returned to their
initial shape absorbing the water that came out of the gel while compressing. This indicates
the large interconnectivity in the cryogels. While preparing the gels, it became clear that the
conformation of HA is the crucial parameter for the cryogelation process, although the exact
mechanism is not yet clear. The optimal HA/EGDE ratio is raised to 8.2 instead of 4.3 in the
previous study.
The preparation method can be repeated using different freezing temperatures in order to
observe the influence on the pores. The durability and degradation rate of the HA cryogels
are also interesting to be investigated. Future studies will show whether or not HA cryogels
are useful in real world applications in the TE field.
SAMENVATTING
Tissue engineerging (TE) is een techniek die de laatste jaren veel aandacht gekregen heeft
aangezien het een alternatief biedt voor de pijnlijke en dure transplantatie operaties.
Hiervoor wordt een driedimensionale scaffold gebruikt die biocompatibel en
biodegradeerbaar moet zijn. Een cryogel is een nieuw soort materiaal dat voor TE kan
gebruikt worden omdat het een macroporeuze structuur is die voldoende mechanisch
veerkrachtig is. Omdat het voldoet aan de eisen van een scaffold, is hyaluronzuur (HA) een
geschikt polymeer om deze scaffold mee te maken. HA is een negatief geladen lineair
polysacharide dat gevonden wordt in onder andere de huid, kraakbeen en gewrichtsvocht.
In deze studie werd de reproduceerbaarheid van HA cryogels, met ethyleen glycol diglycidyl
ether (EGDE) als chemische crosslinker, nagegaan. HA werd opgelost in 1% NaOH vooraleer
de crosslinker werd toegevoegd. Door de hydrogel in te vriezen bij -18°C, gebruik makend
van vloeistofkoeling, ontstond de cryogel. Twee gel series werden bestudeerd: één met een
vaste HA concentratie en variërende EGDE ratio, een andere waarbij de EGDE ratio constant
werd gehouden en de HA concentratie werd gevarieerd. Dezelfde gel series werden bereid
door een nieuwe bereidingsmethode te gebruiken, die probeert om de HA concentratie
nodig voor een goede gel, te verlagen. De HA hydrogel werd gemaakt in Milli-Q water en
werd dan, 30 minuten voordat EGDE werd toegevoegd, in een 50:50 ratio gemengd met een
2% NaOH oplossing. Op de gels van beide bereidingsmethoden werd een mechanische test
uitgevoerd en de gels werden onderworpen aan zwelling/ontzwelling testen. Deze
gebeurden in water/PBS om te zwellen, aceton werd gebruikt om te ontzwellen.
De gels toonden een uitstekend zwelgedrag. Na volledige ontzwelling keerden ze terug naar
hun initieel gewicht door het water binnen één minuut te absorberen. In PBS gebeurde de
zwelling trager. De gels konden 100% worden samengedrukt zonder enige breukvorming en
de gels namen hun oorspronkelijke vorm weer aan door het water, dat uit de gels kwam bij
compressie, terug te absorberen. Dit duidt erop dat de poriën in de cryogels met elkaar zijn
verbonden. Tijdens het bereiden van de gels werd het duidelijk dat de conformatie van HA
een cruciale parameter is voor het bereiden van cryogels hoewel dat het exacte mechanisme
nog niet is achterhaald. De optimale HA/EGDE ratio is in deze studie gestegen naar 8.2 in
vergelijking met 4.3 in de vorige.
De bereiding kan worden herhaald gebruik makend van verschillende vriestemperaturen om
zo de invloed hiervan op de poriën te bestuderen. Het is ook interessant om de
duurzaamheid en degradatiesnelheid van de HA cryogels te onderzoeken. Toekomstige
studies kunnen aantonen of HA cryogels effectief kunnen gebruikt worden voor TE.
ACKNOWLEDGEMENTS
I could not have written this thesis without all the support and encouragement I’ve got
during this period. So before you start to read my hard work, I want to thank a few people:
First of all, Dr. Anna Ström, for being the best supervisor you could imagine. You were
always there to answer questions, correct my report in no time and give advice. Your
enthusiasm was contagious. Anna, without your help my thesis wouldn’t look like this!
I want to thank Prof. Dr. K. Braeckmans and Prof. Dr. A. Larsson for giving me the
opportunity to work on this project at Chalmers University of Technology.
Johanna Eckardt, thank you for showing me the world of Zotero. It made referencing a
piece of cake!
I also want to thank all the Masters in the MasterRoom for giving me the adventure of my
life here in Sweden. Jonathan, Toon, Stu, Ching Chiao, Diego, Tone, Victor, Caroline,
Nicole and Esther: thanks for all the great moments we shared and I hope to see you all
soon!
I know it was boring sometimes, but Nicole, thank you for all the time you spent doing the
swelling test and helping me with some of my experiments.
Mama, papa: thank you for always supporting me throughout my studies and this thesis
work. You were always listening to my frustrations and results, although you probably
didn’t understand everything I was saying But most of all, thank you for letting me go on
Erasmus and thus letting me experience the adventure of my life! Special thanks to my
Brother, Robbe, for reading my thesis and correcting the ‘stupid’ language mistakes.
And last but not least, Charlotte! Before I went on Erasmus I just knew who you were, now
I think I can say that I have a new friend for life. Thank you for all those fun moments we
had, all those moments that we couldn’t stop laughing, all of those moments which I am
glad I have experienced them with you And I also want to thank you for expanding my
dictionary with some of your best West Flemish words!
Tack så mycket Sverige för den bästa tiden i mitt liv!
TABLE OF CONTENTS
1. INTRODUCTION .......................................................................................................... 1
1.1. HYALURONAN .............................................................................................................. 2
1.1.1. Functions and distribution in the body .......................................................... 2
1.1.2. Synthesis and degradation ............................................................................ 4
1.1.3. Applications and use of hyaluronic acid ........................................................ 5
1.1.4. Structural properties .................................................................................... 6
1.1.5. Rheological properties of HA solutions ......................................................... 7
1.2. CRYOGELS .................................................................................................................... 8
1.2.1. Polymeric gels in general .............................................................................. 8
1.2.2. Forming of the cryogels ................................................................................ 9
1.2.3. Properties of cryogels ................................................................................. 11
1.2.3.1. Porosity ....................................................................................................... 11
1.2.3.2. Swelling properties ..................................................................................... 11
1.2.3.3. Mechanical properties ................................................................................ 12
1.3. USE OF CRYOGELS ...................................................................................................... 12
1.3.1. Tissue engineering ...................................................................................... 13
2. OBJECTIVES .............................................................................................................. 16
3. MATERIALS AND METHODS ...................................................................................... 17
3.1. MATERIALS ................................................................................................................. 17
3.2. METHODS .................................................................................................................. 18
3.2.1. Preparation of the cryogels ......................................................................... 18
3.2.2. Variation of cryogel preparation ................................................................. 18
3.2.2.1. Variation 1 ................................................................................................... 18
3.2.2.2. Variation 2 ................................................................................................... 18
3.2.2.3. Variation 3 ................................................................................................... 18
3.2.3. Swelling and de-swelling measurements ..................................................... 19
3.2.4. Uniaxial compression measurements .......................................................... 19
3.2.5. Characterisation of HA ................................................................................ 20
3.2.6. Imaging of the pores ................................................................................... 22
4. RESULTS AND DISCUSSION ....................................................................................... 23
4.1. PREPARATION OF CRYOGELS USING HYALURONAN ................................................. 23
4.1.1. Understanding the role of polymer conformation ....................................... 27
4.2. PHYSICAL PROPERTIES OF THE CRYOGELS................................................................. 29
4.2.1. Rheological properties ................................................................................ 30
4.2.2. Swelling capacity ........................................................................................ 32
4.3. MICROSTRUCTUAL CHARACTERISATION ................................................................... 34
5. CONCLUSION ........................................................................................................... 36
6. REFERENCES ............................................................................................................. 38
ABBREVIATIONS
CD44 Cluster of differentiation 44
CEGDE Concentration of EGDE (for EGDE, see below)
CHA Concentration of HA (for HA, see below)
CLSM Confocal laser scanning microscopy
ECM Extracellular matrix
EGDE Ethylene glycol diglycidyl ether
GAG Glycosaminoglycan
HA Hyaluronan, hyaluronic acid
HAS HA synthase
Mw Molecular weight
NA Numerical aperture
PBS Phosphate buffered saline
RHAMM Receptor for HA-mediated motility
SEM Scanning electron microscopy
TE Tissue engineering
UDP Uridine diphosphate
UFLMP Unfrozen liquid monophase
ΔCEGDE Series of gels with varying EGDE concentrations and a fixed hyaluronan
concentration
ΔCHA Series of gels with varying hyaluronan concentrations and a fixed EGDE
concentration
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1. INTRODUCTION
Hyaluronic acid or hyaluronan (HA) is a linear polysaccharide that is found in tissues and
fluids of our body such as the skin, the vitreous, cartilage and synovial fluid. It is
biodegradable, biocompatible, has viscoelastic properties and takes part in different
processes that occur in the body like wound healing and cell motility and differentiation [1].
