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

1

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.

2

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

3

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

4

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

5

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].

6

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

7

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

8

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

9

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

6. REFERENCES

[1] J. Necas, L. Bartosikova, P. Brauner and J. Kolar, “Hyaluronic acid (hyaluronan): a review,” Vet. Med., vol. 53, no. 8, pp. 397–411, Aug. 2008.

[2] M. Brown and S. Jones, “Hyaluronic acid: a unique topical vehicle for the localized delivery of drugs to the skin,” Journal of the European Academy of Dermatology and Venereology, vol. 19, no. 3, pp. 308–318, 2005.

[3] K. Meyer and J. W. Palmer, “The polysaccharide of the vitreous humor,” J. Biol. Chem., vol. 107, no. 3, pp. 629–634, Dec. 1934.

[4] V. I. Lozinsky, “Cryogels on the basis of natural and synthetic polymers: Preparation, properties and application,” Uspekhi Khimii, vol. 71, no. 6, pp. 559–585, 2002.

[5] F. M. Plieva, I. Y. Galaev, W. Noppe and B. Mattiasson, “Cryogel applications in microbiology,” Trends Microbiol., vol. 16, no. 11, pp. 543–551, Nov. 2008.

[6] V. I. Lozinsky, F. M. Plieva, I. Y. Galaev and B. Mattiasson, “The potential of polymeric cryogels in bioseparation,” Bioseparation, vol. 10, no. 4–5, pp. 163–188, 2001.

[7] E. Jain, A. Srivastava and A. Kumar, “Macroporous interpenetrating cryogel network of poly(acrylonitrile) and gelatin for biomedical applications,” Journal of Materials Science-Materials in Medicine, vol. 20, pp. 173–179, Dec. 2009.

[8] N. Bölgen, I. Vargel, P. Korkusuz, E. Güzel, F. Plieva, I. Galaev, B. Matiasson and E. Pişkin, “Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model,” J Biomed Mater Res A, vol. 91, no. 1, pp. 60–68, Oct. 2009.

[9] F. Menaa, A. Menaa and B. Menaa, “Hyaluronic Acid and Derivatives for Tissue Engineering,” Journal of Biotechnology, no. S1, pp. 1–7, 2011.

[10] T. C. Laurent, U. B. Laurent and J. R. Fraser, “Functions of hyaluronan,” Ann Rheum Dis, vol. 54, no. 5, pp. 429–432, May 1995.

[11] T. Laurent, “Biochemistry of Hyaluronan,” Acta Oto-Laryngol., no. Suppl. 442, pp. 7–24, 1987. [12] T. Laurent and J. Fraser, “Hyaluronan,” Faseb J., vol. 6, no. 7, pp. 2397–2404, Apr. 1992. [13] winanur, “Chemistry and Biology of Hyaluronan,2004,” Docstoc.com. [Online]. Available:

http://www.docstoc.com/docs/74159718/Chemistry-and-Biology-of-Hyaluronan2004. [Accessed: 26-Feb-2013].

[14] J. R. E. Fraser, T. C. Laurent and U. B. G. Laurent, “Hyaluronan: Its nature, distribution, functions and turnover,” J. Intern. Med., vol. 242, no. 1, pp. 27–33, Jul. 1997.

[15] L. Lapcik Jr., L. Lapcik, S. De Smedt, J. Demeester and P. Chabrecek, “Hyaluronan: Preparation, structure, properties, and applications,” Chem. Rev., vol. 98, no. 8, pp. 2663–2684, Dec. 1998.

[16] S. Reitinger and G. Lepperdinger, “Hyaluronan, a Ready Choice to Fuel Regeneration: A Mini-Review,” Gerontology, vol. 59, no. 1, pp. 71–76, 2013.

[17] R. D. Price, M. G. Berry and H. A. Navsaria, “Hyaluronic acid: the scientific and clinical evidence,” Journal of Plastic, Reconstructive & Aesthetic Surgery, vol. 60, no. 10, pp. 1110–1119, Oct. 2007.

[18] E. A. Balazs, “Therapeutic use of hyaluronan,” Structural Chemistry, vol. 20, no. 2, pp. 341–349, Apr. 2009.

[19] F. S. Brandt and A. Cazzaniga, “Hyaluronic acid gel fillers in the management of facial aging,” Clin Interv Aging, vol. 3, no. 1, pp. 153–159, Mar. 2008.

