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ENZYME CO-LOADING INTO

POLYMERIC PARTICLES AND

CAPSULES

Aantal woorden: 15.900

Kenneth Asselman Stamnummer: 00903978

Promotor: Prof. dr. Andre Skirtach

Supervisor: Dr. Bogdan Parakhonskiy

Masterproef voorgelegd voor het behalen van de graad master in de richting Master of Science in de

industriële wetenschappen: biochemie

Academiejaar: 2016 – 2017

ENZYME CO-LOADING INTO

POLYMERIC PARTICLES AND

CAPSULES

Aantal woorden: 15.900

Kenneth Asselman Stamnummer: 00903978

Promotor: Prof. dr. Andre Skirtach

Tutor: Dr. Bogdan Parakhonskiy

Masterproef voorgelegd voor het behalen van de graad master in de richting Master of Science in de

industriële wetenschappen: biochemie

Academiejaar: 2016 – 2017

4

“The author and the promoter give the permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws, more

specifically the source must be extensively specified when using the results from this thesis”.

Promotor: Prof. dr. André Skirtach

Tutor: Dr. Bogdan Parakhonskiy

Author: Kenneth Asselman

Ghent University 20 January 2017

5

Foreword

I would like to thank my promotor Prof. dr. Andre Skirtach to give me the opportunity to work

on this thesis. Further I would like to express my gratitude to Dr. Bogdan Parakhonskiy and

Dr. Timothy Douglas for their help and advice.

Further I would like to thank my parents and friends for their constant support.

6

Abstract In this study, new calcium/magnesium particles were designed and their characteristics (size

shape and crystallinity) were studied. Next, Gellan gum hydrogels were prepared with the

newly synthesized particles with the objective to create hydrogel-particle composites as

novel drug delivery system. The hydrogel properties (gelation time and gel stiffness) were

investigated. First, calcium/magnesium carbonate particles are synthesized by varying

Ca2+:Mg2+ ratio and reaction conditions (time and addition of organic solvent). This was done

to investigate the influence of reaction conditions on the size, shape and crystallinity of the

particles. These tests showed that different types particles are created varying the reaction

conditions: spherical, elliptical, paired, star-like and amorphous particles. The size decreases

three time if an organic solvent is added and if the reaction time increases from minutes to

hours. All samples contain vaterite and/or calcite with an increasing amorphous phase if

Ca2+:Mg2+ ratio decreases. Hydromagnesite is only present in samples containing at least

50% magnesium in micro-particles and in nano-particles with 33,3% magnesium. Next,

gelation time of hydrogels prepared with the newly designed particles decreases if particle

concentration increases or if the Ca2+:Mg2+ ratio decreases. Whereas particles with a 0%

magnesium could not induce gelation and those with 33,3% magnesium need to contain

minimum 20% particles for comparable gelation time as those with a higher magnesium

content but with 4% particles. Also gel stiffness was studied, this increased if the particle

concentration increased or the Ca2+:Mg2+ ratio decreased. With these results, spherical

micro-particles (Ca2+:Mg2+ ratio of 3:1) are more suitable since they have an appropriate

gelation time, high particle concentration to prepare hydrogels as drug delivery system.

Keywords: carbonate particles, hydrogel, Gellan gum, drug delivery system

7

Samenvatting In deze studie werden nieuwe calcium/magnesium deeltjes gecreëerd en werden hun

eigenschappen (grootte, vorm en kristalliniteit) bestudeerd. Daarna werden Gellan gum

hydrogels gesynthetiseerd met de nieuwe deeltjes met als doel om nieuwe drug delivery

systemen te creëren. De eigenschappen (stijfheid en snelheid van gelvorming) van de

gevormde hydrogels werden onderzocht. Eerst werden de verschillende deeltjes

gesynthetiseerd door de reactie omstandigheden (tijd en toevoegen van organisch solvent)

en de Ca2+:Mg2+ ratio te veranderen. Door deze parameters te veranderen kon de invloed

van deze op de grootte, vorm en kristalliniteit van de deeltjes bestudeerd worden. Hieruit

blijkt dat verschillende deeltjes kunnen gecreëerd worden: sferische, elliptische, gepaarde,

ster-vormende en amorfe deeltjes. De grootte van de deeltjes daalt ongeveer drie maal als er

een organisch solvent wordt toegevoegd en als de reactie tijd stijgt van minuten naar uren.

Verder werd er in alle stalen vateriet en/of calciet teruggevonden waarbij de amorfe fase

toeneemt als de Ca2+:Mg2+ ratio daalt. Hydromagnesiet is enkel aanwezig bij deeltjes

gesynthetiseerd met een met een magnesiumgehalte van 50% of hoger bij microdeeltjes en

bij nanodeeltjes met een magnesiumgehalte van 33,3%. Daarna bleek dat de snelheid van

gelvorming daalt als de Ca2+:Mg2+ ratio stijgt, deeltjes die geen magnesium bevatten, kunnen

geen gelvorming induceren. Ook de stijfheid van de gels werd bepaald, de stijfheid van

hydrogels neemt toe als de concentratie van de deeltjes toeneemt of als de Ca2+:Mg2+ ratio

daalt. Uit alle resultaten kan besloten worden dat sferische microdeeltjes met een met een

magnesiumgehalte van 33,3% de voorkeur genieten om hydrogels te synthetiseren. De

reactie omstandigheden zijn beter dan die voor nano-deeltjes en er is een hogere

concentratie mogelijk zonder de gelvorming drastisch te verhogen.

Kernwoorden: drug delivery system, carbonaat deeltjes, Gellan gum, hydrogel

8

Index

1 Introduction ................................................................................................................................... 13

2 Literature ....................................................................................................................................... 15

2.1 Drug delivery ............................................................................................................................. 15

2.2 Hydrogel .................................................................................................................................... 16

2.3 Gellan gum................................................................................................................................. 18

2.4 Ceramic biomaterials ................................................................................................................. 20

2.4.1 Ceramic Particles ................................................................................................................... 21

2.4.2 CaCO3 Particles ...................................................................................................................... 22

2.4.3 MgCO3 Particles ..................................................................................................................... 25

2.5 Particles for gelation.................................................................................................................. 27

3 Goals .............................................................................................................................................. 28

3.1 Characterization of micro- and nanoparticles ........................................................................... 28

3.2 Identification of hydrogel properties ........................................................................................ 28

4 Materials and Methods ................................................................................................................. 29

4.1 Materials .................................................................................................................................... 29

4.2 Methods .................................................................................................................................... 29

4.2.1 Synthesis of the particles....................................................................................................... 29

4.2.2 Characterization of the particles ........................................................................................... 30

4.2.3 Synthesis of Gellan gum ........................................................................................................ 30

4.2.4 Preparation of the hydrogels ................................................................................................ 30

4.2.5 Investigation of the hydrogel properties ............................................................................... 31

5 Results and Discussion .................................................................................................................. 32

5.1 Characterization of the particles ............................................................................................... 33

5.1.1 Yield of the Particles .............................................................................................................. 33

5.1.2 SEM Images ........................................................................................................................... 35

5.1.3 XRD Data ................................................................................................................................ 38

5.1.4 FTIR Data ............................................................................................................................... 40

5.2 Investigation of gel properties .................................................................................................. 43

5.2.1 Gelation time ......................................................................................................................... 43

5.2.2 Rheometry results ................................................................................................................. 45

5.2.3 FTIR Data ............................................................................................................................... 47

6 Conclusion ..................................................................................................................................... 49

References ............................................................................................................................................. 51

Attachments .......................................................................................................................................... 56

9

Attachment 1: Statistics of yield............................................................................................................ 56

Attachment 2: SEM Images ................................................................................................................... 60

Attachment 3: Gel formation ................................................................................................................ 70

Attachment 4: Rheometry Data ............................................................................................................ 71

10

Abbreviations CaP calcium phosphates

CD cyclodextrin

EG ethylene glycol

GDL D-glucono-δ-lactone

GG Gellan gum

HA hydroxyapatite

HAA hyaluronic acid

HEMA hydroxyethyl methacrylate

PAAc poly(acrylic acid)

PAAm polyacrylamide

PAN polyacrylonitrile

PBO poly(butylene oxide)

PCL polycaprolactone

PEG poly (ethylene glycol)

PEMA poly(ethyl methacrylate)

PEO poly(ethylene oxide)

PF propylene fumarate

PGEMA poly(glucosylethyl methacrylate)

PHB poly(hydroxy butyrate)

PHPMA poly(hydroxypropyl methacrylamide)

PLA poly(lactic acid)

PLGA poly(lactic-co-glycolic acid)

PMMA poly(methyl methacrylate)

PNIPAAm poly(N-isopropyl acrylamide)

PNVP poly(N-vinyl pyrrolidone)

PPO poly(propyleneoxide)

PVA poly(vinyl alcohol)

PVAc poly(vinyl acetate)

PVamine poly(vinyl amine)

TCP tricalcium phosphate

11

Tables and Figures

Tables

Table 1: Overview of the hydrophilic polymers used to synthesize hydrogels.

Table 2: Overview of the different solutions used to synthesize (Ca/Mg) CO3

particles.

Table 3: Overview of the solutions used for the synthesize of the small Ca/Mg

particles.

Table 4: Overview of the solutions used to prepare the big (Ca-Mg-Zn)CO3 particles.

Table 5: Summary of shape and size of the different particles.

Table 6: Summary of bands visible in each sample and the corresponding type of

particle.

Table 7: Summary of surface area of the different types of particles. For spherical

particles, surface area is calculated with A = 4 x π x r², for elliptical particles A = π x

length axial side x length radial side, for paired particles A = 2 x π x r² + 2 x π x r x h.

Figures

Figure 1: Chemical structure of GG.

Figure 2: Scanning electron micrograph of CaCO3 polymorphs: aragonite (a), vaterite

(b) and calcite (c).

Figure 3: Confocal fluorescence scanning images of spherical (a), elliptical (b), star-

like (c) and cubic (d) calcium particles. The particles are loaded with TRITC-dextran

molecules (the scale bar is 5 µm). Insets show show scanning electron microscopy

images (the scale bar is 1 µm).

Figure 4: SEM images of the particles from various reaction conditions: (a) stirring

time of 2 min at 313 K for 0 min, (b) stiring time of 4 min at 313 K for 20 min, (c) the

particles by adding the ones from into 100 mL of double deionized water with a

temperature of 353 K for 15 min, (d) for 25 min, (e) large magnification of the red

ellipse in panel d and (f) large magnification of the yellow ellipse in panel d.

Figure 5: Overview of average yield and their standard deviation of the particles.

Figure 6: SEM images of particles, in each image, the scale bar is represented.

Different magnifications are used, 2000x for sample 5, small particles; 5000x for

sample3, 4 and 5, big particles; 10.000x for sample 1 and 4, small particles and

sample 1, big particles; 20.000x for sample 2, big and small particles.

Figure 7: XRD patterns for all samples in the group of big particles (A) and in the

group of small particles (B). Each color corresponds to a type of particle (see legend

in the right top).

Figure 8: FTIR spectra of the different samples. Figure A represents the group of big

particles and figure B the group of small particles.

12

Figure 9: Graphics corresponding to time of gel formation, whereas figure A

corresponds to big particles and figure B to small particles. The axis on the left

represents the particle concentration in the gel for sample 3-5 and the axis on the

right represents the particle concentration in the gel for sample 2 (Figure 9, A). In

Figure 9, B ,the axis on the left represents the particle concentration in the gel for

sample 2 and the axis on the right represents the particle concentration in the gel for

sample 3-5. Whereas the axis on the top represents the time of gelation for sample 3-

5 and the axis on the bottom represents the gelation time for sample 2.