Since its discovery in 1934 by Karl Meyer and John Palmer, a lot of research has been done
on this molecule, partly due to its interest in several cosmetic, medical and pharmaceutical
applications [2, 3].
In recent years, the technique of cryotropic gelation is becoming more popular for different
applications in a lot of branches [4, 5]. With cryotropic gelation, a macroporous gel is formed
at subzero temperatures. The gel is called cryogel after the Greek word κρύος (kryos), which
means frost or ice [6]. This kind of gel possesses interesting and unique properties. The gels
are typically characterised by large interconnected pores making them spongy and elastic
while being mechanically resilient [7]. These are some of the reasons why cryogels have
gained much attention.
Combining both cryogels and HA, the structure could become very interesting to use in
tissue engineering. Patients whose organ is damaged or should be replaced can be helped
using a HA-cryogel scaffold. It is important that this scaffold is biodegradable and
biocompatible and that it preserves its mechanical properties while in the body [8].
Hyaluronan is very suitable for this purpose because of its many functions in the human
body and its properties. Also, the properties of cryogels match with the requirements for
such a scaffold. Since this is such an interesting structure, it is useful to verify if HA cryogels
can be reproduced and which parameters influence the cryogelation process.
In this introduction, both hyaluronan, cryogels and tissue engineering will be further
described as it is the background of this report. In Chapter 2 the main goals are outlined,
followed by a description of the used materials and methods in Chapter 3. The section that
lists and discusses the results can be found in Chapter 4. The last Chapter formulates the
conclusions that can be drawn from this study.
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1.1. HYALURONAN
Figure 1.1: Disaccharide unit of HA [1].
The name hyaluronic acid was proposed by Karl Meyer and John Palmer. They were the first
to isolate the molecule from bovine vitreous in 1934. Hyaluronic acid is composed of D-
glucuronic acid and N-acetyl-D-glucosamine, two sugar molecules, linked through β-1,4 and
β-1,3 glycosidic bonds (Figure 1.1). The structure is the same in all mammals, which indicates
that hyaluronic acid is quite an important molecule. The name hyaluronic acid is derived
from the word hyalos (Greek for glass), because it was discovered in the vitreous body, and
uronic acid. A more correct name according to the nomenclature of polysaccharides is
hyaluronan (HA). At physiological pH, the polyanionic molecule behaves as a salt, sodium
hyaluronate [1, 3]. In a vitreous replacement during eye surgery in the late 1950s, HA was
used for the first time in medicine. Now HA is used in various applications such as
dermatology and wound healing, cardiovascular applications, ophthalmology and orthopedic
applications [1]. It can be found in the human body in the umbilical cord, skin and synovial
fluid. HA for medical applications used to be isolated from human umbilical cord, but is now
isolated from rooster combs and through microbial fermentation of, for example,
Streptococcus bacteria [1, 9].
1.1.1. Functions and distribution in the body
The role of HA in the body is diverse. It is a lubricant, takes part in wound healing and tumor
metastasis and much more. A few of its functions are described below.
HA has a non-ideal osmotic pressure which means that there is an exponential relationship
between the osmotic pressure and the concentration of HA in solution instead of a linear
relation. Due to these properties, HA has excellent buffering abilities and thus can be used as
an osmotic buffer [10]. HA molecules in solution form, at physiological conditions, an
entangled random network with rather small pores. The small pores can both act as a sieve
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for macromolecules and give the HA solution a high flow resistance. Because of the high flow
resistance, HA and other polysaccharides prevent flow of large volumes of fluids through
tissues [11]. Combining these properties of being an osmotic buffer and having high flow
resistance, HA can regulate the water homeostasis in tissues [10].
Whereas small molecules can go through the HA network without any problems, the
diffusion of large molecules is disturbed by obstructions. Hereby, HA and other
polysaccharides of the connective tissue play a role in the transport of molecules through
the extracellular matrix (ECM) and thus regulate the permeability of tissues [10]. Because of
this property, distribution of infective agents in the body can be inhibited and the deposition
of secretory products like collagen fibers can be directed [11]. Also due to the small pores, a
HA network excludes large macromolecules because there is not enough space between the
polymer chains for the large macromolecules. Small molecules can manage to settle in the
pores. It follows that the larger the molecule and the higher the HA concentration (thus the
tighter the chain network), the more the large molecules will be excluded [11, 12].
HA is involved in several cell-biological interactions. It works as an anti-inflammatory agent
and its concentration is increased during inflammation. Some cells like human mesothelial
cells have a coat of HA which provides protection against attacks of for example viruses,
bacteria and cytotoxic lymphocytes. The coat also regulates other cell interactions like
mitosis and preventing cell adhesion [10]. Besides this, it also takes part in cell
differentiation [12]. Aggrecans, which are cartilage proteoglycans, can bind to HA. This
ensures a stabilisation of the cartilage matrix.
There exist a lot of HA-binding receptors of which the most important are CD44 and
RHAMM. CD44 stands for cluster of differentiation 44. Most tissues express this receptor
that has a lot of functions in the body. At cellular level, it plays a role in endocytosis,
maturation and differentiation, anchoring, motility and it induces growth and survival.
Inflammation and immune function, malignancy, tissue homeostasis and organogenesis and
development also belong to the functions of CD44. RHAMM, the receptor for HA-mediated
motility, mediates the adhesion and cell motility [13].
HA is also involved in cancer promotion. Strangely, both HA and the hyaluronidases interact
in the cancer progression. Tumors like epithelial, ovarial, colon, stomach and acute leukemia
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over-express CD44 and RHAMM and elevate the levels of HA. Due to the increased level of
HA in these cells, the matrix is less dense and hereby are the cell’s motility and its ability to
invade raised. Tumor growth and metastasis are speeded up by the over-expression of HA
synthase (HAS) which elevates the HA concentration [1].
Resulting from the different functions of hyaluronan, it is obvious that HA is widely
distributed in the body. It is one of the largest components of the ECM. The major part can
be found in soft connective tissue like skin, vitreous body, synovial fluid and umbilical cord
but also the lungs, liver, kidneys, brain and muscles contain considerable amounts of HA.
Serum levels are low but rise during disease [1, 14].
HA is, thanks to its outstanding rheological properties, an excellent molecule to lubricate
tissues and joints, where it further acts as a shock absorber and prevents mechanical
damage of the joints [15].
1.1.2. Synthesis and degradation
The synthesis of HA occurs inside the plasma membrane. HAS is a membrane-bound integral
enzyme that catalyses the addition of the sugar residues, using the activated uridine
diphosphate (UDP)-derivatives of the sugars as its substrate. There are three isotypes of
HAS: HAS1, HAS2 and HAS3, differing from each other by their kinetics and polymeric
weights. The sugar units are added at the reducing end of the growing chain in the
extracellular space. This in contrast with the other glycosaminoglycans like heparin from
which the chain grows on the non-reducing end [11, 12, 16]. The number of disaccharides
coupled can be more than 10 000 and each disaccharide weighs approximately 400Daltons.
The molecular weight of HA can thus rise up to several million Daltons depending on the
origin of HA and even when extracted from a single source, it demonstrates a large
polydispersity with regards to molecular weight [11]. Since one disaccharide measures about
1nm, a chain of 10 000 disaccharides has the length of 10µm if stretched out [1]. Some
growth factors like epidermal growth factor, platelet-derived growth factor and other
cytokines are known for stimulating the HA synthesis [12, 16].
The major part of the HA is degraded by the liver. Hyaluronidase, β-D-glucuronidase and β-
N-acetyl-hexosaminidase are the responsible enzymes. First, HA is recognised by a specific
receptor which then will take up the HA via endocytosis. In the lysosomes, the enzymes will
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degrade the HA [16]. High molecular weight HA is cleaved into oligosaccharides by
hyaluronidase. The other two enzymes remove the non-reducing ends from the
oligosaccharide. This yields glucuronic acid and N-acetyl-D-glucosamine which are
transferred to the cytosol where they are further degraded [1, 12].
1.1.3. Applications and use of hyaluronic acid
HA was used for the first time in the medical world in the 1950s during an eye surgery.
Nowadays, HA has applications in several other pharmaceutical and medical uses [1].