[20] A. Migliore, F. Giovannangeli, E. Bizzi, U. Massafra, A. Alimonti, B. Laganà, A. D. Picchianti, V. Germano, M. Granata and P. Piscitelli, “Viscosupplementation in the management of ankle osteoarthritis: a review,” Arch Orthop Trauma Surg, vol. 131, no. 1, pp. 139–147, Jan. 2011.

[21] M. Gomes, H. Azevedo, P. Malafaya, S. Silva, J. Oliveira, G. Silva, R. S. João Mano and R. Reis, “16 - Natural Polymers in Tissue Engineering Applications,” in Handbook of Biopolymers and Biodegradable Plastics, Boston: William Andrew Publishing, 2013, pp. 385–425.

[22] W. D. Comper and T. C. Laurent, “Physiological function of connective tissue polysaccharides.,” Physiol Rev, vol. 58, no. 1, pp. 255–315, Jan. 1978.

39

[23] R. L. Cleland and J. L. Wang, “Ionic polysaccharides. III. Dilute solution properties of hyaluronic acid fractions,” Biopolymers, vol. 9, no. 7, pp. 799–810, 1970.

[24] J. E. Scott, C. Cummings, A. Brass and Y. Chen, “Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient network-forming polymer.,” Biochem J, vol. 274, no. Pt 3, pp. 699–705, Mar. 1991.

[25] H. B. Wik and O. Wik, Rheology of hyaluronan, vol. 72. London: Portland Press Ltd, 1998. [26] O. Jeon, S. J. Song, K.-J. Lee, M. H. Park, S.-H. Lee, S. K. Hahn, S. Kim and B.-S. Kim, “Mechanical

properties and degradation behaviors of hyaluronic acid hydrogels cross-linked at various cross-linking densities,” Carbohydrate Polymers, vol. 70, no. 3, pp. 251–257, Oct. 2007.

[27] A. Maleki, A.-L. Kjoniksen and B. Nystroem, “Effect of pH on the Behavior of Hyaluronic Acid in Dilute and Semidilute Aqueous Solutions,” Macromol. Symp., vol. 274, pp. 131–140, 2008.

[28] L. Gatej, M. Popa and M. Rinaudo, “Role of the pH on hyaluronan behavior in aqueous solution,” Biomacromolecules, vol. 6, no. 1, pp. 61–67, Feb. 2005.

[29] V. I. Lozinsky, I. Y. Galaev, F. M. Plieva, I. N. Savinal, H. Jungvid and B. Mattiasson, “Polymeric cryogels as promising materials of biotechnological interest,” Trends in Biotechnology, vol. 21, no. 10, pp. 445–451, Oct. 2003.

[30] J. Jagur-Grodzinski, “Polymeric gels and hydrogels for biomedical and pharmaceutical applications,” Polymers for Advanced Technologies, vol. 21, no. 1, pp. 27–47, 2010.

[31] J. B. Leach, J. B. Wolinsky, P. J. Stone and J. Y. Wong, “Crosslinked alpha-elastin biomaterials: towards a processable elastin mimetic scaffold,” Acta Biomater., vol. 1, no. 2, pp. 155–164, Mar. 2005.

[32] F. M. Plieva, I. Y. Galaev and B. Mattiasson, “Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications,” J. Sep. Sci., vol. 30, no. 11, pp. 1657–1671, Jul. 2007.

[33] F. Ak, Z. Oztoprak, I. Karakutuk and O. Okay, “Macroporous Silk Fibroin Cryogels,” Biomacromolecules, vol. 14, no. 3, pp. 719–727, Mar. 2013.

[34] T. M. A. Henderson, K. Ladewig, D. N. Haylock, K. M. McLean and A. J. O’Connor, “Cryogels for biomedical applications,” J. Mater. Chem. B, Apr. 2013.

[35] S.-L. Loo, W. B. Krantz, T.-T. Lim, A. G. Fane and X. Hu, “Design and synthesis of ice-templated PSA cryogels for water purification: towards tailored morphology and properties,” Soft Matter, vol. 9, no. 1, pp. 224–234, Nov. 2012.

[36] Fatima Plieva, Igor Galaev and Bo Mattiasson, “Production and Properties of Cryogels by Radical Polymerization,” in Macroporous Polymers, CRC Press, 2009, pp. 23–47.

