ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2021

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 2024

Ceramic Core–Shell Particles

Synthesis and Use within Dentistry

CAMILLA BERG

ISSN 1651-6214ISBN 978-91-513-1163-0urn:nbn:se:uu:diva-437696

Dissertation presented at Uppsala University to be publicly examined in Häggsalen,Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, Friday, 7 May 2021 at 09:15 for thedegree of Doctor of Philosophy. The examination will be conducted in English. Facultyexaminer: Doctor Nicola Döbelin (RMS Foundation).

AbstractBerg, C. 2021. Ceramic Core–Shell Particles. Synthesis and Use within Dentistry. DigitalComprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 2024. 67 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1163-0.

Dentin hypersensitivity is one of the most prevalent conditions related to oral health, affectinga large share of the adult population. Shortcomings with the available treatment options arerelated to non-ideal particle sizes and degradation properties. An improved clinical outcomecould possibly be obtained using a bioactive occluding agent that can offer a high, continuousrelease of ions, as well as having a particle size that allows for penetration into the dentin tubules.

The work in this thesis focused on the development and investigation of a synthesis approachfor calcium phosphate core–shell particles and the use of those in the treatment of dentinhypersensitivity. The overall aim was to increase the knowledge about the synthesis andto evaluate the in vitro performance of amorphous calcium magnesium phosphate (ACMP)particles when used as an occluding agent.

The synthesis of the core-shell particles was based on precipitation reactions in aqueoussolutions and the synthesized materials were studied in terms of morphological, structural,and compositional aspects. Resulting particles had diameters ranging from 400 nm–1. 5 µm(depending on reaction conditions), with morphologies and structures that were shown tocorrelate with the ionic radius and the concentration of the substituting ion. This insight resultedin the possibility to control the outcome of the reaction and to extend the synthesis to otheralkaline earth phosphates. The mechanism of formation was suggested to be the simultaneousprecipitation of primary nanoparticles (NPs) and the formation of gas bubbles that could functionas soft templates.

A study of the degradation properties together with a series of in vitro studies, using a dentin-disc model, indicated that the ACMP particles may be a promising candidate for clinical use.The material was shown to offer a rapid and continuous release of Ca2+, Mg2+, and phosphate,aiding surface, as well as intratubular occlusion and mineralization. Additional use of a fluoridetoothpaste resulted in incorporation of F– in the mineralized material. This could enhance thein vivo performance due to the known benefits of including F– in dental tissues, e.g. decreasedsolubility. The ACMP particles were, furthermore, shown to be more efficient in terms of degreeof occlusion when compared to other similar products available on the market. The intratubularmineralization was additionally mitigating the effect of an acid attack, which is of importancefor a long-lasting effect in clinical use.

Keywords: Calcium phosphate, Core-shell particles, Substituting ions, Dentinhypersensitivity, Occlusion, Mineralization

Camilla Berg, Department of Materials Science and Engineering, Applied Material Science,Box 534, Uppsala University, SE-751 21 Uppsala, Sweden.

© Camilla Berg 2021

ISSN 1651-6214ISBN 978-91-513-1163-0urn:nbn:se:uu:diva-437696 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-437696)

Till Mamma

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Berg, C., Yu, S., Engqvist, H., Xia, W. “Bubble-assisted fabrica-

tion of calcium phosphate core–shell particles”. In manuscript.

II Berg, C., Engqvist, H., Xia, W. ”Ion substitution induced for-mation of spherical ceramic particles”, Ceramics International, 45 (2019) 10385–10393.

III Berg, C., Unosson, E., Engqvist, H., Xia, W. ”Amorphous cal-

cium magnesium phosphate particles for treatment of dentin hy-persensitivity: a mode of action study”, ACS Biomaterials Sci-ence & Engineering, 6 (2020) 3599–3607

IV Berg, C., Unosson, E., Riekehr. L., Xia, W., Engqvist, H. ”Elec-

tron microscopy evaluation of mineralization on peritubular den-tin with amorphous calcium magnesium phosphate micro-spheres”, Ceramics International, 46 (2020) 19469–19475

V Berg, C., Unosson, E., Engqvist, H., Xia, W. “Comparative study

of technologies for tubule occlusion and treatment of dentin hy-persensitivity”. Under review.