DRUG DELIVERY
Hydrogels of HA can be used as trans-dermal and dermal drug delivery systems, which
provide controlled release through the skin into the systematic circulation. The controlled
release reduces the dose and side effects of the drug and keeps a coherent efficacy. They are
also used to selectively apply a cytotoxic drug into HA receptor-expressing cancer cells [9].
Besides trans-dermal and dermal drug delivery, HA is used in several other drug delivery
systems such as nasal, vaginal, parenteral and corneal delivery [15, 17]. Also, it has been
observed that HA appears in the lymph nodes after intravenous injection. It can be
interesting to couple drugs to HA to get the drug in the lymph nodes since the lymphatic
system is often used for the dissemination of malignant tumors [15].
OPHTALMOLOGY
During ophthalmic procedures, a HA gel is often used to prevent dehydration of the cornea.
HA also has applications in the treatment of dry eye and Sjögren’s syndrome, because it
hydrates and protects the eye surface. Due to this property, moisturizing eye drops often
contain HA. Eye drops with HA as polymer have the advantage of having pseudoplastic
properties which cause less irritation and are more comfortable than other polymers. Using
a gel prolongs the drug release and increases the ocular residence thus improving the
bioavailability of HA [15, 17].
HA protects the endothelium, which covers the inner surface of the cornea, from damage
during transplantation of the cornea. Once the endothelium is damaged, it cannot
regenerate and the cornea will become non-transparent. This is why it is important to
protect it. The same applies for cataract surgery [18]. In addition, HA is an excellent carrier
for antibiotics [17].
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VISCOAUGMENTATION (DERMAL FILLERS)
During the aging process, the volume of the skin is reduced due to changes in the
microscopic and macroscopic structure of the skin and so affects the look of the human face.
Surgical techniques used to be the procedure for facial rejuvenation but it increases the loss
of volume. Dermal fillers are the perfect solution because they provide volume due to the
water retention capacity of HA. HA fillers are the most popular because they have most of
the properties an ideal dermal filler should have. They are biocompatible, non-immunogenic,
non-allergenic, safe, easy to distribute and remove and so on [17, 19].
VISCOSUPPLEMENTATION
Viscosupplementation can be defined as the use of an HA solution or gel for the replacement
and supplementation of the pathological synovial fluid [18]. In osteoarthritis, the viscosity of
the synovial fluid is decreased which increases the chance of a cartilage injury. By applying
exogenous HA in the joint, the viscosity, elasticity and the other functions of the synovial
fluid are restored [20].
TISSUE ENGINEERING
Tissue engineering is used for the regeneration of damaged tissues and to replace organs
that are failing or malfunctioning. Degradable biomaterials like HA are very suitable for this
purpose [21]. In paragraph 1.3., this application will be further explained in detail.
1.1.4. Structural properties
HA is a long unbranched polysaccharide, more specifically a glycosaminoglycan (GAG). Unlike
all the other GAGs like heparin and chondroitin, HA is not sulfated. Besides this difference,
the synthesis of HA takes place in the plasma membrane instead of in the endoplasmatic
reticulum and Golgi apparatus [14]. HA consists of a repeating sequence of a disaccharide
containing an uronic acid, D-glucuronic acid, and an aminosugar, N-acetyl-D-glucosamine [1,
12]. Under physiological conditions, the carboxy groups of the glucuronic acid units are
entirely ionised [22]. Due to its polyelectrolyte nature, HA acts as a flexible coiling molecule
at neutral pH and moderate ionic strength [23].
As seen in Figure 1.1, the sugar units are linked through β-1,4 and β-1,3 glycosidic bonds.
Due to the β-conformation, all the functional groups (e.g. hydroxyl groups) are in equatorial
position which makes the molecule sterically and thus energetically stable. When brought in
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solution, hydrogen bonds parallel with chain axis, the molecular structure and interaction
with the solvent causes the HA molecule to form a rigid helix. The equatorial placed
functional groups form a hydrophilic side in solution which can be hydrated. This will ensure
that the molecule can retain 1000 times its own weight in water [1, 12].
HA molecules have large hydrophobic patches regularly repeated at opposite faces of the
molecule which in solution can interact with each other. HA molecules thus can self-
aggregate through hydrophobic interactions [24].
1.1.5. Rheological properties of HA solutions
Solutions of HA behave as non-Newtonian and pseudoplastic materials [1, 25, 26]. The
viscosity of the solutions is influenced by the concentration of polymer, ionic strength, pH
and shear rate.
It is seen that the pH has an influence on the degradation of HA due to the cleavage of the
glycosidic bonds. At pH values below 1.6 and above 12.6, degradation of HA occurs. Between
those values there is only little degradation and the rheological properties stay almost the
same [27, 28]. The hydrogen bonds between HA chains create a helix structure of HA hereby
providing chain stiffness. At high pH the hydrogen meshwork disaggregates which makes the
viscosity decrease [24]. So, high pH causes a decrease in viscosity due to breaking of the
hydrogen bonds and cleavage of the glycosidic bonds. The degree of degradation is much
higher at high pH than at very low pH. Also the molecular weight and the radius of gyration
are reduced both related to the breakage of hydrogen bonds and the cleavage of the
backbone [27, 28]. At a pH of 2.5, the solution behaves differently than at other pHs and
becomes a highly elastic paste. It is assumed that at pH 2.5, the repulsive forces of the
ionised carboxyl groups and attractive interactions are in balance forming a distinct rigidity
in the HA chains and aggregation of HA chains. This causes the paste like character of the
solution rather than covalent crosslinks or entanglement between the HA molecules [15].
The viscosity will also decrease when the shear rate is increased. This phenomenon is seen
for example when a HA solution is pushed through a syringe. At high shear rates, the
entanglements and the intermolecular interactions between the HA chains disrupt. This is
why a HA solution with a pH between 1.6 and 12.6 shows shear thinning. In the pH range
lower than 1.6 and greater than 12.6, the viscosity decreases even at low shear rates and
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this because the pH causes the cleavage of the glycosidic bonds which was mentioned
before. At a pH of 13, the network is fragmented and completely gone resulting in a
Newtonian viscosity [27, 28]
The last parameter that influences the viscosity is the ionic strength. Solutions with high
ionic strength will be less viscous than low ionic strength solutions [15].
HA does not form strong gels but entangled solutions. At a concentration of 1 mg/mL, the
HA chains start to entangle with each other [22]. This means that the interactions are mainly
topological or weak transient aggregates. This results in a sample that cannot support its
own weight. The HA will eventually be diluted by the surrounding (body) fluids thus
disentanglement will occur resulting in a largely reduced elasticity and viscosity when no
covalent crosslinks are present.
1.2. CRYOGELS
Polymeric gels are already used in different fields of science, for example biotechnology,
biomedicine and pharmaceutics, but they encounter some problems. Due to these problems
and the different biological targets that have been found already, other gels with other
properties are required. Cryogels can provide these different properties. The gels are called
after their production process, namely a freezing technique. Hence, the gels are called
cryogels which is derived from the Greek word for ice or frost, kryos [29].
1.2.1. Polymeric gels in general
A gel can be defined as an immobilised solvent-polymer system. The polymer strains
crosslink with each other hereby forming a three-dimensional network. Liquids make the
network swell but the polymeric network does not dissolve in it. For preparing the gel either
synthetic or biological polymers can be used. Gels are typically divided as chemical, physical
or entangled networks. A chemically crosslinked gel is formed by adding a chemical agent,
which through the formation of chemical bonds, will provide crosslinks. A physically
crosslinked gel is crosslinked via weak interactions, such as ionic interactions and hydrogen
bonds. If the gel is made by the entanglement of the polymer strand, it is typically referred
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to as an entangled network [30]. This means that at rest, the sample is characterised as a
solution but as it is disturbed, it will have elastic properties.
As mentioned before, HA has poor biomechanical properties and is hydrophilic and thus
highly soluble in water. These problems will be solved by covalently crosslinking the gel using
e.g. ethylene glycol diglycidyl ether (EGDE). EGDE is non-cytotoxic and hydrophilic and it can
form crosslinks in three different ways, depending on the pH (Figure 1.2). Under alkaline
conditions, EGDE will react with the hydroxyl groups and under acidic conditions with the
carboxyl groups. At neutral pH, EGDE would react with an amine but this will not happen as
HA does not have amine groups [31].
Figure 1.2: Crosslinking reactions of EGDE [31]
1.2.2. Forming of the cryogels
Figure 1.3 shows schematically the steps required for the formation of a cryogel. A cryogel is
formed by freezing a system, capable of forming gels, at a temperature a few degrees below
the crystallisation point of the solvent. Except for thermotropic gels, every gel-forming
system can be used. When frozen, the gels consist of two phases: the unfrozen liquid
monophase (UFLMP) and crystals of the solvent, in this case water. The solvent crystals grow
during freezing and will eventually meet each other resulting in a frozen network of crystals.