Figure 10: Overview of average storage modulus measured in each hydrogel for big

(A) and small (B) particles. For each sample, standard deviation is shown for a single

point.

Figure 11: FTIR spectra for the hydrogels. The spectra of the hydrogels prepared

with big particles are represented in Figure 11, A and those of the hydrogels prepared

with small particles in Figure 11, B.

Enzyme co-loading into polymeric particles and capsules 13

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1 Introduction Bone regeneration is a serious issue in medicine. To overcome bone defects, defects are

filled with a material or scaffold that supports development of new tissue. Until new tissue is

formed, the scaffold has to degenerate slowly, and disappear completely until the defect is

filled with bone tissue.

Nowadays, autograft and allograft tissue is used to regenerate bone defects. Since

autografting has several disadvantages (costs, patient pain, limited supply), allografting is

used as an alternative. However, also this technique has limitations: uncertainty of disease

transmitting and biocompatibility. For these reasons novel methods, like injectable materials,

are introduced. This is a minimal invasive procedure, resulting in more patient comfort.

Second, outside the body, the material is liquid and solidifies in situ. Thereby, the defect is

completely filled since the injected material can adjust its shape.

Bone filling materials include bone cements consisting of calcium phosphates. However, this

material has some drawbacks, for example, biological active components or cells cannot be

incorporated and they don’t degenerate fast enough to make room for a new bone ingrowth.

An alternative to the above mentioned bone cements are hydrogels. Hydrogels are three-

dimensional cross-linked networks that contain a large amount of water. Therefore,

substances can be transported in and out the gel by diffusion, and maintain viability of the

cells. With the same process toxic and undesirable products can be removed. Also, such

biological active substances as calcium phosphates and calcium carbonate particles or

capsules can be incorporated to stimulate bone growth and cell proliferation. These particles

can also be loaded with drug (e.g. enzymes).

Calcium carbonate is widely used in bone regeneration, however, recent studies focus

mostly on enrichment with magnesium, iron and zinc. Magnesium promotes cell adhesion,

while iron has an influence on the cell metabolism and zinc has antibacterial effects.

In this study, (Ca2+/Mg2+) micro- and nanoparticles are prepared and mixed with Gellan gum

(GG) solution to create in-situ forming injectable hydrogel solutions. The particles are

multifunctional: first, they cross-link GG and induce hydrogel formation through release of

divalent cations (Ca2+, Mg2+). Since the used particles are also ceramics, they would

enhance mechanical properties of the hydrogel. The particles also induce proliferation,

adhesion and differentiation of bone forming cells.

The aim of this work is to obtain nano- and micro-sized ceramic particles with an appropriate

size, shape and chemical composition to manufacture in-situ forming injectable hydrogel

composites.

14 Enzyme co-loading into polymeric particles and capsules

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The experimental part is composed of two parts. In the first part, ceramic particles are

synthesized and their size, shape and chemical composition will be evaluated to create

hydrogels. The goal is to compare nano- and micro- particles with different Ca2+/Mg2+ ratio’s

and determine their properties.

Second, hydrogel-particle composites are prepared with the ceramic particles from the first

stage. The influence of gelation time, hydrogel strength and hydrogel on cell viability is

evaluated.


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2 Literature

2.1 Drug delivery

In the last years, substantial research has been done to create novel drug delivery systems.

There are several reasons to develop new platforms: (i) by creating new drug delivery

systems the efficacy and safety of the administration of the drug can improve; it can also lead

to new therapies, (ii) the production of novel drugs by genetic engineering can require more

complex delivery methods, (iii) drug response can be affected by the release pattern

(continuous vs. pulsatile), (iv) by the advances in materials and biotechnology, new systems

can be developed (Langer, 1990).

Novel drug delivery systems include several approaches, like chemical modification, drug

entrapment in small vesicles later injected in the bloodstream or drug entrapment into

polymeric materials placed in body compartments. Whereas conventional forms of drug

delivery rely on pills, ointments, eye drops and intravenous solutions (Langer, 1990). Drug

delivery is also a very important area for tissue regeneration. Bone defects, for example, are

managed using traditional methods like allografting and autografting cancellous bone (Burg,

Porter, & Kellam, 2000), using vascularized grafts from fibula and iliac crest, bone marrow

replacement and the use of bone cement fillers. Since these techniques encounter several

shortcomings (costs, patient pain, limited supply, uncertainty of disease transmitting and

biocompatibility), alternative techniques (e.g. hydrogels) are being investigated (Burg et al.,

2000).

One of the novel approaches in drug delivery systems for bone defects is the use of

hydrogels because of their unique properties (porous structure, biocompatibility,

biodegradability and deformability). They have a highly porous structure, which permits

loading of drugs into their matrix. In addition, the rate of drug release depends on the

porosity; which is influenced by the diffusion rate of the molecule through the gel matrix

(Hoare & Kohane, 2008). Another advantage of hydrogels is that they are injectable. Before

injection, hydrogels are aqueous solutions which would become a gel under physiological

conditions. Using this method bioactive compounds or cells can be integrated providing an

advantage of minimal surgical wounds. This is done by mixing before injection and the

injected material can fill gaps irrespective of the shape of the defect (Yu & Ding, 2008).

An ideal injectable hydrogel should have the following characteristics: (i) the sol state should

have a low viscosity (this allows a smaller pinpoint injection), (ii) gelation should happen after

the injection, (iii) the gels should be biodegradable or (gradually) dissolvable, (iv) the polymer

and the products should be biodegradable (Yu & Ding, 2008).


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2.2 Hydrogel

Hydrogels are two- or multicomponent systems in a three-dimensional network containing

cross-linked polymer chains, where the space between the macromolecules is filled with

water (Ahmed, 2015). They can absorb water if hydrophilic groups (-OH, -SO3H, -CONH,

etc.) are present. In that case the hydrogels can be hydrated for more than 90% wt. Their

ability to absorb water can also be limited (<5-10% wt.) if they contain groups with

hydrophobic characteristics (e.g. PLA and PLGA) (Hamidi, Azadi, & Rafiei, 2008). According

to the type of cross-linking, hydrogels can be classified into two groups: a chemical gel where

the network is cross-linked via a covalent bond and a physical gel where a physical

association between nanoparticles and polymer chains forms the gel (like molecular

entanglements, and/or ionic, H-bonding or hydrophobic forces). Although in some cases,

both types of cross-linking can occur in the same gel (Hoffman, 2012; Yu & Ding, 2008).

During the swelling process, the most polar, hydrophilic groups will be hydrated. The portion

of water used, is defined as primary bound water. If these groups are hydrated, the network

swells and the hydrophobic groups are exposed. These groups will interact with water

through hydrophobic interactions, while the portion of water needed for this process is

referred to secondary bound water. (Patel & Mequanint). The primary and secondary bound

water together are defined as total bound water. After these interactions, more water can be

absorbed through the osmotic driving force of the network. However, this additional swelling

is opposed by the presence of crosslink junctions through an elastic network retraction force.

Finally, an equilibrium swelling level will be established if there is a balance between the

refraction force and the dilution force. The water absorbed beyond the total water is defined

as free or bulk water (Patel & Mequanint).

Three different types of polymers are used to synthesize hydrogels: natural, semi or semi-

synthetic polymers.

Table 1 on the following page gives an overview of hydrophilic polymers used for hydrogels

(Hamidi et al., 2008).


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Table 1: Overview of the hydrophilic polymers used to synthesize hydrogels. (Hamidi et al., 2008)

Hydrophilic polymers used in preparation of hydrogels

Natural polymers and their derivatives

Anionic polymers: HAA, alginic acid, pectin, carrageenan, chondroitin sulfate, dextran sulfate,

GG

Cationic polymers: chitosan, polylysine

Amphipathic polymers: collagen (and gelatin), carboxymethyl chitin, fibrin

Neutral polymers: dextran, agarose, pullulan

Synthetic polymers

Polyesters: PEG–PLA–PEG, PEG–PLGA–PEG, PEG–PCL–PEG, PLA–PEG–PLA, PHB,

P(PF-co-EG)6acrylate end groups, P(PEG/PBO terephthalate)

Other polymers: PEG-bis-(PLA-acrylate), PEG6CDs, PEG-g-P(AAm-co-Vamine),

PAAm, P(NIPAAm-co-AAc), P(NIPAAm-co-EMA), PVAc/PVA, PNVP, P(MMA-co-HEMA),

P(AN-co-allyl sulfonate), P(biscarboxy-phenoxy-phosphazene), P(GEMA-sulfate)

Combinations of natural and synthetic polymers

P(PEG-co-peptides), alginate-g-(PEO–PPO–PEO), P(PLGA-co-serine), collagen-acrylate,

alginate-acrylate, P(HPMA-g-peptide), P(HEMA/Matrigel®), HA-g-NIPAAm

An aqueous environment protects cells and sensitive drugs incorporated in the hydrogel and

allows transportation of substances in and out of the gel (Gkioni, Leeuwenburgh, Douglas,

Mikos, & Jansen, 2010). The viability of the cells can be maintained through diffusion of

substances like oxygen and nutrients. The same process will remove toxic and undesirable

products (T. L. A. Douglas, Aparicio, & Ginebra, 2015).

Hydrogels have a wide range of applications, in the last years a lot of research has been

done in their use as drug delivery system, tissue engineering and regenerative medicines

(Ahmed, 2015).

Advantages and disadvantages

Hydrogels have several benefits: they are porous, their porosity can be influenced by

changing the density of the crosslinks. The porosity also permits loading of drugs into the gel.

They are generally highly biocompatible, which is promoted through the high water content

and their similar structure to native extracellular matrix. Another advantage is that the

biodegradability can be regulated easily. This can be done via hydrolytic, enzymatic or

environmental (e.g. pH, temperature) pathways. Hydrogels can deform easily and adopt to

the shape of surface, which they are applied to (Hoare & Kohane, 2008).

However, there are also some limitations. If a gel is loaded, then it can dissolve premature or

flow away from a targeted site because of its low tensile strength. There are also problems

related to drug delivery. In the case of hydrophobic drugs, the quantity and homogeneity of

the drug loaded into the gel can be limited. If the hydrogel contains high amounts of water

and has large pores, hydrophobic drugs will be released in the time span of hours to days.


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Also, the ease of application may be limited: some hydrogels are deformable and can be

injected, while other have to be placed surgically (Hoare & Kohane, 2008).

2.3 Gellan gum

Gellan gum (GG) is an anionic polysaccharide that is produced by the gram-negative

bacterium Sphingomonas paucimobilis during aerobic fermentation. GG forms a linear

structure consisting of β-D-glucose, β-D-glucuronic acid and α-L-rhamnose in a molar ratio of

2:1:1 and two acyl groups (acetate and glycerate) bound to the glucose residue adjacent to

glucuronic acid (Figure 1) (T. E. Douglas, Wlodarczyk, et al., 2014).

Figure 1: Chemical structure of GG (T. E. Douglas, Wlodarczyk, et al., 2014).

There are two forms of GG, an acetylated and a deacetylated form. Both of these forms can

gelate, but the mechanical properties vary from soft and elastic (acetylated form) to hard and

brittle (fully deacetylated) (Oliveira et al., 2010).

GG has a lot advantages: it is injectable, thermos-settable, not expensive, not derived from

animals and has a high affinity for mono- and divalent cations (e.g. Na+, Mg2+, Ca2+)(T. E.