[37] M. Sheridan, L. Shea, M. Peters and D. Mooney, “Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery,” Journal of Controlled Release, vol. 64, no. 1–3, pp. 91–102, Feb. 2000.

[38] J. A. Stammen, S. Williams, D. N. Ku and R. E. Guldberg, “Mechanical properties of a novel PVA hydrogel in shear and unconfined compression,” Biomaterials, vol. 22, no. 8, pp. 799–806, Apr. 2001.

[39] F. J. O’Brien, “Biomaterials & scaffolds for tissue engineering,” Mater. Today, vol. 14, no. 3, pp. 88–95, Mar. 2011.

[40] T. Dvir, B. P. Timko, D. S. Kohane and R. Langer, “Nanotechnological strategies for engineering complex tissues,” Nat Nano, vol. 6, no. 1, pp. 13–22, Jan. 2011.

[41] F. G. Heineken and R. Skalak, “Tissue Engineering: A Brief Overview,” J Biomech Eng, vol. 113, no. 2, pp. 111–112, May 1991.

[42] M. N. Collins and C. Birkinshaw, “Hyaluronic acid based scaffolds for tissue engineering—A review,” Carbohydrate Polymers, vol. 92, no. 2, pp. 1262–1279, Feb. 2013.

[43] R. Glicklis, L. Shapiro, R. Agbaria, J. C. Merchuk and S. Cohen, “Hepatocyte behavior within three-dimensional porous alginate scaffolds,” Biotechnology and Bioengineering, vol. 67, no. 3, pp. 344–353, 2000.

40

[44] Yoshito Ikada, Ed., “Chapter 1 Scope of Tissue Engineering,” in Interface Science and Technology, vol. 8, Elsevier, 2006, pp. 1–89.

[45] Y. Zhang and M. Zhang, “Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering,” Journal of Biomedical Materials Research, vol. 55, no. 3, pp. 304–312, 2001.

[46] Y. Hwang, C. Zhang and S. Varghese, “Poly(ethylene glycol) cryogels as potential cell scaffolds: effect of polymerization conditions on cryogel microstructure and properties,” J. Mater. Chem., vol. 20, no. 2, pp. 345–351, 2010.

[47] M. B. Dainiak, I. U. Allan, I. N. Savina, L. Cornelio, E. S. James, S. L. James, S. V. Mikhalovsky, H. Jungvid and I. Y. Galaev, “Gelatin-fibrinogen cryogel dermal matrices for wound repair: Preparation, optimisation and in vitro study,” Biomaterials, vol. 31, no. 1, pp. 67–76, Jan. 2010.

[48] S. Bhat, A. Tripathi and A. Kumar, “Supermacroprous chitosan-agarose-gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering,” J. R. Soc. Interface, vol. 8, no. 57, pp. 540–554, Apr. 2011.

[49] N. Boelgen, F. Plieva, I. Y. Galaev, B. Mattiasson and E. Piskin, “Cryogelation for preparation of novel biodegradable tissue-engineering scaffolds,” J. Biomater. Sci.-Polym. Ed., vol. 18, no. 9, pp. 1165–1179, Sep. 2007.

[50] V. I. Lozinsky, “Polymeric cryogels as a new family of macroporous and supermacroporous materials for biotechnological purposes,” Russian Chemical Bulletin, vol. 57, no. 5, pp. 1015–1032, May 2008.

[51] Y. Dehdari, “Synthesis and Characterization of Highly Porous and Elastic Hydrogels of Hyaluronic acid.” University of Gothenburg, Sweden, 2013.

[52] F. Meyer, D. Lohmann and W.-M. Kulicke, “Determination of the viscoelastic behavior of sodium hyaluronate in phosphate buffered saline with rheo-mechanical and rheo-optical methods,” Journal of Rheology, vol. 53, no. 4, pp. 799–818, 2009.

[53] E. R. Morris, D. A. Rees and E. J. Welsh, “Conformation and dynamic interactions in hyaluronate solutions,” Journal of Molecular Biology, vol. 138, no. 2, pp. 383–400, Apr. 1980.

[54] Y. Minaberry, D. A. Chiappetta, A. Sosnik and M. Jobbagy, “Micro/Nanostructured Hyaluronic Acid Matrices with Tuned Swelling and Drug Release Properties,” Biomacromolecules, vol. 14, no. 1, pp. 1–9, Jan. 2013.