These crystals will act as pore-forming agents. The chemical reaction of gel-formation occurs
in the UFLMP, in which the polymer and crosslinker are concentrated [32]. This is called
cryo-concentration. Due to the cryo-concentration of the polymer and the crosslinking agent
in the UFLMP, the critical polymer concentration of gelation is decreased and thus there is
10
less polymer needed to form a gel. By changing the solvent, the concentration and molecular
weight of polymer or crosslinker, freezing temperature, freezing rate and the chain
conformation of the polymer, the volume of the UFLMP will change therefore changing the
cryo-concentration [4, 29]. Because the system is semi-frozen, the polymer chains can move
from the pores to the pore walls ensuring the mechanical strength of the gel [32]. On
thawing the gels, the network of solvent ice crystals form angular, interconnected pores
filled with the solvent. Because of the surface tension of the gel phase, the macropores are
bent and have a round shape. Besides the interconnected pores, the pore walls and polymer
phase also contain pores. These micropores are formed due to the high concentration of
polymer and crosslinker when they were frozen [4]. The dimension and shape of the pores
are determined by many factors of which concentration of precursors and regimes of
cryogenic treatment are the most important ones. Cryogels contain a heterophase as well as
a heteroporous structure as a result of the two phase system of the cryogel and the
existence of both macro and micro pores [29].
Figure 1.3: Formation of a cryogel [32]
11
1.2.3. Properties of cryogels
1.2.3.1. Porosity
The porosity of a cryogel is an important factor, considering the properties of the gel, and it
is affected by different parameters which are explained below.
The first two important parameters are the freezing temperature and freezing rate. The
lower the freezing temperature, the smaller the pores will be. This is due to the fact that at
lower temperatures, the crystallisation of the solvent occurs much faster resulting in a large
number of small ice crystals and thus smaller pores as the ice crystals act as pore forming
agents [33, 34]. The large number of small pores give rise to an increased pore volume,
interconnectivity and porosity [35]. If at lower temperatures the pores are smaller, it follows
that when the freezing temperature is increased (which slows down the cooling rate) the
pores are bigger. There is an optimum temperature at which the pore size reaches a
maximum. Another result of the lower freezing temperature is that the UFLMP becomes
more concentrated. This leads to thinner and denser pore walls [34].
The pore size, density and interconnectivity are also affected by the concentration and the
molecular weight of the polymer, in this case HA, and the crosslinker concentration. A low
molecular weight polymer forms a gel with larger pores [34]. The result of a higher polymer
concentration is increased mechanical strength and thicker pore walls, but the pores will be
smaller and there will be less interconnectivity. The explanation can be found in the fact
that due to the more concentrated initial solution, there is less solvent that can freeze. This
will result in smaller pores but thicker pore walls. Also the type and concentration of the
crosslinker used for the gel will affect the pore size and thickness [35, 36].
1.2.3.2. Swelling properties
When a cryogel is immersed in water, the gel will swell. The rate at which the water is taken
up by the cryogel depends on different parameters. The total monomer or polymer
concentration, the thickness of the pore walls, preparation temperature and the amount of
crosslinks in the gel affect the rate. As the concentration of initial monomer or polymer is
increased, the pore wall thickness increases too, which will result in a lower swelling degree.
As mentioned before, the pore size differs by changing the preparation temperature. When
the gels are made using the optimum temperature, the pores will have their maximum size
and the swelling will occur faster than when the gels have smaller pores. The crosslinker
12
concentration has the opposite effect. It will influence the stiffness of the gel and thus the
swelling properties. The more crosslinks, the lower the swelling degree [7, 34, 35].
Another parameter that influences the swelling rate is the dimension of the gel. In this case
it would be the diameter since the cryogels are cylinders. The larger the diameter, the
slower the swelling and de-swelling will occur. The most important parameter however is
the interconnectivity. The more interconnected the pores are, the faster the water will flow
through the gel [7].
The water in a swollen cryogel can be polymer-bound, capillary or free water. The water can
be squeezed out of the gel easily since most of the water is capillary-bound or free water
and is therefore located within the macropores rather than within the polymer material
making up the pore wall [34, 35].
1.2.3.3. Mechanical properties
Pores do not only have an effect on the swelling properties. Both the thickness of the pore
walls as the density and the size of the pores change the mechanical properties of the
cryogels [36]. The smaller the pores, the higher the mechanical strength [7]. The elasticity
will increase since the density of the pore walls is increased [34]. A cryogel can be
compressed up to high values without any crack formation. The release of the water from
the pores prevents this. After the large deformation, the shape of the gel is restored by
absorbing the water again [33].
1.3. USE OF CRYOGELS
The cryogenic process is becoming a technique that is frequently used for different
purposes. It has applications in a broad range of branches like the food industry,
pharmaceutical and medical world and in scientific fields such as microbiology and
biochemistry/biotechnology. New food forms, tissue engineering, drug delivery, filters,
catalyst systems, chromatography, immobilization of molecules and cells and processing cell
and virus suspensions are only a few examples for which cryogels are used now and the list
of applications will only grow. Cryogels gain this much of interest because of their
macroporosity and elasticity, which are really unique properties [4, 5, 29].
13
One important application of cryogels within the medical and pharmaceutical field, namely
tissue engineering, will be further described below as this is the most interesting application
for HA cryogels.
1.3.1. Tissue engineering
When an organ is damaged or malfunctioning, a surgery to replace the organ is executed.
This transplantation surgery is not only costly but faces a lot of other problems: such a
surgery is not without danger, there is a shortage of donors and of organs that can be used
for transplantation and the organ can be rejected by the body. Also bone repair faces
problems. If the cartilage is severely damaged, the joint can be replaced, debridement can
be applied or chondrocytes can be transplanted. These are painful surgeries and the
recovery for the patient takes a long time. Tissue engineering can be used in both cases as
an alternative using a scaffold made out of natural or synthetic polymers [9, 37-39]. Figure
1.4 illustrates the principle of tissue engineering.
Figure 1.4: Principle of tissue engineering. Adopted from Dvir et al., 2001 [40]
14
Tissue engineering was defined for the first time during a meeting of the National Science
Foundation in 1987 as follows:
"the application of principles and methods of engineering and life sciences toward the
fundamental understanding of structure-function relationships in normal and
pathological mammalian tissues and the development of biological substitutes to
restore, maintain, or improve tissue functions" [41].
What this practically means is that when cells are seeded on a biological scaffold together
with growth factors and in some cases drugs, a new tissue will grow. The scaffold is used to
act as a matrix that resembles the ECM, both in structure and properties, till the body can
make one itself. It is also a structure that allows cells to proliferate, migrate and differentiate
in order to form a new tissue that has the same properties and functions as the initial tissue
in the human body [42, 43]. To mimic the ECM, the scaffold must meet a few criteria. First of
all, the scaffold should be biocompatible and biodegradable. Both the scaffold itself and its
degradation products should not cause inflammation or toxicity. Depending on what the
application of the scaffold is, the degradation rate has to be different. The rate of
degradation should resemble the rate at which the new tissue is formed. A HA scaffold is
degraded by hyaluronidases (see also section 1.1.2.). Also the mechanical strength of the
scaffold is important. It should be mechanically stable enough to bear the pressure that can
occur in a tissue and at the same time be able to pass on this mechanical force to the cells as
they need it in order to grow and differentiate [42, 44, 45]. The mechanical strength of the
scaffold thus needs to mimic the mechanical properties of the original tissue [39]. In
addition, the surface available for cell attachment should be large enough and should allow
the cells to grow. Pores increase the surface-volume ratio so a final critical requirement is
porosity and interconnectivity. Pores should be both large and small. Depending on which
tissue is engineered and the type of cells that is used, there exists an optimal pore size
range. In general, the optimum varies between 100 and 500µm. When the porosity is more
than 90%, the scaffold reaches its ideal porosity [39, 44]. Micropores provide a large enough
surface for the cells to adhere. The macropores will ensure cell infiltration and
vascularisation [42, 44, 45]. Vascularisation is necessary to succeed the transplantation of
cells in a scaffold but is often a problem. That is why it is necessary to make sure the scaffold
15
has macropores that facilitate the vascularisation and to implant growth factors into the
scaffold to make sure angiogenesis occurs [37]. In addition to the macropores,
vascularisation needs an interconnected network. Interconnectivity is also of great
importance for the diffusion of oxygen, nutrients and waste materials. In order to keep the
new forming cells alive, oxygen and nutrients need to be able to reach the cells and waste
products need to be removed [42, 44, 45].