Douglas, Piwowarczyk, et al., 2014). As biomedical application, GG has several benefits: it

lacks toxicity, can be processed under mild conditions, can be used in a minimally invasive

manner, and, due to the glucuronic acid, it has a structural similarity to native cartilage

glycosaminoglycans (Oliveira et al., 2010).

The gel is formed using the following process: GG has a coil form at high temperatures. If the

temperature decreases, a thermally reversible coil to double-helix transition occurs. After this

prerequisite form, junction zones are formed; these are oriented bundles consisting of anti-

parallel double helices. In the final step, the junction zones are linked together with untwined

zones of polysaccharide chains (these are in the form of extended helical chains). This leads

to the formation of a three-dimensional network (Oliveira et al., 2010).

Gel formation depends on the polymer concentration, temperature and the presence of

mono- and divalent cations. If structurally stable gels are synthesized, the presence of

cations is critical. At low GG concentrations, an ordered structure can be formed through


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helix formation and partial aggregation. But the helical aggregates don’t form a gel since they don’t form a continuous network. The carboxyl groups repel each other (through electrostatic

interactions), hindering the aggregation and tight binding of the helices. Introduction of

cations shield the repulsion and solves the problem of formation of aggregated and tight

bound helices (Oliveira et al., 2010). Gel strength depends on other parameters like acetyl

content, type and concentration of ions, pH and hydrophilic ingredients (Bajaj, A., Survase,

Saudagar, & Singhal, 2007).

GG is widely used in the food and pharmaceutical industry due to its minimal toxicity. It is

also used in biomedical application, for example, GG is a versatile encapsulating agent and

active ingredient in drug delivery systems. GG can be also used for wound healing and more

recently in tissue engineering (Ferris, Gilmore, Wallace, & Panhuis, 2013). The major

drawback of GG is its mechanical properties (i.e. stiffness) (Coutinho et al., 2010). There are

multiple solutions available to overcome this problem, for example, T. E. Douglas,

Piwowarczyk, et al incorporated bioglass particles in GG (T. E. Douglas, Piwowarczyk, et al.,

2014). Another solution is the incorporation of polydopamine, which enhances the stiffness

of hydrogels (T. E. Douglas, Wlodarczyk, et al., 2014). Another example is mineralization of

hydrogels with zinc-phosphate (by using alkaline phosphate) (T. E. Douglas et al., 2015) or

mineralization with ceramics such as calcium carbonate (CaCO3), magnesium enriched

CaCO3 and magnesium carbonate mediated by using urease (Timothy Douglas et al.). So

ceramics represent an interesting solution for enhancement of mechanical properties of

hydrogels.


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2.4 Ceramic biomaterials

According to Barsoum & Barsoum, ceramics can be described as “solid compounds that are

formed by the application of heat, and sometimes heat and pressure, comprising at least one

metal and a nonmetallic elemental solid or a nonmetal, a combination of at least two

nonmetallic elemental solids, or a combination of at least two nonmetallic elemental solids

and a nonmetal.” Ceramics are used due to their positive effects in regard with interactions

with human tissues (Barsoum & Barsoum, 2002).

The ceramics comprise of calcium phosphates, silica, alumina, zirconia and titanium dioxide.

The characteristics of these ceramics are: low or non-existing biodegradability, good body

response and high mechanical strength. Because of the poor degradability, these materials

don’t appear to be used in tissue engineering. However, this problem can be overcome by

introducing porosity (Habraken, Wolke, & Jansen, 2007).

Calcium phosphates (CaP) are one of the most widely studied ceramics. They include

hydroxyapatite (HA) (Ca10(PO4)6(OH)2), tricalcium phosphate (TCP), and tetracalcium

phosphate. Calcium phosphates have several benefits: protein free, minimal immunologic

reactions and no foreign body reactions or systemic toxicity have been reported (Burg et al.,

2000). They also have a good biocompatibility due to their resemblance to bone mineral

(chemical and crystal) structure. CaP have been reported to support the attachment,

differentiation, and proliferation of cells (osteoblasts and mesenchymal). The drawbacks of

CaP are low fracture toughness, high stiffness and brittleness. The mechanical properties of

CaP depend on the amorphous phase, micro-porosity and grain size. The mechanical

stiffness decrease, if the volume of amorphous phase, the porosity and grain size increase

(Chen, Zhu, & Thouas, 2012).

One of the most important groups is biological HA, it contains minor trace elements like

carbonate (CO3), magnesium (Mg) and sodium (Na). Several studies have shown that the

formula of biological (or carbonated) HA is approximate to (Ca, X)10(PO4, HPO4, CO3)6(OH,

Y)2 where X can be cations like Mg or Na who can replace Ca and Y can be anions like F or

Cl who can replace OH. Since carbonated HA contains CO3, the use of CO3 in biomaterials is

reported (Rahaman, 2014).

Another group of ceramics used are Na-containing silicate bioactive glasses. The basic

constituents of these are, SiO2, P2O5, Na2O and CaO. The benefits of these glasses are

excellent bioactivity and the ability to chemically bind the host bone through a carbonated

phosphate surface layer. They have the ability to induce bone growth processes like enzyme

activity, revascularization, osteoblast adhesion and differentiation, gene expression and

growth factor production. A key reason to use this material is that the range of some

chemical properties can be controlled and thus the rate of bio resorption (Chen et al., 2012).


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More recently, bioactive glasses were doped with magnesium (Mg), zinc (Zn) or strontium

(Sr). The release of these elements would accelerate the proliferation of bone marrow

mesenchymal stem cells and the ALP activity in vitro (Wang, Li, Ito, & Sogo, 2011).

It can be noted that a particulate form of ceramic materials containing calcium or magnesium

ions would be useful for spreading the ceramics in the doped materials. Magnesium or

calcium ions could also be useful to prepare calcium carbonate particles and magnesium

carbonate particles.

2.4.1 Ceramic Particles

Ceramic particles can be added to the polymer (hydrogel) to increase its mechanical stiffness

and/or make it more osteoconductive.

Typically the particles are be prepared by mixing aqueous solutions containing reactants.

The particles synthesized during this work contain calcium, magnesium and a mixture of

calcium and magnesium ions.

Calcium

The divalent cation Ca2+ plays an important role in human health. Bone consists of calcium.

The calcium inside bone is a reserve for a calcium deficiency in the blood. It maintains the

balance between bone formation and resorption. It has a vital role in metabolism, blood

clotting, nerve impulse conduction, cellular communication and muscle contraction (Weaver

& Heaney, 2007). As it is mentioned above, the addition of calcium ions will result in cross-

linking of the hydrogel.

Magnesium

Magnesium is a cofactor in a lot enzymatic reactions which are involved in nucleic acid,

protein and lipid synthesis and directly stimulates osteoblast proliferation (Castiglioni,

Cazzaniga, Albisetti, & Maier, 2013; Suchanek et al., 2004). Magnesium is important for the

bone strength and growth, muscle contraction and several metabolic reactions (Song, 2007).

Magnesium introduction in HA has a positive effect on the osseo-integrational properties

(Cacciotti, 2016).


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2.4.2 CaCO3 Particles

Calcium carbonate (CaCO3) has become one of the most popular cores for polymer particle

templating. These particles have several benefits: a simple preparation procedure (Y. I.

Svenskaya et al., 2016), low costs, good biocompatibility (Bogdan V Parakhonskiy et al.,

2013) and mild decomposition conditions (Y. Svenskaya et al., 2013). The main advantage of

CaCO3 is the possibility to control size and shape of the particles during synthesis. Because

the synthesis of calcium carbonate particles is sensitive to reagent concentration,

temperature, intensity and duration of stirring the reaction mixture (Trushina, Sulyanov,

Bukreeva, & Kovalchuk, 2015; Volodkin, 2014), these parameters need to be controlled

throughout the reaction.

There are three anhydrous polymorphs form of CaCO3: vaterite, calcite and aragonite. The

least stable form is vaterite, whereas calcite is the most stable one. However, the most

attractive form for technical applications with micrometer-sized particles is vaterite, because

of its highly porous structure and a large surface area (Volodkin, 2014). Variation of the

synthesis parameters allows for the preparation of particles in the size range from 500

nanometers to 10 micrometers. The metastable vaterite particles can re-crystallize in an

aqueous solution to calcite (which has a lower solubility) (B. V. Parakhonskiy et al., 2014).

Figure 2: Scanning electron micrograph of CaCO3 polymorphs: aragonite (a), vaterite (b) and calcite (c). (Volodkin, 2014)


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Only vaterite particles can be prepared by mixing a supersaturated salt solution at an optimal

temperature of 20-50 °C. After the synthesis, a channel-like porous structure appears with a

pore size of 20-40 nm (Volodkin, 2014).

Controlling the size of the particles is important for its applications. If the particles are used in

drug delivery systems, they have to move through blood vessels. Therefore, the size of the

particles has to be sufficiently small (Trushina et al., 2015).

CaCO3 with a defined size (3-20 µm) can be prepared by mixing of CaCl2 and Na2CO3 under

a vigorous stirring. The mixing of these salts results in the formation of nuclei, followed by the

growth of vaterite crystals. If the amount of salt and concentrations are not changed, then a

higher number of nuclei are initially formed, crystal size will be smaller. Also, the stirring time

and stirring speed have an influence, the higher these parameters are, the smaller the

crystals will be (Volodkin, 2014).

However, it is impossible to increase the time of the reaction, because of the recrystallization

process from vaterite to a more stable calcite. For this reason, Bogdan V Parakhonskiy,

Haase, & Antolini (Bogdan V Parakhonskiy, Haase, & Antolini, 2012) suggested to add

organic solvents (ethylene glycol (EG)) to increase the viscosity and decrease the solubility

of the vaterite particles. This results in a decreasing recrystallization rate to calcite and

provides a better ion distribution via extended stirring time. Porous vaterite particles with a

size of 400 nm have been reported using these method (Bogdan V Parakhonskiy et al.,

2012).

Such small particles can be used as drug delivery system and successfully immobilize drugs

(Y. Svenskaya, Gorin, Parakhonskiy, & Sukhorukov, 2015). They can also penetrate through

cell membranes (B. Parakhonskiy et al., 2015).

Shapes of CaCO3

By controlling the crystallization reaction, isotropic (spherical and cubical) and anisotropic

(elliptical and star-like) particles can be synthesized. Cubic particles are prepared, if vaterite

recrystallizes to calcite. To prevent the formation of calcite (or cubical particles) EG can be

added. EG reduces solubility of the particles, so that no calcite will be formed (B. V.

Parakhonskiy et al., 2014).


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Figure 3: Confocal fluorescence scanning images of spherical (a), elliptical (b), star-like (c) and cubic (d) calcium particles. The particles are loaded with TRITC-dextran molecules (the scale bar is 5 µm). Insets show show scanning electron microscopy images (the scale bar is 1 µm). (B. V. Parakhonskiy et al., 2014)

B. V. Parakhonskiy et al determined the influence of time, solvent (water vs 80% EG) and

ratio of salt concentrations (CaCl2 and Na2CO3) on the synthesis of CaCO3 particles (B. V.

Parakhonskiy et al., 2014). Spherical particles can be created by mixing the two salts in

equal concentrations. Elliptical particles can be produced by varying the ratio of the salt

concentrations. Star-like particles are formed by aggregation of the vaterite crystals on a

common nucleus, which is the result of competition between growth and nucleation

processes (B. V. Parakhonskiy et al., 2014).

Comparison between the particles shows that vaterite particles have a better loading

efficiency than calcite particles. Star-like particles have the highest surface area followed by

elliptical and spherical particles (B. V. Parakhonskiy et al., 2014). The shape of the particles

also has an influence on the activity of encapsulated molecules. Star-like particles have a

significantly higher enzymatic activity (of encapsulated molecules) than elliptical, spherical

and rhomboidal particles, while their loading capacity is just slightly higher (Donatan et al.,

2016).