Reprints were made with permission from the respective publishers.

Authors contributions

The author’s contributions to the papers included in this thesis are: Paper I Part of planning, experimental work and evaluation. Major part of writing. Paper II Major part of planning, experimental work, evaluation, and writing. Paper V Major part of planning, experimental work, evaluation and writing. Paper IV Part of planning and experimental work. Major part of evaluation and writing. Paper V Part of planning, experimental work and evaluation. Major part of writing.

Other work by the author

X. Bai, W. Liu, L. Xu, Q. Ye, H. Zhou, C. Berg, H. Yuan, J. Li and W. Xia. “Sequential macrophage transition facilitates endogenous bone regeneration induced by Zn-doped porous microcrystalline bioactive glass”. Journal of Ma-terials Chemistry B. Accepted.

Contents

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

2. Dental anatomy ......................................................................................... 14 2.1 Dentin ................................................................................................. 15

2.1.1 Dentin hypersensitivity ............................................................... 16

3. Calcium phosphates in dentistry ............................................................... 18 3.1 Ionic substitutes in calcium phosphates ............................................. 19

3.1.1. Anionic substitutes ..................................................................... 19 3.1.2 Cationic substitutes ..................................................................... 19

3.2 Synthesis of nanostructured calcium phosphates ............................... 21 3.2.1 Synthesis of calcium phosphate core–shell particles .................. 22 3.2.2. Gas bubbles as soft templates .................................................... 22

4. Summary of aims and objectives .............................................................. 24

5. Preparation and characterization methods ................................................ 25 5.1 Synthesis of ceramic core–shell particles ........................................... 25 5.2 Dentin occlusion/mineralization ......................................................... 26

5.2.1 Degradation properties ................................................................ 27 5.2.2 In vitro evaluation of dentin occlusion/mineralization ............... 28

5.3 Characterization .............................................................................. 29 5.3.1 Imaging ....................................................................................... 29 5.3.2 Elemental composition ............................................................... 31 5.3.3 Structural characterization .......................................................... 32

6. Synthesis of core–shell particles ............................................................... 33 6.1 Mechanism of formation .................................................................... 33 6.2 The role of the substituting ion ........................................................... 36

6.2.1 Effect of ionic radius .................................................................. 36 6.2.2 Effect of concentration ............................................................... 38

7. Dentin occlusion and mineralization using ACMP particles .................... 41 7.1 Mode of action ................................................................................... 41 7.2 Mineralization on peritubular dentin .................................................. 44 7.3 Effect of fluoride treatment ................................................................ 46 7.4 Comparison to similar products available on the market ................... 48

8. Concluding remarks .................................................................................. 50

9. Future outlooks ......................................................................................... 52

Svensk sammanfattning ................................................................................ 54

Acknowledgements ....................................................................................... 57

References ..................................................................................................... 60

Abbreviations

ACP Amorphous calcium phosphate ACMP Amorphous calcium magnesium phosphate β-TCP β-Tricalcium phosphate ICP-OES Inductively coupled plasma optical emission spectroscopy EDX Energy dispersive X-ray spectroscopy FA Fluorapatite FIB Focused ion beam HA Hydroxyapatite ITD Intertubular dentin nanoHA Nanocrystalline hydroxyapatite NPs Nanoparticles PTD Peritubular dentin SAXS Small angle X-ray scattering SEM Scanning electron microscopy STEM Scanning transmission electron microscopy TEM Transmission electron microscopy TGA Thermogravimetric analysis WH Whitlockite XRD X-ray diffraction

13

1. Introduction

Oral diseases and injuries remain to be one of the major public health prob-lems. Demographic transition and lifestyle changes, including sugar-rich diets and consumption of tobacco and alcohol, have shown to be factors affecting oral health [1]. A consequence of this is that the demand for dental materials is increasing, and they are used for the treatment of a range of oral conditions. Among these, dentin hypersensitivity is one of the most prevalent conditions, affecting a large share of the adult population worldwide [2].