Polymeric hydrogels have been used for a long time in tissue engineering because of their
many advantages. Hydrogels are hydrophilic, provide mass transport, are biodegradable,
easy to produce, their structure mimics the ECM and hydrogels can be applied in an easy
way. A hydrogel would be even more interesting if it had pores and interconnectivity and if
their mechanical strength was better [38, 42, 46]. This is why cryogels gain more and more
attention in the field of tissue engineering. They have all of these properties. Currently, there
is a lot of research going on the use of cryogels in tissue engineering. The articles [47-50] are
only a few examples.
The scaffolds will contain living cells and thus will potentially result in cell growth and cell
repair. In order to be successful, it is of great importance that the best polymer is chosen in
order to build up the scaffold. HA, alginate and chitosan are only a few of the polymers that
can be used for building a scaffold. These polymers are interesting since they have ECM-like
properties or are components of the ECM [42]. An alginate three-dimensional scaffold has
already been used to replace a damaged liver [43].
HA is very useful in tissue engineering since it has many advantages required for this
application. HA is a component of the ECM, is biodegradable and biocompatible, is non-
allergenic and it interferes in a lot of processes in the body which already have been
discussed in section 1.1.1. [17, 42].
16
2. OBJECTIVES
In recent years, much research has been done on the use of tissue engineering to replace
organ and cartilage transplantations. These transplantation surgeries are dangerous and
painful and there is a risk that the organ is rejected by the human body. A scaffold is
necessary to seed on cells, growth factors and in some cases drugs. Since tissue engineering
is gaining a lot of interest, the development of good scaffolds cannot stay behind.
HA and cryogels are perfect for this purpose. HA is a non-immunogenic, biodegradable and
biocompatible polysaccharide. Cryogels are macroporous gel structures with interesting and
unique properties. They are spongy and elastic but still mechanically resilient. If HA and
cryogels are combined, we get a structure that matches perfectly with the requirements for
a tissue engineering scaffold.
This is why we wanted to investigate if the cryogels of hyaluronan, which were prepared in a
previous study by Yasamin Dehdari [51], were reproducible in our lab. We also wanted to
investigate the gel functionalities obtained in that study. Secondly, we wanted to optimise
the cryogelation process in order to lower the hyaluronan concentration required to make a
cost-efficient gel. To lower this concentration, we were interested in the molecular
mechanism of cryogelation of covalently crosslinked hyaluronan.
Two series of gels were prepared following the protocol of Yasamin Dehdari. One series had
a fixed HA concentration with a varying crosslinker ratio, the other series was made with
varying HA concentrations and a fixed crosslinker ratio. HA was dispersed in 1% NaOH before
adding the crosslinker ethylene glycol diglycidyl ether (EGDE). Using liquid cooling, the gels
were prepared at -18°C for four days. Afterwards, new protocols were drafted to optimise
the gelation process and to see the influence of different parameters (retained molecular
weight of HA and its conformation as well as ionic strength of the media) on this process.
The properties of the gels were analysed using large deformation and swelling tests. The
microstructure was visualised with confocal laser scanning microscopy.
17
3. MATERIALS AND METHODS
3.1. MATERIALS
Hyaluronic acid sodium salt from Streptococcus equi was purchased from Sigma-Aldrich,
Czech Republic (53747).
Phosphate Buffered Saline, PBS, (P4417) and Rhodamine B (R6626) were purchased from
Sigma-Aldrich, Sweden. Rhodamine B is a fluorescent cationic molecule with molecular
structure C28H31ClN2O3 and a molecular weight of 479.01 g/mol. The fluorescence emission
wavelength is 627 nm and excitation wavelength 554 nm.
Figure 3.1: Structure of rhodamine B
Ethylene glycol diglycidyl ether (EGDE), the crosslinker, was purchased from Polysciences
Inc., Warrington, USA (cat#01479). EGDE has a molecular weight of 174.2 g/mol.
Figure 3.2: Structure of ethylene glycol diglycidyl ether
Sodium hydroxide (NaOH) was supplied by Sigma-Aldrich, Sweden (S5881). It was used for
making the different solutions for adjusting the pH and the ionic strength of the gels.
Sodium chloride (NaCl) 99+% was supplied by Aldrich, Germany (lot S20690-264) and was
used to adjust the ionic strength of the gels.
Fisher Scientific supplied the aceton (A/0600/21). It was used to de-swell the cryogels during
the swellingtest.
To ensure liquid cooling, the gels were put in glycerol 87% BioChemica (A0970). It was
purchased from AppliChem (Darmstadt, Germany).
18
3.2. METHODS
3.2.1. Preparation of the cryogels
These gels were made following the protocol of Yasamin Dehdari. The amount of HA
required for the gels was weighed on a Shimadzu AUW220D scale (Japan). The HA was then
dissolved in 1% NaOH while stirring on a magnetic stirrer. For complete dispersion and
dissolving of the HA and to prevent its degradation, the solution was placed in a refrigerator
at 4°C overnight (about 15 hours). The HA-1% NaOH solution has a pH of 13. To make the
crosslinks, EGDE was added under stirring on a magnetic stirrer. The gel was stirred for five
minutes and after waiting for ten minutes, the gel was transferred into syringes of 1mL. The
syringes were placed in glycerol, which was pre-cooled to -18C and then placed into a
freezer (Labconco FreeZone® Stoppering Tray dryer model 7948030, USA) for four days.
After freezing, the syringes were put into water to thaw at room temperature. When thawed
for about three hours, the cryogels were taken out of the syringes and were placed in Milli-Q
water (18.2MΩ cm at 25°C) to reach the equilibrium swollen state.
3.2.2. Variation of cryogel preparation
HA was weighed on a Shimadzu AUW220D scale and then dissolved in Milli-Q water. This
solution was kept at 4°C for at least 12 hours to allow full dissolution of the HA. Different
preparation methods were used to determine the method that yields the most cost-efficient
HA gel. The methods are described below.
3.2.2.1. Variation 1
After a night at 4°C, the pH of the gels was adjusted to pH 13 with 1M NaOH before adding
the crosslinker. The exact pH was measured with a Jenway 3510 pH meter (Staffordshire,
UK). After stirring for five minutes and a resting period of ten minutes, the same protocol as
described in section 3.2.1. was followed.
3.2.2.2. Variation 2
These gels were made following the same procedure as described in section 3.2.2.1., but
before adding the 1M NaOH, the ionic strength was adjusted to 0.2M using NaCl.
3.2.2.3. Variation 3
These gels were prepared by weighing the amount of HA needed for the gel and then
dissolving this in half of the end volume of water. After a night in the refrigerator, the same
19
volume of 2% NaOH was added and then stirred. Before adding the crosslinker, the gel
solution was placed at 4°C for 30 minutes. Under five minutes of stirring, the crosslinker was
added to the hydrogel. After a resting period of ten minutes, the gels were transferred in
1mL syringes and added to the freezer as above.
3.2.3. Swelling and de-swelling measurements
To determine the swelling and de-swelling rate, the gels were cut in cylinders of
approximately the same height and were immersed in water to reach the equilibrium
swollen state with weight m0. The gels were de-swelled by immersion in aceton, during
which their weight reduction was measured every minute using a Sartorius CP323P scale
(Goettingen, Germany). After complete shrinkage of the cryogels (when the weight
reduction levelled out), the gels were re-immersed in Milli-Q water or PBS and their swelling
ratio and speed were determined by measuring the gel weight every minute. The PBS
solution was made by dissolving 1 tablet of PBS in 200mL Milli-Q water which then contains
0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride. The pH
of the PBS solution is 7.4 and simulates human body fluid. The measurements of each gel
sample are performed in triplicate.
The relative weight was plotted against the time. The relative weight mrel was calculated as
follows:
(3.1)
where
3.2.4. Uniaxial compression measurements
The cryogels that were in the syringes were cut in their swollen state into cylinders of
approximately 9mm of height. On these cylinders, uniaxial compression tests were
performed using an Instron 5565A system (USA) with a load cell of 2kg at room temperature.
The sample was compressed up to 60% strain at a compression rate of 5% s-1. The tests were
performed in triplicate. The compressive extension (mm), force F (N) and compressive strain
mrel = relative mass
m0 = original mass of the gel (g)
mt = mass at a certain time during de-swelling or swelling (g)
20
(%) were calculated by a software program (Bluehill®2) provided by Instron. The
deformation ratio λ was calculated as:
(3.2)
where
The uniaxial normal stress σ was defined as the force F over a certain area A as shown in
Equation (3.3). Since the gel samples are cylinders, the surface area A can also be written as
seen in Equation (3.4).