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2.4.3 MgCO3 Particles

Magnesium carbonate has been widely used in various industrial applications

(pharmaceuticals, rubber industry, cosmetics, precursor for magnesium based chemicals,

etc). It includes magnesite (MgCO3), nesquehonite (MgCO3.3H2O), hydromagnesite

(Mg5(CO3)4(OH)2.4H2O), lansfordite (MgCO3.5H2O) and artinite (Mg5(CO3)4(OH)2.4H2O),

whereas magnesite is the most stable Mg-carbonate (Hänchen, Prigiobbe, Baciocchi, &

Mazzotti, 2008; Zhang et al., 2006).

However, it is impossible to form magnesite at ambient temperature. The minimum reported

temperature to form magnesite was 60-100°C and needs an elevated CO2 pressure

(Giammar, Bruant Jr, & Peters, 2005). When aqueous solutions are used at a temperature of

25°C and a moderate CO2 pressure close to ambient pressure, nesquehonite is formed

(Zhang et al., 2006). If the temperature is higher (approximately 40°C), mostly

hydromagnesite is formed (Hänchen et al., 2008). Magnesite can also be formed from a

transition of hydromagnesite. At a temperature below 150°C and a low pressure, this

transition can take days. However, it can be accelerated by using a higher temperature, high

CO2 pressure and introducing a high salinity (Hänchen et al., 2008).

Shapes of MgCO3

Zhang et al created various morphologies of magnesium carbonate depending on the

reaction characteristics (time, pH, etc), Figure 4. At a temperature lower than 328 K and a

low pH, needle-like MgCO3.xH2O (whereas x depends on the reaction characteristics) is

formed, Figure 4 (b). At higher temperatures (higher than 333 K) and a high pH, spherical

Mg5(CO3)4(OH)2.4H2O can be synthesized. (Zhang et al., 2007)

Spherical-like particles with a diameter of 15-17 µm can be synthesized by mixing two

aqueous solutions (Mg(NO3)2 and K2CO3) for 0.5 min at 353 K (Zhang et al., 2007).

One important parameter in this reaction is time. Zhang et al have shown that at a low time

the particles form agglomerates built of fine grains. If time increases, a serpentine strip

occurs and if time increases further, spherical-like particles are produced, Figure 4 (d). Two

other important parameters are temperature and stirring time. The particles have a

morphology ranging from spherical to nest-like and a decrease in diameter, with an

increasing temperature. An increasing stirring time decreases the particles size, and the

morphology changes from spherical-like to nest-like and further from nest-like to cake-like

and dumbbell-like (Zhang et al., 2007).


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Figure 4: SEM images of the particles from various reaction conditions: (a) stirring time of 2 min at 313 K for 0

min, (b) stiring time of 4 min at 313 K for 20 min, (c) the particles by adding the ones from into 100 mL of double deionized water with a temperature of 353 K for 15 min, (d) for 25 min, (e) large magnification of the red ellipse in panel d and (f) large magnification of the yellow ellipse in panel d (Zhang et al., 2007).


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2.5 Particles for gelation

Particles can be used to induce gelation of hydrogels. Carbonate particles have been

reported to induce gelation of different types of hydrogels. Pectin hydrogels prepared with

carbonate particles cause gelation when acidification is introduced. Baldursdóttir & Kjøniksen

have synthesized (Baldursdóttir & Kjøniksen, 2005) these gels using D-glucono-δ-lactone

(GDL) for acidification. Moreira et al used NaHCO3 instead of GDL for acidification (Moreira

et al., 2014).

Carbonate particles are also used to synthesize alginate hydrogels. Kuo & Ma reported the

preparation of alginate hydrogels, using both CaCO3-GDL and CaCO3-GDL-CaSO4 systems

(Kuo & Ma, 2001).

Gellan gum gelation can be introduced using microparticles and bioglasses. TE Douglas et al

have reported the synthesis of GG using magnesium/calcium carbonates and calcium and

magnesium carbonates (TE Douglas et al., 2016). Bioglasses consisting of oxides (SiO2,

CaO, Na2O and P2O5) in several proportions can induce GG gelation due to the slow release

of divalent ions (T. E. Douglas, Piwowarczyk, et al., 2014; Gorodzha et al., 2016). Grasdalen

& Smidsrød studied the effect of the type of cation and ionic strength on gelation of GG

(Grasdalen & Smidsrød, 1987). They found that for divalent cations the gel strength

increased in following order:

Mg2+, Ca2+, Sr2+, Ba2+ < Zn2+ < Cu2+ < Pb2+

During this work, GG gelation will be introduced due to addition of calcium,

calcium/magnesium and magnesium carbonate particles.


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3 Goals

3.1 Characterization of micro- and nanoparticles

The goal of the first part is to design new particles consisting of calcium/magnesium

carbonate particles by varying reaction parameters (reaction time, addition of organic solvent

and Ca2+/Mg2+ ratio). The influence of these parameters will be evaluated regarding the size,

shape, porosity and crystallinity of the particles. The size and shape of the particles will be

evaluated using electron microscopy. Whereas the crystallinity will be evaluated using X-ray

diffraction and Fourier transform infrared spectroscopy.

3.2 Identification of hydrogel properties

In the second part, the influence of size, shape, crystallinity and concentration of the particles

will be evaluated regarding hydrogel gelation. First, the influence of the type of particle and

particle concentration will be studied regarding the time of hydrogel gelation and hydrogel

strength. In the final steps, the influence of particle distribution in the hydrogel on cell viability

will be evaluated using microCT and cell viability tests.


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4 Materials and Methods

4.1 Materials

Sodium carbonate (Na2CO3), calcium chloride (CaCl2), magnesium chloride (MgCl2), calcium

carbonate (CaCO3), Gellan gum (Gelzan™ CM) and ethylene glycol are all purchased from

Sigma-Aldrich without any further purification. Ethanol is purchased from Chem-Lab.

4.2 Methods

4.2.1 Synthesis of the particles

Synthesis of big (Mg-Ca)CO3 particles

Following solutions are used to synthesize the particles (Table 2):

Table 2: Overview of the different solutions used to synthesize big (Ca/Mg)CO3 particles.

Solution CaCl2 (mol/L) MgCl2 (mol/L)

1 0,333 0,000

2 0,248 0,083

3 0,165 0,165

4 0,083 0,248

5 0,000 0,333

Big particles are synthesized by mixing 2 mL of 0,33 M Na2CO3 with 2 mL of one of the

solutions from Table 2. The mixture is mixed using a magnetic stirrer at 750x during 1 minute

at room temperature. Afterwards the mixture is poured in 2 mL reaction tubes and

centrifuged for 5 minutes at 3000 RPM. The supernatant is removed and the pellet is washed

twice with ethanol (70%) and centrifuged (3000 RPM, 5 min.) after washing. The samples are

dried for 10 hours at 70°C for storage.

Later during this work, particles synthesized using solution 1-5 will be referred to as resp.

sample 1-5.

Synthesis of small (Mg-Ca)CO3 particles

Table 3: Overview of the solutions used for the synthesis of small (Ca/Mg)CO3 particles.

Solution CaCl2 (mol/L) MgCl2 (mol/L) Ethylene Glycol (mL)

1 0,333 0,000 10,0

2 0,248 0,083 10,0

3 0,165 0,165 10,0

4 0,083 0,248 10,0

5 0,000 0,333 10,0

To synthesize small particles, 2 mL Na2CO3 + 10 mL ethylene glycol are added to one of the

solutions from Table 3. The mixture is mixed using a magnetic stirrer at 750x during 3 hours.

After mixing, the solution is poured into tubes of 50 mL and centrifuged for 5 minutes at 3000

RPM. After centrifugation, the supernatant is removed and the pellet will be washed twice


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with ethanol (70%) like the big particles. Before storage the samples are dried for 10 hours at

70°C.

Later during this work, particles synthesized using solution 1-5 will be referred to as resp.

sample 1-5.

4.2.2 Characterization of the particles

Scanning electron microscopy (SEM)

The size and shape of the synthesized particles are evaluated by SEM. A

MIRA/TESCAN instrument is used for this purpose. The acceleration voltage is 30 kV and

the magnification ranges from 2-200 kx. Before analysis, a drop of 10 µL is put on the silica

plate and dried under vacuum.

X-Ray Diffraction (XRD)

Powder x-ray diffraction analysis of the polycrystalline samples was performed with a Rigaku

Miniflex-600 diffract meter (Rigaku Corporation, Tokyo, Japan). The XRD data were recorded

using Cu-Kα radiation (40 kV, 15 mA, Ni-Kβ filter) in the 2θ range 10–80° at a scan speed

1°/min. The crystalline phases were identified with the use of integrated X-ray powder

diffraction software (PDXL: Rigaku Diffraction Software) and ICDD PDF-2 datasets (Release

2014 RDB).

FTIR

A FTIR spectrometer from Bruker (VERTEX 70) in reflection mode is used to obtain infrared

spectra from the different types of particles. The spectra are recorded in a range of 150-4200

cm-1. The spectrum of each sample is measured 15 times and the average spectrum is

calculated.

ANOVA Analysis

ANOVA (Analysis of variances) is an analysis tool to test whether there are statistically

differences between the means of two groups in an experiment. This analysis is performed

with SPSS Statistics which is a software package for statistical analysis.

4.2.3 Synthesis of Gellan gum

Gellan gum was dissolved in double-distilled water at a concentration of 0.875 % (w/v). The

solution was autoclaved for 15 minutes at 121 C.

4.2.4 Preparation of the hydrogels

Hydrogels prepared during this work contain resp. 2%, 4% and 30% of the particles. The

preparation is carried out in 2 mL reaction tube (Greiner Bio-One). 300 µL of distilled water is

added to the particles. The particles are dispersed using an ultrasonic bath (Sonorex Super


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10P, Bandelin) for 30 seconds at room temperature. Afterwards 500 µL of Gellan gum is

added and mixed with the particles by shaking the reaction tubes.

4.2.5 Investigation of the hydrogel properties

Rheological properties

A rheometer (AR-1000 N, TA Instruments) is used to study the visco-elastic properties of the

hydrogels. The rheometer consist of a stainless steel plate and an acrylic cone of 40 mm.

The storage (G’) and loss modulus (G’’) are recorded at 22°C with a frequency of 1 Hz and

an angular frequency of 6,283 rad/s.

FTIR

A FTIR spectrometer from Bruker (VERTEX 70) in reflection mode is used to obtain infrared

spectra from the different types of particles. The spectra are recorded in a range of 150-4200

cm-1. Before measuring the spectra, the hydrogels are dried on a heater at 65°C for 30

minutes. The spectrum of each sample is measured 15 times and the average spectrum is

calculated.

ANOVA Analysis

ANOVA (Analysis of variances) is an analysis tool to test whether there are statistically

differences between the means of two groups in an experiment. This analysis is performed

with SPSS Statistics which is a software package for statistical analysis.


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5 Results and Discussion In the first part, the newly synthesized particles are characterized. On the one hand, a

comparison of the particles is performed between samples containing particles prepared in

different reaction conditions (reaction time and absence or presence of an organic solvent)

but with the same Ca2+:Mg2+ ratio. On the other hand, samples containing particles with a

different Ca2+:Mg2+ ratio but prepared in the same reaction conditions (reaction time and

absence or presence of an organic solvent) are compared. First, the influence of the

Ca2+:Mg2+ ratio and reaction conditions (reaction time and absence or presence of an organic

solvent) on the size and shape of the particles is investigated by taking SEM images of the

particles. Next, the crystallinity of the synthesized particles is studied using X-ray diffraction

and Fourier transform infrared spectroscopy.