(3.3)
where
(3.4)
where
The mechanical strength was expressed with the elastic modulus G’, which is calculated as:
(3.5)
where
3.2.5. Characterisation of HA
The molecular weight of HA was defined using an automated Ubbelohde viscometer (Schott-
Geräte, Germany). A Type No. 531 0a capillary was used and the measurements were carried
out at 25°C. Each sample was run five times. To determine the initial molecular weight,
λ = deformation ratio
l = deformed length of the gels (mm)
lo = initial length of the gels (mm)
σ = normal stress (Pa)
F = force (N)
A = surface area (m²)
A = surface area (m²)
D0 = diameter of the initial gel (mm)
G’ = elastic modulus (Pa)
σ = normal stress (Pa)
λ = deformation ratio
21
solutions with different concentrations of HA in PBS were used. The Hagenbach corrections
were applied on the running times before calculating the relative viscosity ηrel, which was
given as:
(3.6)
where
The intrinsic viscosity [η] (dL/g) was determined by plotting [
and
against
the concentration c (g/dL). When c was extrapolated to zero, the intrinsic viscosity was
determined.
The intrinsic viscosity [η] can also be determined by calculating Equation (3.7) or (3.8). The
accuracy increases when both equations are calculated.
(3.7)
(3.8)
The molecular weight of HA was calculated using the Mark-Houwink-Sakurada equation,
shown in Equation (3.9).
(3.9)
where K = a constant = 0.00034 dL/g
a = Staudinger index = 0.79
M = molecular weight (g/mol)
These values were adopted from Meyer et al. 2009 [52] and are valid when the test is
performed at 25°C and at an ionic strength concentration of 0.15M.
ηrel = relative viscosity
η = viscosity of the HA solution (Pa.s)
η0 = viscosity of the pure solvent (in this case PBS) (Pa.s)
t = running time of the HA solution (s)
t0 = running time of the pure solvent (PBS)
22
The HA that was used is characterised with an intrinsic viscosity of 21.14dL/g and molecular
weight of 1 169kDa.
3.2.6. Imaging of the pores
The cryogels were stained with a 0.01% w/v solution of rhodamine B and were rinsed with
Milli-Q water afterwards to get rid of the dye in the pores. The gels were kept in the dark
between these actions to prevent bleaching of the gels by light. The analyses were
performed at room temperature using Leica confocal laser scanning microscopes of model
TCS SP5 II or SP2 AOBS (Heidelberg, Germany). The light source was a HeNe laser with an
emission maximum of 594nm, and the signal emitted at a wavelength interval of 605 to
685nm was recorded. The formats of the images were 512x512 or 1024x1024. These were
recorded using a 20x water objective (NA of 0.50) and computer zooming was done at 1x, 2x
and 4x.
23
4. RESULTS AND DISCUSSION
4.1. PREPARATION OF CRYOGELS USING HYALURONAN
The first aim of this study was to prepare cryogels from HA according to the protocol
developed by Yasamin Dehdari [51] and outlined in detail in the materials and methods
section. The reason for this was to investigate the reproducibility of the protocol and the gel
functionalities obtained.
Briefly, hyaluronan was dissolved in 1% NaOH and the solution was kept at 4°C for 15 hours
in order to fully hydrate. The crosslinker (EGDE) was added and the mixture was put into
syringes. Hereafter, the syringes were immersed in glycerol (-18°C) and kept at -18°C for 4
days. It is important to immerse the syringes in the glycerol to ensure liquid cooling since no
gel formation was observed when air cooling was used [51]. Once the cryogels were set,
they were allowed to completely swell in water before any investigation was done on them.
Two series of gels were made: one with varying HA concentrations but constant crosslinker
ratio and one with a constant HA concentration but varying EGDE ratios.
The elastic modulus (G’) was plotted against the HA (CHA) or EGDE concentration (CEGDE) as
shown in Figures 4.1 and 4.2. G’ was calculated using Equation (3.5) as described in the
materials and methods section. Figure 4.1 shows HA concentrations in a range between 2
and 9% w/v and the concentration of EGDE was fixed at a ratio of 4.3. The elasticity of these
gels increases with increasing CHA but at a concentration of 9%, there is a reduction of
strength as G’ drops. G’ of the 2% HA gel is not shown in the graph as it deformed under its
own weight. The increase in elastic moduli can be explained following the literature which
says that the higher the polymer concentration, the higher the mechanical strength. This
increase in mechanical strength is typically related to the thicker pore walls and the smaller
pores that are formed because of a more concentrated initial solution [36]. It was speculated
in the report of Yasamin Dehdari that the reduction in G’ was related to the non-constant
ratio of crosslinker that was used, thus resulting in fewer crosslinks per HA molecule. This
explanation is not applicable in this study since the crosslinker ratio was kept constant with
respect to the CHA. More detailed studies need to be done in order to explain the drop in G’
upon increasing the polymer concentration from 7 to 9% but the reduction could be related
24
to changes in microstructure and then specifically pore sizes, as will be discussed in more
detail under section 4.3.
Although the absolute values of G’ differ between the current study and the one performed
by Yasamin Dehdari, similar trends are observed. Two explanations can be given to the
observed differences in absolute values. The first one is that in the work performed by
Yasamin Dehdari, a home-made “Instron” device was used and the crosshead speed was not
defined, whereas in this study an automatic system is used with a defined crosshead speed
of 5mm s-1. It is well known that the recorded strength of the gels is dependent on the cross
head speed (personal communication with Dr. A. Ström), which indeed was observed in this
study (results not shown). The second explanation could be related to the fact that HA was
kept for 24 hours in 1% NaOH instead for 15 hours, as in this study. The Mw of the HA will be
reduced to a larger extend in the study performed by Yasamin Dehdari compared to the
current study.
0
0,5
1
1,5
2
2,5
3
3,5
0 1 2 3 4
G' (kPa)
% EGDE w/v
Figure 4.2: Elastic moduli (G’) of the
cryogels plotted against the EGDE
concentration (% w/v). The HA
concentration was kept constant at 7.3%
w/v. G’ increases up to 0.89% w/v and
after that point, G’ does not change that
much. The crosslinker probably reached its
saturation point.
Figure 4.1: Elastic moduli (G’) of the
cryogels plotted against the HA content (%
w/v). The EGDE concentration was fixed at
a ratio of 4.3. G’ increases as the
concentration of HA increases too. At 9%
w/v, the elasticity drops.
0
0,5
1
1,5
2
2,5
3
3,5
0 5 10
G' (kPa)
% HA w/v
25
Besides a series with varying HA concentration, a series was made where the HA
concentration was kept at 7.3% w/v and the amount of crosslinker varied between 0.25 and
3% w/v, resulting in a crosslinker ratio between 2.4 and 29.2 respectively. The result of this
analysis is shown in Figure 4.2. When these results are plotted in a graph, there is an
increase in mechanical strength up to the point of 0.89% w/v. After this concentration, the
mechanical strength does not change significantly. The drop in G’ at 0.75% is probably an
outlier as the trend of the curve is clear. More measurements should be performed to
confirm this. The crosslinker probably reached its saturation point at a concentration around
0.89% w/v and increasing the crosslinker concentration does not affect the mechanical
strength anymore. Here, the values were also higher than in Yasamin’s study, which could be
explained by the different crosshead speed used while testing the strength. The optimum
HA/EGDE ratio obtained in this study was 8.2 compared to the value of 4.3 obtained in the
previous study [51].
For the two series, the extremes were used to perform a swelling test. It takes between 15
and 20 minutes for the gels to fully de-swell in aceton. When re-immersed in water, the
swelling of the gels happens really fast. Within a minute, the gels are back at their initial
relative weight. As they all contain macropores which are interconnected, the water can
0
0,2
0,4
0,6
0,8
1
1,2
0 10 20 30 40
mrel
Time (min)
Figure 4.3: Swelling – de-swelling graph of a 3% ( ) and 9% ( ) HA
cryogel. The relative mass (mrel) is plotted against the time. The ratio of
EGDE is 4.3. The de-swelling occurred in aceton, the swelling in Milli-Q
water. After re-immersing, all the gels swell back to their initial relative
mass within a minute.
26
flow through the cryogels without any hindrance. Since the extreme values showed similar
behaviour in Figures 4.3 and 4.4, the values in between these extremes were not measured.
To make a prediction about how the cryogels will behave in the human body, a swelling test
in PBS was performed on the extreme values of each series. The gels, swollen in water, were
immersed in aceton to fully undergo de-swelling which was reached after about 20 minutes.
When immersed in PBS to swell, the swelling occurred slower than when immersed in water
as seen in Figure 4.5. This can be explained by the fact that in water, the negative charges of
the HA chains repel each other which results in a fast swelling rate. In PBS, the ions of the
PBS solution cover the charges so there is no repulsion between the charges. It is also
observed that while none of the gels swell to 100% of their initial weight, as is the case of
swelling in water, they do swell to different degrees in PBS as illustrated in Table 4.1. The
swelling degree increases if the crosslinker ratio of the gels containing 7.3% HA increases
too. For the gels containing the same amount of HA, it applies, that the more crosslinks and
thus the lower the ratio, the lower the swelling degree [34]. For the gels with the same ratio
but a different CHA, this trend is not observed.
0
0,2
0,4
0,6
0,8
1
1,2
0 10 20 30 40 50 60
mrel
Time (min)
Figure 4.4: Swelling – de-swelling graph of a 3% EGDE ( ), 0.89%EGDE ( )
and 0.75% EGDE ( ) cryogel. The HA concentration was 7.3% for all the
gels. The de-swelling occurred in aceton, the swelling in Milli-Q water. The
relative mass (mrel) is plotted against the time. The percentages are in %
w/v. The gels swell within a minute after re-immersing in water and go back
to their initial relative mass.
27
The findings of Yasamin Dehdari were perfectly reproducible in this study so we can
conclude that the methodology is robust.
4.1.1. Understanding the role of polymer conformation
Another aim of this study was to optimise the cryogelation process in order to lower the HA
concentration required to make a cost-efficient gel. The effect of three different parameters
that potentially could influence the gel preparation was investigated. These parameters are:
retention of the molecular weight (Mw) of HA, the conformation of HA and the ionic
strength of the preparation media.
0
0,2
0,4
0,6
0,8
1
1,2
0 10 20 30 40
mrel
Time (min)
Gel Ratio HA / EGDE % of its initial weight
9% HA 4.3 45%
3% HA 4.3 45%
3% EGDE 2.4 50%
0.89% EGDE 8.2 60%
0.75% EGDE 9.7 70%
Table 4.1: Percentages of the initial weight after swelling in PBS.
Figure 4.5: De-swelling and swelling curve of a few NaOH-gels where the
relative mass is plotted against the time in minutes. The green symbols
depict a 3% HA ( ) and 9% HA ( ) gel, with a fixed EGDE ratio of 4.3. The
blue symbols show a 0.75% ( ), 0.89% ( ) and 3% ( ) gel with a
constant CHA of 7.3%. The percentages are w/v %. The swelling occurred
slower and the gels do not return to their initial relative weight.
28
Retention of HA Mw
It is known that HA degrades when in contact with a pH <4 or >11 and that the degradation
of Mw occurs faster at high pH. It is also proven that at a pH of 13, the degradation starts
immediately and lasts for a few days [27, 28]. For the crosslinker to react, a pH of at least 10
is necessary. It is thus highly probable that Mw degradation will occur, so it is advisable to
retain the time at which the HA mixture is exposed to high pH to a minimum or to reduce
the pH. It is generally known that a polymer with higher Mw gives rise to a more stiff gel than
a lower Mw gel. For this reason, the first variation of preparing the gels was to dissolve the
HA in Milli-Q water and to adjust the pH of the hydrogel to 13 using 1M NaOH just before
adding the crosslinker, in order to reduce the time at which the hydrogel is exposed to such
a high pH. Since the gels contain a high CHA, the viscosity was high too. It was difficult to
measure the pH properly and it was not possible to stir with a magnetic stirrer. When the
gels came out of the freezer, they were soft and did not form a proper gel that could stand
on its own. Large deformation could not be performed on these gels. This could potentially
be explained by the high viscosity of the hydrogel and the difficulty to obtain efficient mixing
between EGDE and water. This will probably result in the creation of fewer crosslinks,
however, as the reaction time is long (four days), it is unlikely that the crosslinker did not
have time to diffuse evenly in the media. Furthermore, it was difficult to measure the pH of
the solution. Although a theoretical value of pH was obtained by calculating the amount of
base added to the solution, the buffering effect related to HA was not taken into account.
This may mean that the pH is actually lower than pH 13, thus indicating that a high pH of >12
is necessary for cryogels of HA to be obtained as suggested by a previous study as well [51].
Influence of ionic strength
The next step was adjusting the ionic strength. The ionic strength in the gels dissolved in 1%
NaOH was 0.25 whereas the gels adjusted with 1M NaOH had an ionic strength of only 0.08.
It was important to retain a constant ionic strength as it will have an influence on the
conformation of HA and the freezing point of the UFLMP. These gels were prepared
following the same preparation method as described in the paragraph above but before
adding EGDE, a calculated amount of NaCl was mixed in the gel. Whereas you expect the
viscosity to decrease, the opposite happened. The ions cover the negative charges on the HA
29
which allows the HA chain to coil more, normally resulting in a lower viscosity as a coil
causes lower viscosity than a more rigid structure. The gels coming out after cryogelation did
not retain their shape under their own weight and thus were too soft to handle.
Conformation of HA
At a pH of 12.1 or more, the hydroxyl groups on the HA backbone start to ionise resulting in
a conformation change due to breaking of the internal hydrogen bonds. The rather stiff HA
molecule will become a more flexible coil. The Mw will only degrade to a large extend when
exposed to an extremely high pH for hours [53]. It is likely that the Mw is not reduced largely
as the gel is only for a short time at room temperature and high pH (<30 minutes). However,
it is not sure whether or not degradation occurs while freezing. The gels were prepared by
mixing HA and a 2% NaOH solution in a ratio of 50:50 in order to obtain a final 1% NaOH and
HA solutions ranging from 2% to 9%. The NaOH was added only 30 minutes before adding
the required amount of EGDE to reduce the time that the HA was exposed to NaOH at T =
4°C from 15 hours to 30 minutes. This methodology ensures similar ionic strength and pH as
the methodology employed previously, while retaining the Mw of HA. With this preparation
method (hereafter referred to as gels prepared by using method 2), proper gels were
obtained. Their rheological and swelling properties are outlined in section 4.2. The results
obtained suggest that the conformation of the HA in solution is crucial for its potential to
form cryogels using EGDE as done in this study. It could be speculated that the breakage of
internal hydrogen bonds is necessary for the hydroxyl groups to be available for reaction
with the EGDE crosslinker. Above a pH of 12.5, the viscosity decreases drastically and it is
likely that the stiff conformation will become more like a random coil [28, 53].
4.2. PHYSICAL PROPERTIES OF THE CRYOGELS
From now on, the cryogels where the HA is dissolved in 1% NaOH will be called gels made by
method 1, the ones where HA is dissolved in Milli-Q water and the NaOH is added 30
minutes before adding the EGDE are called method 2. The influence of HA and EGDE
concentration on rheological properties and swelling capacity of the gels was investigated
comparing gels of both methods.
30
4.2.1. Rheological properties
Again, two series of gels were made. For the first series, the CHA was kept at 3% and the CEGDE
varied between 0.10 and 0.71%, resulting in a HA crosslinker ratio between 30 and 4.3
respectively. The other series consists of gels with concentrations in the range of 2 to 9%
whereas the crosslinker ratio was kept at 4.3. All the percentages are w/v %.
Uniaxial compression analysis was performed on both gel series and the results were plotted
in a stress-strain curve in order to calculate the elastic moduli of the cryogels. As an
example, Figure 4.6 shows a stress (kPa) – strain (%) curve of a cryogel made by method 2.
The HA content is 3% w/v with a 4.3 crosslinker ratio. The curve was obtained by
compressing the gel up to 60% strain. The gels could have been compressed up to 100%
strain without any crack development, but this was not possible with the Instron as the
water that came out of the gel made it slip. After the compression, the gels reabsorbed the
water returning to their initial shape.
A compression cycle of three compressions was performed on some of the gels, resulting in
graphs that overlapped each other. This can indicate a good durability of the gels but it has
to be further investigated. Furthermore, it could be mentioned that if the water was
removed from the compression plate, the gel did not return to its initial shape.
0
0,5
1
1,5
2
2,5
3
0 10 20 30 40 50 60
Stress (kPa)
Strain (%)
Figure 4.6: Stress-strain curve of a 3% HA cryogel with a crosslinker
ratio of 4.3. The gel was made by method 2. The gel was compressed
up to 60% strain but could have been compressed up to 100%. After
compression, the gel adopted its original shape again.
31
The rheological properties of the ΔCHA gels are compared in Figure 4.7 where G’ (kPa) is
plotted against the HA content (%). The graph of the gels made by method 2 shows an
increasing elasticity as the CHA increases. The values of these gels are higher, meaning that
they are more elastic. The difference between the gels made by method 1 and method 2
becomes bigger as the CHA increases. As opposed to the method 1-gels, there is no drop in
elasticity at 9%. The trend of this curve follows the literature better which says that the
higher CHA, the thicker the pore walls will be and thus the higher the mechanical strength
[35, 36]. The fact that the values are higher than for the method 1-gels could be related to
the Mw. The Mw is higher in the method 2-gels because there the degradation is less due to
the little time they were exposed to high pH.