In the second part, hydrogels are prepared with the designed particles. Gelation time is

investigated for the different samples with varying particle concentrations. This test is

conducted to study the influence of size and shape of the particles and particle concentration

on the gelation time. Also gel stiffness is measured by rheometry tests. During these tests,

influence type of particle and particle concentration is investigated.


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5.1 Characterization of the particles

5.1.1 Yield of the Particles

After each synthesis, the reaction yield of the particles is determined. Figure 5 gives an

overview of the average reaction yield and standard deviation of respectively big and small

particles (Attachment 1, Table 1). Statistical analysis is done using SPSS Statistics 24 to

compare the reaction yield of each sample within and between the group of small and big

particles.

Figure 5: Overview of average yield and their standard deviation of the particles.

One-way ANOVA is performed to compare yield within both groups with a significant

difference for p<0,05. Analysis shows that in the group of big particles, Ca2+:Mg2+ ratio has

no influence on the reaction yield and thus on the ion distribution. In the other group,

according to ANOVA analysis (Attachment 1, Table 5) there is a difference between the

samples in this group (p<0,05). However, if a post-hoc analysis is done (Attachment 1, Table

6) no difference can be noticed (p>0,05). This can be explained due to the fact that with

ANOVA the significance is slightly smaller (p=0,049) than p=0,05. So also in this group, there

is no influence of Ca2+:Mg2+ ratio on the ion distribution in these reactions.


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Also, comparison of reaction yield between samples from both groups is done. This analysis

(ANOVA) shows that no difference can be determined between the same samples in both

groups. This shows that the reaction yield is independent of reaction time, Ca2+:Mg2+ and

addition of organic solvent.

The reaction yield from sample one in the group of big particles (59,59 %) is lower than the

reaction yield from the particles synthesized by Y. I. Svenskaya et al (74 % with a magnetic

stirrer and 85,9% with ultrasonic agitation) (Y. I. Svenskaya et al., 2016).


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5.1.2 SEM Images

Sample Big Particles Small Particles

1

2

3


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4

5

Figure 6: SEM images of particles, in each image, the scale bar is represented. Different magnifications are

used, 2000x for sample 5, small particles; 5000x for sample3, 4 and 5, big particles; 10.000x for sample 1 and 4, small particles and sample 1, big particles; 20.000x for sample 2, big and small particles.

Size and shape of the particles are determined based on images in Figure 6 and additional

images (Attachment 2). ImageJ is used to determine size and standard deviation of the

different types of particles (Table 5), for this purpose size of 100 particles of each different

type in each sample is determined.

Comparison between the groups of big and small particles shows that in sample 1 spherical

vaterite particles occur in both groups whereas in sample 1 in the first group also cubical

calcite particles are present. In sample 2 in the group of big particles, spherical particles

appear and in the other group elliptical particles. Sample 3 contains spherical vaterite

particles (in the group of small particles) and star-forming and two-connected to each other

particles, which we will refer to throughout this work as paired (Figure 6) particles in the

group of big particles. The former two types of particles are aggregates formed by vaterite,

whereas the paired particles seem to exist out of two spherical particles and the star-forming

particles out of multiple vaterite particles forming several layers (Attachment 2, Figure 4).


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Samples 4 and 5 consist of an amorphous phase for both big and small particles. However,

in sample 4 in the group of small particles, star-like particles are detected in the amorphous

phase suggesting the presence of vaterite.

This shows that with an increasing Ca2+:Mg2+ ratio, the morphology of the particles changes.

However, if the ratio is increased above 1:1, than amorphous particles are prepared.

Table 5 summarizes size and standard deviation of the different types of particles. For

spherical particles, diameter of the particles is measured. For the elliptical and paired

particles, the axial and radial sides are determined. For the group of big particles, sizes are in

the range of 1,6 – 8,2 µm. Whereas for the other group, sizes are in a lower range: 0,6 – 2,5

µm. Addition of organic solvent and an increasing reaction time decreases particle size.

Table 5: Summary of shape and size of the different particles.

Big Particles Small Particles

Size (µm) SD (µm)

Size (µm) SD (µm)

Sample 1 Sample 1

Big Spherical 3,3 ± 0,4 Big Spherical 1,3 ± 0,2

Small Spherical 1,6 ± 0,3 Small Spherical 0,6 ± 0,1

Sample 2 Sample 2

Spherical 2,7 ± 0,5 Axial Side 1,7 ± 0,3

Radial Side 2,5 ± 0,4

Sample 3 Sample 3

Paired Particles 8,2 ± 3,2 ± 21,3 ± 0,4 Spherical 1,3 ± 0,3

Star-Forming 7,1 ± 3,2 ± 1,1 ± 0,5

Sample 4 Sample 4

Amorphous - - Amorphous - -

Sample 5 Sample 5

Amorphous - - Amorphous - -


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5.1.3 XRD Data

Figure 7: XRD patterns for all samples in the group of big particles (A) and in the group of small particles (B).

Each color corresponds to a type of particle (see legend in the right top).

In the group of big particles, samples 1 and 2 show peaks at 2θ values 21,0; 25,0; 27;2; 32,8;

43,9; 49,1; 50,1 and 55,8; all corresponding to vaterite (Le Bail, Ouhenia, & Chateigner,

2011). Also calcite peaks occur in these samples at 29,5; 36,0; 39,5 and 47,7 (Sitepu, 2009).

This suggests that in both samples vaterite and calcite is present. In the SEM images

however, calcite particles (cubical) are only detected in sample 1. In sample 3 peaks occur at

27,2 and 32,8 suggesting the presence of vaterite and also at 36,0 a peak is shown,

corresponding to calcite. Next to these, peaks at 31,9 and 45,5 are present, which are

characteristic for NaCl (Strel’tsov, Tsirel’son, Ozerov, & Golovanov, 1987). Also at 15,3 and

38,3 peaks can be found. Both peaks are characteristic for hydromagnesite (Botha &

Strydom, 2003). However, in the SEM images no calcite is present in this sample. Samples 4

A

B


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and 5 show a very broad peak at approximately 26,0 suggesting the presence of an

amorphous phase. SEM images of these samples show an amorphous phase confirming the

XRD spectra. In sample 5 however, a peak at 15,3 suggests the presence of hydromagnesite

(Botha & Strydom, 2003). TE Douglas et al also found hydromagnesite in particles created

with varying Ca2+:Mg2+ ratio’s. However, this was indicated by a peak shift from 29,5 to 29,9

and shifts in the range of 2θ of 30-50. (TE Douglas et al., 2016)

In the group of small particles, samples 1 and 3 have a similar XRD spectrum, probably due

to a mix-up of the samples. Both spectra show peaks at 21,0; 25,0; 27,2; 32,8; 42,7; 43,9;

49,0; 50,1 and 55,8 all characteristic for vaterite (Le Bail et al., 2011). This suggests that only

vaterite is present in this sample. Regarding this result, the XRD spectrum probably refers to

sample 1, since in the group of big particles only calcite and vaterite peaks are present in

sample 1. Whereas in sample 3 also peaks of hydromagnesite occur in the group of big

particles. However, also the SEM images show similar particles in both samples. This is

probably also the result of a mix-up between the samples. In sample 2, peaks are shown at

values characteristic for calcite (Sitepu, 2009). This suggests that the particles in sample 2

recrystallized from vaterite to calcite before the XRD measurement. However these peaks

also show a shift of ± 0,4° in the range 2θ of 30-50. Also at 23,3 and 48,2 occur, suggesting

the presence of resp. hydromagnesite (Botha & Strydom, 2003) and magnesian calcite

(Paquette & Reeder, 1990). This is characteristic for the presence of magnesian calcite

(Alruopn, 1977). In the SEM images only elliptical particles are present, which are no

magnesian calcite since they have a rhombohedral structure. Samples 4 and 5 both show a

very broad peak at approximately 26,0 indicating an amorphous phase is present in these

samples. Also in the SEM images an amorphous is detected, confirming the XRD spectra.

This shows that hydromagnesite can be found in particles with a Ca2+:Mg2+ ratio of 1:1 if they

are synthesized with a low reaction time and without organic solvent. If an organic solvent is

added and reaction increased, magnesian calcite is found in particles with a Ca2+:Mg2+ ratio

of 3:1.


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5.1.4 FTIR Data

Figure 8: FTIR spectra of the different samples. Figure A represents the group of big particles and figure B the

group of small particles.

Figure 8 shows all FTIR spectra for the different samples. Table 6 gives an overview of all

bands in the samples.

-1

-0.5

0

0.5

1

1.5

2

200700120017002200

Re

lati

ve T

ran

smit

tan

ce (

%)

Wavelength (cm-1)

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Calcite

200700120017002200

Re

lati

ve T

ran

smit

tan

ce (

%)

Wavelenght (cm-1)

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

B

A


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Table 6: Summary of bands visible in each sample and the corresponding type of particle.

Big Particles

Sample Wavenumber (cm-1)

Calcite Vaterite Other

1 1400 1089 871 - - - - - - - - -

2 1400 - 871 712 1078 - 2359 2344 1105 860 668 610

3 1400 - 871 712 1078 - 2359 2344 1105 860 668 610

4 1400 - - - - - 2359 2344 1105 860 668 610

5 1400 - - - - - 2359 2344 1105 860 668 610

Small Particles

Sample Wavenumber (cm-1)

Calcite Vaterite Other

1 1400 - 871 - 1078 746 - - - - - -

2 1400 - 871 - 1078 - 2359 2344 1040 860 668 610

3 1400 - - - 1078 - 2359 2344 1040 860 668 610

4 1400 - - - 1078 - 2359 2344 1040 860 668 610

5 1400 - 871 - 1078 - 2359 2344 1040 860 668 610

Comparison between the samples in the group of big particles shows that in all samples

calcite is present, since all samples show a band at 1400 cm-1 (ʋ3 antisymmetric stretching)

(Andersen & Brecevic, 1991). Samples 1,2 and 3 also show a band characteristic for calcite

at 871 cm-1 (ʋ2 antisymmetric bending) (Andersen & Brecevic, 1991). However, additional

calcite bands occur. Only sample 1 shows a band at 1089 cm-1 (ʋ1 symmetric stretching),

while samples 2 and 3 have a band corresponding to calcite at 712 cm-1 (ʋ4 symmetric

bending) (Andersen & Brecevic, 1991). This corresponds to XRD data where calcite is found

in sample 1, 2 and 3. Whereas in samples 4 and 5 no calcite peaks are present due to the

amorphous phase. The intensity of band at 1400 cm-1 is similar in samples 2, 3 and 4 and

lower in samples 1 and 5. This indicates that the presence of calcite is lower in sample 1 and

5 than in samples 2, 3 and 4 who have more or less the same amount of calcite. According

to the FT-IR spectra, vaterite is only present in sample 2 and 3 according to a band at 1078

cm-1 (combination of ʋ4a and lattice mode 332) (Andersen & Brecevic, 1991). Since the

intensity of this band is approximately the same in both samples, this suggests that both

have the same amount of vaterite. Whereas according to XRD data, vaterite is present in

sample 1,2 and 3. All samples (except sample 1) show additional bands at 2359 ,2344, 860,

668 and 610 cm-1. The first two bands (2359 and 2344 cm-1) correspond to CO2 (Zhang et al.,

2006). The bands at 860 and 668 cm-1 are characteristic of ʋ2 bending and ʋ4 bending of

free CO32-. (Andersen & Brecevic, 1991; Kloprogge, Wharton, Hickey, & Frost, 2002) The last

bands, 1105 and 610

cm-1 refer to resp. ʋ1 mode (Zhang et al., 2006) and ʋ4 of CO32- in MgCO3 (Umbreit &

Jedrasiewicz, 2000; Zhang et al., 2006). The presence of MgCO3.3H2O (nesquehonite) in

sample 2,3,4 and 5 is confirmed by the presence of bands 1105, 860 and 610 cm-1 (Zhang et

al., 2006). This is in contrast with the results obtained from the XRD data where


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hydromagnesite is found in sample 3 and 5. The results differ from results obtained by TE

Douglas et al. Instead of nesquehonite, they found hydromagnesite, confirmed by bands at

1472, 1416 and 792 cm-1 (TE Douglas et al., 2016).

In the group of small particles, calcite is present in each sample. All samples show a band at

1400 cm-1. Next to this, in samples 1 and 3, a band occurs at 1089 cm-1. Also a third band

represents calcite, 871 cm-1, present in samples 2 and 5. Comparison of intensity of the

bands at 1400 cm-1 shows that sample 5 contains more calcite than the other four samples.

The band has a higher intensity in sample 3 than samples 2 and 4 (more or less same

intensity) and sample 1. This indicates that further, the amount of calcite decrease in

following order:

3 > 2 and 4 > 1. Whereas the intensity of the band at 871 cm-1 is higher in sample 1 than in

samples 2 and 5 (who have more or less the same intensity). These results are in contrast

with the XRD data where only in sample 2 calcite is found. All samples show a band at 1078

cm-1, corresponding to vaterite. Whereas only in sample 1, a band occurs at 746 cm-1 (ʋ4a

bending), characteristic of vaterite (Andersen & Brecevic, 1991). According to the intensity of

band 1078 cm-1 the amount of vaterite present in the different samples decreases from

5 > 4 and 3 > 2 > 1. In the XRD spectra however, vaterite is only detected in sample 1 (and

only 1 peak in sample 2). All samples (except sample 1) also show additional bands at 2359

and 2344 cm-1 referring to the presence of CO2. Further bands occurred at 1040, 860, 668

and 610 cm-1. Representing resp. ʋ1 mode of CO32-, ʋ2 bending and ʋ4 bending of free CO3

2-

and ʋ4 of CO32- in MgCO3. Presence of nesquehonite in samples 2,3,4 and 5 could be

proved by bands 860 and 668 cm-1, however in contrast to the group of big particles no band

is found at 1105 cm-1.


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5.2 Investigation of gel properties

5.2.1 Gelation time

First, gelation time is studied. This is done by preparing a hydrogel and measuring time when

Gellan gum is added to the particles until a solid hydrogel is formed. Pictures are taken

before and after gel formation for big and small particles (Attachment 3, Figure 1).

In sample 1 for both small and big particles, no gel formation is introduced. As a control, a

sample containing Gellan gum and 100% commercial CaCO3 is prepared. Also in this

sample, no hydrogel is formed. The other samples all have the ability to form a gel, however,

in sample 2, a higher particle concentration is necessary than that reported for the other

samples. According to TE Douglas et al this is due to a higher release of Mg and Ca ions (in

sample 1 < 2 < 3 < 4 < 5) to crosslink GG (TE Douglas et al., 2016). This is induced through

a higher solubility resulting from an increased amorphicity (in sample 2 < 3 < 4 < 5) of the

crystals.

Figure 9 shows graphics of gel formation with particle concentration in function of gelation

time. In Attachment 3, Table 1 and 2 the data are found for gelation time of the different

types of particles. Graphics show that within a group of particles (small or big particles),

gelation time decreases if particle concentration in the gel increases. If more particles are

available, more ions can be released to introduce gel formation. Gelation time also

decreases if there is more magnesium present in the sample (gelation time decreases in

sample 3 to sample 5 for an equal particle concentration).

Comparison between both groups shows that samples containing small particles induce

gelation much quicker than samples containing big particles. This is due to lower surface

area of small particles 5 (Table 7), therefore ions are more easily released than from big

particles.

Table 7: Summary of surface area of the different types of particles. For spherical particles, surface area is

calculated with A = 4 x π x r², for elliptical particles A = π x length axial side x length radial side, for paired and star-forming particles A = 2 x π x r² + 2 x π x r x h.


Sample 1 A (µm²) Sample 1 A (µm²)

Big Spherical 136,8 Big Spherical 21,2

Small Spherical 32,2 Small Spherical 4,5

Sample 2 Sample 2

Spherical 91,6 Elliptical 13,4

Sample 3 Sample 3

Paired Particles 98,5 Spherical 21,2

Star-Forming 87,4


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Figure 9: Graphics corresponding to time of gel formation, whereas figure A corresponds to big particles and

figure B to small particles. The axis on the left represents the particle concentration in the gel for sample 3-5 and the axis on the right represents the particle concentration in the gel for sample 2 (Figure 9, A). In Figure 9, B ,the axis on the left represents the particle concentration in the gel for sample 2 and the axis on the right represents the particle concentration in the gel for sample 3-5. Whereas the axis on the top represents the time of gelation for sample 3-5 and the axis on the bottom represents the gelation time for sample 2.

15

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5.2.2 Rheometry results

Figure 10 shows average storage modulus as a function of average time. Both are

determined out of three measurements of each sample. Next to the average, standard

deviation is calculated for storage modulus and time (Attachment 4, Table 1 and Table 2).

Figure 10: Overview of average storage modulus measured in each hydrogel for big (A) and small (B) particles.

For each sample, standard deviation is shown for a single point.

First, within both groups, ANOVA analysis is done with a significant difference for p<0,05 to

compare hydrogel strength for the different types of hydrogels. In the group of big particles,

hydrogel strength increases from 5 2% < 4 4% < 5 4% < 3 4% < 2 30% (the first number

represents the sample of particles used and the second one the concentration of particles in

the hydrogel). According to ANOVA analysis (Attachment 4, Table 3), only between

0

1000

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3000

4000

5000

6000

7000

0 200 400 600 800 1,000 1,200

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rag

e M

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ulu

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a)

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Sample 5 2%

Sample 3 4%

Sample 4 4%

Sample 5 4%

A

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5000

6000

0 200 400 600 800 1000 1200

Sto

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Sample 4 2%

Sample 5 2%

Sample 3 4%

Sample 4 4%

Sample 5 4%

B


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hydrogels 5 4% and 3 4% no significant difference is detected. This suggests that if the

particle concentration is increased from 2 to 4%, the hydrogel strength increases

significantly. In the group of small particles, hydrogel strength increases from

4 2% < 5 2% < 4 4% < 2 30% < 3 4% < 5 4%. ANOVA analysis shows that no significant

difference is detected between hydrogels 5 2%,4 4%, 2 30%, 3 4%. This confirms that if

particle concentration is increased (from 2 to 4%) the hydrogel strength increases

significantly. These results also show that Ca2+:Mg2+ ratio has no influence on gel strength. In

the group of big particles hydrogel strength increases 4 < 5 < 3 for samples with the same

amount of particles and in the other group from 4 < 3 < 5.

Hydrogels containing similar samples of particles and particle concentration from both groups

are compared using independent T-tests with a significant difference for p<0,05. This

analysis shows that significant differences between hydrogels containing small and big

particles are present. Since hydrogel strength is stronger for small particles in hydrogels

consisting of resp. particles from samples 5 containing 4 and 2% of the particles in the

hydrogel and particles from sample 4 containing 4% particles in the hydrogel. Whereas the

hydrogel strength is stronger for big particles in hydrogels consisting of resp. particles from

samples containing 30% of the particles in the hydrogel and particles from sample 3

containing 4% of the particles in the hydrogel. These results indicate that particle size (and

thus surface area) have no influence on hydrogel strength.


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5.2.3 FTIR Data

Figure 21: FTIR spectra for the hydrogels. The spectra of the hydrogels prepared with big particles are

represented in Figure 11, A and those of the hydrogels prepared with small particles in Figure 11, B.

Figure 11 shows FTIR spectra obtained for hydrogels containing 4 and 30% particles

prepared in point 5.2.1. In the group of big particles (A), all hydrogel samples show two

bands at 2359 and 2344 cm-1 both characteristic with CO2 (Zhang et al., 2006). They also

show in the range of 1900 to 1500 cm-1 series of bands with two bands at 1652 (H-O-H

bending of water molecules) (Andersen & Brecevic, 1991) and 1558 cm-1 (O-C-O vibration)

(Li et al., 1989) having a higher intensity then the rest. Further, all samples also have three

similar bands at 871, 860 and 668 and 227 cm-1. The first band is a characteristic of ʋ2

antisymmetric bending of calcite, the second and third are characteristic of ʋ2 bending and

ʋ4 bending of free CO32- (Andersen & Brecevic, 1991) and the last band is a characteristic of

-2.2

-1.7

-1.2

-0.7

-0.2

0.3

0.8

1.3

1.8

2.3

2007001200170022002700

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lati

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nte

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ty

Wavenumber (cm-1)

Sample 2 30%

Sample 3 4%

Sample 4 4%

Sample 5 4%

GG

-0.2

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%)

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Sample 2 30%

Sample 3 4%

Sample 4 4%

Sample 5 4%

B

A


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calcite (Andersen & Brecevic, 1991). However, there are also differences between the

samples. The hydrogels prepared with particles from sample 2 and 3 both show a band at

1400 and 610 cm-1. The first band can corresponds to ʋ3 antisymmetric stretching of calcite

(Andersen & Brecevic, 1991). However, this band can is also a characteristic of Gellan gum.

The band at 610 cm-1 corresponds to ʋ4 of CO32- in MgCO3 (Umbreit & Jedrasiewicz, 2000).

The hydrogel containing particles from sample 2, also shows a band at 712 cm-1 representing

ʋ4 symmetric bending of calcite (Andersen & Brecevic, 1991). The spectrum from the

hydrogel containing particles from sample 3 shows two bands at 1067 and 1030 cm-1

corresponding to amorphous calcium carbonate (Andersen & Brecevic, 1991). In the last

spectrum (hydrogel prepared with particles from sample 5), bands are noticed at 1472 and

1416 cm-1 corresponding to ʋ3 antisymmetric stretching of carbonate groups. At 1125, 875

and 792 cm-1 three bands are present, which are characteristics of resp. ʋ1 and ʋ2 mode

CO32- (Umbreit & Jedrasiewicz, 2000) and of ʋ2 symmetric bending of carbonate groups. The

bands at 1472 and 1416 and 792 cm-1 suggest the presence of magnesium carbonate

hydromagnesite (Frost, 2011; White, 1971).

In the group of small particles, all hydrogel samples show bands at 2359 and 2344 cm-1

which are characteristic of CO2. Also series of bands are present in the range of 1900-1500

cm-1, with bands at 1652 (H-O-H bending of water molecules) (Andersen & Brecevic, 1991)

and 1558 cm-1 (O-C-O vibration) (Li et al., 1989) having the highest intensity. In this serie

also a band at 1600 cm-1 which is characteristic of Gellan gum. All samples also show bands

at 1030 corresponding with amorphous calcium carbonate (Dupuis, Ducloux, Butel, & Nahon,

1984). They also show a band at 668 cm-1 (ʋ2 antisymmetric bending of calcite). Further,

shows the spectrum from hydrogel containing particles from sample 2 bands at 1080, 871

and 860 cm-1. The two last bands correspond to ʋ2 antisymmetric bending of calcite and ʋ2

bending of free CO32-. The spectrum of the hydrogel with particles from sample 3 contains

bands at 1067, 1030 and 871 cm-1. These bands are characteristics of resp. amorphous

calcium carbonate and ʋ2 bending of free CO32- (Andersen & Brecevic, 1991). In the spectra

from the hydrogels containing particles from samples 4 and 5, a broad band is detected in

the range of 1200-900 cm-1 with a band at approximately 1030 cm-1 (amorphous calcium

carbonate) and shoulders at 1148, 1070 (amorphous carbonate) and 990 cm-1 (bicarbonate

ion (Zhang et al., 2006)). These hydrogels also show a band at 850 cm-1 characteristic for ʋ2

mode of CO32- (Zhang et al., 2006). In the spectrum of the hydrogel containing particles from

sample 5 an additional band is detected at 880 cm-1 corresponding to carbonate bending.