The G’ of the ΔCEGDE is not plotted because the gels were too weak to measure on. In the
visual analysis of these gels, it is observed that the gels with 0.10% and 0.20% (ratio of 30
and 15) are too elastic and soft to handle. These concentrations were considered to be too
low. Upward of this concentration, the obtained gels became stronger as the CEGDE
increased. However, the gels did not look as elastic and mechanically strong as the ΔCEGDE
series made by method 1.
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
0 2 4 6 8 10
G' (kPa)
% HA w/v
Figure 4.7: The mechanical strength, represented as the elastic modulus G’
(kPa), of both the Δ CHA series of the method 1-gels ( ) and the method 2-
gels ( ) is plotted against the CHA. The CEGDE was fixed at a ratio of 4.3. The
method 2-gels are a little stronger and there is no drop in G’.
32
4.2.2. Swelling capacity
If the CHA or CEGDE increases, the swelling degree will be lower [35]. An increase in CEGDE
means a decrease in the molar ratio. Since all the gels were made in the same syringes, the
swelling degree can be measured by comparing the diameter of the swollen gels. The
diameters are shown in Table 4.2 and in Figure 4.8, a visual comparison is made. In the
ΔCEGDE series we can see from the diameter of the gels that the lower the crosslinker
concentration, the more the gels swell as would be expected from a network with fewer
crosslinks. This trend is not that clear for the ΔCHA series. This suggests that the
concentration of crosslinks has a bigger impact on the swelling degree than the polymer
concentration.
When comparing the swelling test of both the method 1-gels and the method 2-gels, which
are shown in Figures 4.9 and 4.10, the curves look similar so the gels behave in a same way.
nratio EGDE
Diameter NaOH (mm)
Diameter H2O (mm)
2.22 9 n.m. 2.68 9 8 3.34 9 9 3.99 9 8 7.54 9 7 8.96 8.5 n.m.
13.22 10 9 26.39 11 11-12
% HA nratio Diameter NaOH (mm)
Diameter H2O (mm)
9 3.98 9 9 7 3.98 9 9 5 3.98 9 9 3 3.98 9 9 2 3.98 9 9
Note: n.m. means no measurements since these gels
were not made as method 2-gel.
Figure 4.8: Visual comparison of three gels made by
method 2. The higher the crosslinker concentration, the
less the gels can swell. 0.2% (left), 0.31% (middle) and
0.51% (right) w/v EGDE
Table 4.2: Diameters of the swollen gels of both gel series. Both the diameters of the method 1-gels as
the method 2-gels are listed in this table.
33
Figure 4.9: De-swelling and swelling curve of the crosslinker series
where mrel is plotted against the time in minutes. Both the crosslinker
series of the method 1-gels (green/blue) as the method 2-gels
(yellow/orange) are shown. The CHA of the method 1-gels is 7.3% w/v
whereas the CHA of the method 2-gels is 3%. = 0.71%, = 0.31%,
= 3%, = 0.89% and = 0.75%. Both gel series swell back to their
initial weight within a minute.
Only the extreme concentrations of each series are submitted to the test and since these
look similar, it was not necessary to measure the concentrations between these extremes as
they will behave similarly.
0
0,2
0,4
0,6
0,8
1
1,2
0 10 20 30 40 50 60
mrel
Time (min)
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10 15 20 25 30 35 40
mrel
Time (min)
Figure 4.10: De-swelling and swelling curve of the varying CHA series
where mrel is plotted against the time in minutes. Both the HA series of
the method 1-gels (green/blue) as the method 2-gels (yellow/orange)
are shown. The ratio of EGDE is 4.3. = 3% HA, = 9% HA, = 3%
HA and = 9% HA. As in Figure 4.9, the gels swell within a minute
returning to their initial weight.
34
4.3. MICROSTRUCTUAL CHARACTERISATION
In this study was chosen to use Confocal Laser Scanning Microscopy (CLSM) for imaging the
porous structure because the structure of the swollen sample can be analysed. Scanning
Electron Microscopy (SEM) has been used in previous studies to investigate the structure but
for this technique, the sample needs to be freeze dried with the risk that the structure of the
cryogel will change.
CLSM was performed on three different gels in order to investigate the porous structure of
the cryogels. Images of a 3% and 7% method 2-gel are shown in Figure 4.11. The pores of the
3% gel are more rounded and have about the same size throughout the sample, which is
about 100 µm, whereas the pore size and shape of the 7% varies more. The 7% gel has large
pores of about 200 µm alternated with small ones, which are sometimes smaller than 100
µm. Comparing the pore walls, the walls of the 7% gel are thicker. Generally at a high
monomer concentration, the initial solution gets more concentrated and thus there is less
solvent that can freeze resulting in smaller pores with thicker pore walls [35, 36]. It was
observed by Minaberry et al. (2013) that this general explanation only applies for other
polymeric materials and not for HA cryogels. HA cryogels have a larger average pore size if
the CHA increases [54]. This possibly explains the drop in G’ of the 9% gel in Figure 4.1. It is
known that the smaller the pores, the higher the mechanical strength [7]. As the pore size
will increase, it is likely that the mechanical strength will drop.
Figure 4.12 depicts a 7.3% method 1-gel. The structure of the pores looks different. They are
more needle-shaped compared with the 7% method 2-gel. It is likely that this is due to
molecular weight degradation in the method 1-gel. Cryogels made out of low molecular
weight polymers give rise to larger pores [34]. Although the shape is different, the pore walls
are similar as the pore walls of the 7% method 2-gel.
An SEM image is also shown in Figure 4.12 to compare (courtesy Y. Dehdari). Comparing
both images, the structure looks similar showing a needle-shaped structure. So probably
freeze drying does not affect the structure of these gels due to the thick pore walls in the
cryogels. A disadvantage of CLSM is that the resolution is not high enough to see the
interconnectivity between the pores [35] so this property cannot be compared. More CLSM
35
and SEM pictures can be compared in order to determine the best technique to image the
pores and interconnectivity of the cryogels.
Figure 4.11: Confocal Laser Scanning Microscopy (CLSM) images of a 3% w/v (left) and 7% w/v (right)
method 2-gel. The EGDE ratio was 4.3 in both samples. The pores of the 3% gel are smaller and more
rounded than the 7% gel. The pore walls in the 7% gel are thicker.
Figure 4.12: CLSM image (left) and SEM image (right) (courtesy Y. Dehdari, [51]) of a 7.3% w/v method 1-gel
with 1.68% w/v EGDE (= ratio 4.3). In both images, the pores are large and needle-shaped.
36
5. CONCLUSION
It was possible to prepare covalently crosslinked HA cryogels following Yasamin Dehdari’s
protocol. The HA was dissolved in 1% NaOH and EGDE was used as crosslinker. The gels have
similar interesting properties as the ones in the previous study being mechanically resilient
and having a fast swelling rate in water. The fast swelling rate indicates large pores and
interconnectivity, which are the typical properties of cryogels. The swelling in PBS occurred
slower than in water, indicating that the swelling in the body will occur slower too.
The optimal cryogel determined in the previous study was composed of 7.3% w/v HA and
1.68% w/v EGDE, which is a 4.3 ratio. In this study, the ratio is raised to 8.2 with a 7.3% w/v
HA and 0.89% w/v EGDE cryogel. This means that a lower concentration of EGDE can be used
to form the most cost-efficient gel.
When optimising the cryogelation process, a new preparation method was developed. A HA
hydrogel and a 2% NaOH solution were mixed in a 50:50 ratio 30 minutes before adding
EGDE, resulting in the desired HA concentration and 1% NaOH and ensuring similar ionic
strength and pH as the initial method. 2% w/v is the lowest HA concentration that yields a
gel with this method and the optimum EGDE ratio is between 8.2 and 4.3.
The molecular mechanism behind the cryogelation process is now better understood and
molecular weight, ionic strength and conformation of HA are found to be three important
parameters that could potentially influence the gelation process. It is seen that the
conformational change of the HA molecule is the crucial parameter in the cryogelation,
potentially allowing for the crosslinking reaction to occur. The conformational change,
related to the breakage of internal hydrogen bonds at pH >12.1, further results in a decrease
in the radius of gyration of HA. This in turn causes an increase in critical overlap
concentration and thus an increase in polymer concentration necessary to form a gel. This
could be an explanation to why a relatively high concentration of 2% w/v hyaluronan is
necessary.
Future work can be done on determining the durability of the gels by performing freeze and
compression cycles on the gels. It is also interesting to investigate the influence of the
temperature on the pores and another cost-efficient crosslinker can be used. Every tissue
37
has different requirements and specifications and it could be useful to investigate with
which tissue HA cryogels match. Since the scaffold is applied in the body, the degradation
rate of HA cryogels is also an interesting topic to investigate.
38
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