Since next to this band, also a band at 850 cm-1 is present, this suggests the presence of

hydromagnesite (Zhang et al., 2006).


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6 Conclusion

The aim of this work was to design a new type of calcium/magnesium carbonate particles

with characteristics suitable to create in-situ forming hydrogel composites with GG. Two main

groups of particles were created by varying the reaction time and adding an organic solvent.

Different types of particles were created by varying the Ca2+:Mg2+ ratio. The size, shape, and

crystallinity of these particles were studied using SEM, XRD and FTIR. Results show that

different types of particles are synthesized by decreasing the Ca2+:Mg2+ ratio: spherical,

elliptical, star-forming and paired particles. However, if the ratio is lower than 1:1, amorphous

particles are formed. Size can be decreased 3x by adding an organic solvent and increasing

the reaction time. All samples comprise of vaterite and/or calcite particles, independent of the

reaction time, addition of organic solvent and Ca2+:Mg2+ ratio. However, hydromagnesite

particles are formed at a low reaction time (minutes), in the absence of an organic solvent,

and at a Ca2+:Mg2+ ratio of 1:1. Hydromagnesite particles are formed at a higher Ca2+:Mg2+

ratio, if the reaction time increases (to hours) and an organic solvent is added.

Next, hydrogel formation stimulated by the presence of the described above particles were

studied. The particles containing only calcium are not capable to cross-link GG due to a slow

release of Ca2+ ions, while particles containing either magnesium or both magnesium and

calcium are capable to cross-link GG. Our results show that gelation time increases if either

particle concentration increases or Ca2+:Mg2+ ratio decreases. Rheometry results showed

that the mechanical stiffness of the hydrogel increases with increasing particle concentration,

whereas at a specified particle concentration the mechanical stiffness increases with

decreasing the Ca2+:Mg2+ ratio. The influence of crystallographic structure of the particles

(vaterite, calcite, hydromagnesite, magnesian calcite, and amorphous) was studied for

hydrogels containing a specific type and concentration of particles: a) 4% particles with a

Mg2+ percentage of 50%, 66,6, and 100%, and b) 30% of particles with a Mg2+ percentage of

33,3 %. FTIR spectra showed the presence of calcite, but not vaterite, in all hydrogels.

However, only in the hydrogel consisting of 4 % particles with 100% magnesium,

hydromagnesite particles are found.

As a result of this work, micro-particles prepared with a Ca2+:Mg2+ ratio of 2:1 are preferred

as drug delivery system. These particles contain a higher particle concentration than

particles with a lower Ca2+:Mg2+ ratio, without changing gelation time significantly. The high

particle concentration is beneficial since more particles can be loaded than in hydrogels

prepared with less particles with a lower Ca2+:Mg2+ ratio having a comparable gelation time

and lower gel stifness. Since micro-particles induce gelation slower than nano-particles, they

are the preferred system to create injectable in-situ hydrogels. Another benefit is that they

have more preferable reaction conditions (low reaction time and no organic solvent). Future

research should focus on the optimization of drug delivery systems with micro-particles

having a Ca2+:Mg2+ ratio of 2:1. However, more characterizations should be done. First,

microCT and cell viability tests should be performed to study the particle aggregates and the


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influence of hydrogels on cells. Further, the hydrogel properties have to be studied under

physiological conditions for their application as drug delivery system. Also influence the

loading capacity and enzyme activity have to studied.

51

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56

Attachments

Attachment 1: Statistics of yield

Overview of average yield and standard deviation per sample

Table 1: Overview of average yield and standard deviation for the synthesis of different samples.


Sample Number Yield (%) SD Yield (%) SD

1 59,59 ± 9,56 48,55 ± 9,59

2 54,21 ± 13,90 59,49 ± 11,00

3 71,91 ± 16,70 47,95 ± 8,90

4 60,32 ± 13,11 62,07 ± 20,56

5 51,81 ± 10,87 54,89 ± 18,01

Comparison between big and small particles

Table 2: Normality test for average yield of each sample. Sample number represents the number and resp.

Ca:Mg Ratio in the sample. Part represents the type of particle, number 1: small particles and number 2: big particles. If the Significance is higher than 0,05; the yield distribution is normal. Here sample has a normal distribution of the yield, thus an ANOVA analysis is done to compare the samples with each other.

Tests of Normality

sample part

Kolmogorov-Smirnova Shapiro-Wilk

Statistic df Sig. Statistic df Sig.

1,00 yield 1,00 ,159 7 ,200* ,951 7 ,736

2,00 ,256 6 ,200* ,903 6 ,394

2,00 yield 1,00 ,133 31 ,173 ,967 31 ,446

2,00 ,181 9 ,200* ,914 9 ,342

3,00 yield 1,00 ,151 11 ,200* ,971 11 ,899

2,00 ,303 6 ,090 ,832 6 ,111

4,00 yield 1,00 ,157 10 ,200* ,931 10 ,453

2,00 ,196 8 ,200* ,934 8 ,556

5,00 yield 1,00 ,220 10 ,186 ,901 10 ,227

2,00 ,139 8 ,200* ,980 8 ,963

*. This is a lower bound of the true significance.

a. Lilliefors Significance Correction

57

Table 3: ANOVA test to compare the yield of the reactions between samples of big and small particles.

ANOVA

yield

sample Sum of Squares df Mean Square F Sig.

1,00 Between Groups 394,196 1 394,196 4,299 ,062

Within Groups 1008,677 11 91,698

Total 1402,873 12

2,00 Between Groups 193,924 1 193,924 1,424 ,240

Within Groups 5176,639 38 136,227

Total 5370,564 39

3,00 Between Groups 2229,408 1 2229,408 15,293 ,001

Within Groups 2186,712 15 145,781

Total 4416,120 16

4,00 Between Groups 13,642 1 13,642 ,044 ,837

Within Groups 5007,006 16 312,938

Total 5020,648 17

5,00 Between Groups 42,244 1 42,244 ,180 ,677

Within Groups 3747,499 16 234,219

Total 3789,743 17

Comparison within groups

Table 4: Homogeity test shows that the variances witihin both groups are the same.

Test of Homogeneity of Variances

yield

part

Levene

Statistic df1 df2 Sig.

Small

Particles

2,473 4 64 ,053

Big

Particles

1,263 4 32 ,305

58

Table 5: ANOVA test to compare yield of each sample within the group of big and small particles. Analysis shows

that there is a difference between samples in the group of small particles (since Sig. <0,05). However, post-hoc analysis shows that there is no difference (Table 6, Attachment 1).

ANOVA

yield

part Sum of Squares df Mean Square F Sig.

Small

Particles

Between Groups 1853,882 4 463,471 2,536 ,049

Within Groups 11698,298 64 182,786

Total 13552,180 68

Big

Particles

Between Groups 1635,341 4 408,835 2,410 ,070

Within Groups 5428,235 32 169,632

Total 7063,577 36

Table 6: Post-hoc tests to compare mean yield of each sample within the group of small particles. The test shows

that there is no difference between samples (Sig. >0,05) despite having a Sig. <0,05 for the ANOVA analysis.

Multiple Comparisons

Dependent Variable: yield

part (I) sample (J) sample

Mean

Difference (I-

J) Std. Error Sig.

95% Confidence Interval

Lower

Bound

Upper

Bound

1,00 Tukey

HSD

1,00 2,00 -10,93267 5,65761 ,311 -26,8140 4,9486

3,00 ,59571 6,53676 1,000 -17,7534 18,9448

4,00 -13,52629 6,66265 ,264 -32,2288 5,1762

5,00 -6,34229 6,66265 ,875 -25,0448 12,3602

2,00 1,00 10,93267 5,65761 ,311 -4,9486 26,8140

3,00 11,52839 4,74481 ,121 -1,7906 24,8474

4,00 -2,59361 4,91680 ,984 -16,3954 11,2082

5,00 4,59039 4,91680 ,883 -9,2114 18,3922

3,00 1,00 -,59571 6,53676 1,000 -18,9448 17,7534

2,00 -11,52839 4,74481 ,121 -24,8474 1,7906

4,00 -14,12200 5,90724 ,131 -30,7040 2,4600

5,00 -6,93800 5,90724 ,766 -23,5200 9,6440

4,00 1,00 13,52629 6,66265 ,264 -5,1762 32,2288

2,00 2,59361 4,91680 ,984 -11,2082 16,3954

3,00 14,12200 5,90724 ,131 -2,4600 30,7040

5,00 7,18400 6,04625 ,758 -9,7882 24,1562

5,00 1,00 6,34229 6,66265 ,875 -12,3602 25,0448

2,00 -4,59039 4,91680 ,883 -18,3922 9,2114

3,00 6,93800 5,90724 ,766 -9,6440 23,5200

4,00 -7,18400 6,04625 ,758 -24,1562 9,7882

59

60

Attachment 2: SEM Images

Big Particles

Magnification: 10.000x Magnification: 20.000x

Magnification:100.000x

Figure 3: SEM Images of sample 1, with magnifications of 10.000x, 20.000x and 100.000x.

61


Magnification: 50.000x Magnification: 100.000 x

Figure 4: SEM Images of sample 2, with magnifications of 5.000x, 20.000x, 50.000x and 100.000x.

62


Magnification: 20.000x


63

Magnification:1.000x Magnification: 5.000x



64


Figure 7: SEM Images of sample 5, with magnifications of 2.000x and 10.000x.

65

Small Particles




66




67




68




69


Figure 12: SEM Images of sample 5, with magnifications of 5.00x and 10.000x.

70

Attachment 3: Gel formation

Before Gelation After Gelation

Big Particles

Small Particles

Figure 1: Images of the different samples particles before and after gelation.

Table 1: Overview of gelation for the different types of nano-particles, with a varying concentration.

Sample 2 Sample 3 Sample 4 Sample 5

Concentration

(%)

Time

(s)

Concentration

(%)

Time

(s)

Concentration

(%)

Time

(s)

Concentration

(%)

Time

(s)

30 1 4 1 4 1 4 1

25 3 2 1 2 1 2 1

20 10 0,2 5 0,2 5 0,2 5

0,1 0,1 70 0,1 40

Table 2: Overview of gelation for the different types of micro-particles, with a varying concentration.

Sample 2 Sample 3 Sample 4 Sample 5

Concentratio

n (%)

Time

(sec.)

Concentratio

n (%)

Time

(sec.)

Concentratio

n (%)

Time

(sec.)

Concentratio

n (%)

Time

(sec.)

30 1320 0,5 4620 0,1 3900 0,1 2340

25 3600 1 459 0,5 780 0,5 150

20 5700 2 140 1 245 1 113

16 10800 4 50 2 120 2 100

4 40 4 30

71

Attachment 4: Rheometry Data

Small Particles

Table 3: Overview of average Storage Modulus and average timre and standard deviation for each sample in the group of small particles.

S3 4% S4 4% S5 4% S5 2% S4 2% S2 30%

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD

20,44 0,09 2396 364,87 24,03 6,11 2408,67 188,33 41,55 0,21 3920,00 1079,88 34,48 6,04 2217,67 323,01 41,34 0,23 1165,33 68,01 31,14 0,20 3073,67 247,08

74,55 0,12 2506 365,57 74,56 0,07 2486,33 190,69 85,10 0,19 4152,33 1153,62 74,62 0,08 2294,33 317,86 85,01 0,14 1227,33 69,53 64,11 0,09 3077,67 234,89

134,50 0,12 2596 366,28 134,48 0,08 2552,67 191,47 141,55 6,16 4390,00 1234,80 134,50 0,06 2377,33 327,47 137,95 6,04 1286,33 68,01 127,53 6,01 3058,67 209,69

194,56 0,04 2670 366,99 194,52 0,14 2602,67 189,49 194,59 0,14 4570,67 1287,03 190,99 5,96 2447,00 334,83 194,54 0,18 1336,67 74,66 191,01 6,07 3025,67 198,11

254,47 0,12 2733 367,70 254,56 0,08 2647,33 191,27 254,62 0,01 4736,00 1343,17 251,02 6,03 2506,67 346,28 254,47 0,13 1384,33 75,70 251,14 6,07 2995,00 187,65

314,56 0,03 2790 368,40 314,55 0,09 2686,00 189,86 314,54 0,11 4889,00 1392,82 310,97 6,00 2557,67 357,34 314,49 0,16 1427,67 76,22 311,09 6,05 2963,33 183,75

374,44 0,11 2835 367,70 374,59 0,02 2716,67 192,79 374,60 0,06 5018,67 1433,37 370,95 5,98 2593,67 368,92 374,48 0,11 1464,00 76,74 371,03 5,99 2930,67 181,78

434,61 0,02 2879 364,87 434,61 0,08 2746,00 192,49 434,62 0,06 5128,67 1476,99 431,00 5,96 2639,67 384,59 434,45 0,09 1496,67 79,36 431,02 6,09 2898,00 182,20

494,50 0,21 2920 361,33 494,53 0,03 2771,00 193,46 494,61 0,03 5228,00 1509,03 491,03 6,03 2671,33 384,23 494,50 0,11 1527,00 79,88 491,10 6,10 2868,33 180,34

554,54 0,10 2959 359,92 551,07 6,04 2792,00 189,99 554,59 0,08 5322,67 1538,97 550,97 5,99 2704,67 393,68 554,49 0,06 1554,67 80,93 551,08 6,11 2839,33 183,91

614,56 0,14 2994 356,38 614,50 0,05 2814,67 192,26 614,59 0,02 5403,00 1567,11 610,96 6,04 2740,67 409,71 614,51 0,08 1581,00 81,46 614,51 0,01 2772,33 146,66

674,51 0,12 3023 349,31 671,08 6,02 2832,33 191,84 674,59 0,04 5481,00 1592,59 671,01 5,95 2762,33 405,94 674,46 0,10 1605,00 83,05 674,46 0,04 2753,33 159,79

734,48 0,00 3054 347,19 734,49 0,03 2851,33 192,80 734,58 0,06 5551,00 1618,65 731,06 6,00 2780,00 401,98 734,46 0,13 1628,67 84,11 731,03 6,02 2733,33 167,39

794,53 0,13 3086 345,07 791,05 6,11 2867,33 192,63 794,63 0,05 5614,67 1641,76 791,01 6,00 2798,00 397,09 794,50 0,15 1649,67 85,70 791,07 5,98 2713,00 180,03

854,57 0,16 3113 342,24 844,04 0,02 2879,67 193,46 851,10 6,09 5662,33 1668,42 851,05 6,04 2814,67 391,44 851,00 6,00 1669,33 85,17 850,98 5,96 2695,67 190,48

914,56 0,05 3141 337,29 911,01 6,04 2897,67 192,81 911,08 6,10 5712,67 1696,20 911,02 6,00 2829,67 386,36 911,00 6,03 1688,33 85,17 911,00 6,03 2677,33 199,66

974,51 0,06 3166 330,93 964,07 0,07 2908,33 191,68 974,63 0,06 5772,67 1718,28 967,58 6,03 2840,67 384,78 974,48 0,15 1705,67 87,31 971,13 6,07 2660,67 209,69

1029,25 7,42 3189 323,15 1027,57 6,09 2922,33 190,68 1034,63 0,06 5827,67 1740,41 1027,47 6,00 2856,67 382,44 1034,47 0,12 1723,00 87,84 1031,07 6,03 2645,00 218,00

1094,50 0,14 3217 319,61 1084,03 0,06 2934,33 189,93 1094,67 0,12 5876,00 1757,67 1087,57 6,09 2865,33 372,91 1091,10 6,06 1734,00 94,30 1091,03 6,00 2627,00 227,42

1149,30 7,35 3235 311,13 1144,07 0,06 2945,67 189,17 1154,63 0,12 5921,33 1774,87 1151,00 6,06 2879,33 368,58 1154,53 0,15 1751,00 92,68 1151,03 6,09 2613,67 235,12

72

Big Particles

Table 4: Overview of storage modulus and time measurements for each sample of big particles. Average time and storage modulus are calculated from three measurements

for each single sample.

B3 4% B4 4% B5 4% B5 2% B2 30%

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Time (s)

G' (Pa)

Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD

27,62 6,02 3.034,27 2.501,38 20,48 0,05 780,23 785,26 34,51 6,03 2.998,00 685,61 20,55 0,12 35,31 35,50 37,94 6,16 6.932,00 5.452,70

71,08 6,08 3.139,83 2.493,06 67,45 6,07 846,17 845,74 74,57 0,10 3.046,33 713,81 63,97 0,09 50,37 48,99 81,64 12,27 6.444,67 4.533,37

131,06 6,04 3.264,87 2.466,67 127,45 6,10 892,43 878,24 134,58 0,11 3.110,00 728,45 124,01 0,07 74,19 71,89 138,21 6,24 6.257,00 4.093,23

201,61 12,17 3.400,20 2.386,31 187,40 6,04 935,83 915,51 194,59 0,14 3.166,67 727,43 184,00 0,05 98,60 95,54 201,66 12,25 4.429,33 1.041,45

261,55 12,03 3.499,00 2.344,46 247,52 5,96 975,33 946,03 254,59 0,12 3.219,33 720,65 243,96 0,02 123,58 118,70 258,18 6,22 4.558,67 1.154,72

321,59 12,14 3.591,33 2.296,75 307,42 6,03 1.007,90 972,40 314,48 0,14 3.272,33 700,50 304,03 0,08 149,51 142,94 307,62 6,25 4.591,33 1.161,85

381,62 12,12 3.679,33 2.245,10 367,49 6,10 1.040,43 993,38 374,60 0,09 3.321,00 689,61 363,98 0,04 175,52 168,02 371,10 6,05 4.567,33 1.103,50

441,65 12,16 3.760,67 2.192,37 427,46 6,01 1.066,07 1.015,04 434,55 0,05 3.369,00 666,58 423,98 0,04 200,82 193,29 431,04 5,97 4.539,67 1.099,34

501,59 12,14 3.783,67 2.077,84 487,43 6,03 1.091,63 1.030,39 494,52 0,15 3.411,33 643,45 484,02 0,08 225,61 218,47 491,03 6,07 4.537,67 1.062,08

561,66 12,27 3.870,33 2.036,84 547,44 6,03 1.117,37 1.045,95 554,53 0,09 3.455,33 621,43 543,96 0,03 249,03 241,94 554,57 10,49 4.556,00 1.064,44

618,19 6,08 3.935,67 1.999,19 607,52 5,98 1.139,77 1.056,72 610,96 6,02 3.493,00 595,46 603,98 0,07 273,19 266,76 614,57 10,40 4.557,67 1.051,99

678,13 6,06 4.017,00 1.955,88 667,47 6,11 1.161,67 1.066,29 671,09 5,98 3.532,67 570,99 674,52 18,13 306,01 306,09 667,60 6,21 4.582,67 1.067,45

738,18 6,05 4.083,33 1.903,71 727,45 5,98 1.182,93 1.077,25 730,99 6,03 3.566,67 553,41 730,93 12,01 325,88 326,18 731,09 6,10 4.613,67 1.091,45

798,22 6,18 4.149,00 1.855,63 784,03 0,05 1.195,47 1.087,64 794,49 0,03 3.605,67 541,32 790,97 12,10 343,82 342,60 794,75 10,67 4.621,00 1.088,23

854,72 0,10 4.209,67 1.826,78 847,51 6,06 1.214,63 1.093,19 847,58 6,14 3.642,67 524,71 850,96 12,05 366,38 367,12 851,09 6,11 4.629,33 1.085,13

911,09 6,08 4.265,00 1.776,16 907,49 5,98 1.231,77 1.099,26 907,59 6,01 3.677,67 508,71 910,97 12,02 388,34 391,53 911,00 6,05 4.645,67 1.101,09

974,63 0,21 4.326,33 1.730,35 967,51 6,02 1.247,83 1.104,33 971,11 6,06 3.711,00 493,08 967,49 6,09 407,00 410,74 971,10 6,13 4.665,00 1.110,83

1.034,67 0,06 4.382,00 1.683,99 1.027,47 6,18 1.264,57 1.110,01 1.027,57 6,09 3.732,67 481,87 1.030,93 12,01 430,48 438,25 1.027,63 5,95 4.643,33 1.149,63

1.091,17 6,12 4.432,33 1.630,33 1.087,57 6,00 1.280,03 1.114,76 1.087,57 6,09 3.760,33 473,00 1.087,47 6,00 448,67 457,77 1.091,07 6,12 4.627,33 1.123,72

1.147,60 6,15 4.480,00 1.585,24 1.147,50 6,06 1.295,00 1.118,48 1.151,03 6,00 3.792,00 462,45 1.147,50 6,06 467,23 478,41 1.154,57 0,06 4.626,00 1.110,72

73

Statistic analysis

Table 3: Results from ANOVA for the group of big particles, this shows that only the hydrogels containing

particles from samples 3 and 5 with a particle concentration of 4% don’t show a significant difference in hydrogel strength..

Storage Modulusa

Sample +

Particle

Concentration N

Subset

1 2 3 4

Tukey

HSDb,c

5 2% 60 256,9767

4 4% 60 1098,3533

5 4% 60 3444,1833

3 4% 60 3865,1917

2 30% 60 4881,2667

Sig. 1,000 1,000 ,488 1,000

Means for groups in homogeneous subsets are displayed.

Based on observed means.

The error term is Mean Square(Error) = 2033721,980.a

a. partgroup = Big Particles

b. Uses Harmonic Mean Sample Size = 60,000.

c. Alpha = ,05.

Table 4: Results from ANOVA for the group of small particles, this shows that the hydrogels containing particles

from samples 2,3,4 and 5 with a particle concentration of 30% and 4% don’t show a significant difference in hydrogel strength..

lossmodulusa

parttype N

Subset

1 2 3

Tukey HSDb,c

4 2% 60 1530,2833

5 2% 60 2719,8500

4 4% 60 2763,1500

2 30% 60 2831,0833

3 4% 60 2865,3167

5 4% 60 5208,9167

Sig. 1,000 ,844 1,000

Means for groups in homogeneous subsets are displayed.

Based on observed means.

The error term is Mean Square(Error) = 451901,283.a

a. partgroup = Small Particles

b. Uses Harmonic Mean Sample Size = 60,000.

c. Alpha = ,05.

74

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