The early techniques used for pain-relief related to dentin hypersensitivity were based on in-office treatments (at a dental clinic). To increase efficiency, simplify treatments, and reduce cost, more efforts have recently been put into the development of at-home treatments [3]. These products often include some kind of bioactive ceramics with the purpose to occlude exposed dentin tubules and induce mineralization to offer pain relief. Currently, there are several such products available on the market, but their efficiency is sometimes poor due to non-ideal particle sizes and their degradation properties. This thesis con-tributes to the development and investigation of nanostructured calcium phos-phate core–shell particles to overcome these shortcomings. Calcium phos-phates resemble the mineral component in teeth, which together with their bi-oactive and osteoconductive properties, make them interesting alternatives for use within dentistry [4,5].

Synthesis of nanostructured calcium phosphates, such as core–shell parti-cles, can be performed by seeking inspiration from the formation of calcified materials in nature. Teeth are, for instance, formed by controlled nucleation, crystallization, and self-assembly of nanosized particles where ion substitu-tion plays an important role in the process [6,7]. In this thesis, the role of sub-stituting ions in synthetic calcium phosphates was studied to determine how they can be used to induce the formation, and the resulting properties of core–shell particles. In relation to this, the function of in situ formed gas bubbles was furthermore investigated in terms of templating functions and their role in the formation of ceramic core–shell particles. The development of synthesis strategies of calcium phosphate core–shell particles is not only interesting for the use of those within dentistry and the treatment of dentin hypersensitivity. Finding a robust synthesis approach could allow for future tailoring of the characteristics of the core–shell particles to target other applications within biomedicine.

14

2. Dental anatomy

Human teeth are essential for chewing, but also important for speech. There is some resemblance between teeth and bone, where calcium phosphate makes up the mineral component in both, but the two hard-tissues are quite different in terms of hierarchical structure. The anatomy of human teeth is shown in Figure 1. Any part of the teeth visible in the mouth is referred to as the crown, and the non-visible parts are known as the root.

Teeth are attached with periodontal ligaments to the alveolar bone found on the maxilla and the mandible. Covering the alveolar bone is the gingivae, which is a mucosal soft-tissue. The status of the gingivae is important for oral health since it is strongly correlated to tooth loss and oral infections/inflam-mations [8]. The center part of the tooth is the pulp, which is mainly comprised of nerve bundles and blood vessels. It is covered by dentin, one of the three different types of mineralized tissues found in the tooth. Dentin will be de-scribed more in detail in section 2.1 of the thesis. The other two types of min-eralized tissues are the cementum and the enamel. The cementum covers the dentin in the root section, and it is on this structure that the periodontal liga-ments attach. Its main function is, therefore, to maintain the integrity of the root and the position of the teeth. The outermost part of the teeth, visible by inspection, is the enamel. Enamel is a highly mineralized tissue, serving as a protective cover for the rest of the tooth, and is comparably resistant to low pH and mechanical wear [9,10].

Figure 1. Schematic image of the dental anatomy. Adapted with modifications from [11].

15

2.1 Dentin Dentin is found between the pulp and the cementum or the enamel. It has a lower hardness compared to enamel and is more resilient, but it is generally more sensitive to lowered pH [12,13]. Dentin has a peculiar tubular structure with channels (dentin tubules) extending out from the pulp towards the tooth surface. The tubules, 1.5–4.5 µm in diameter, are filled with pulpal fluid and resides the odontoblasts [14,15]. The odontoblasts are large columnar cells that have their cell body close to the pulp and the odontoblast process (Tome’s fibre) extending out in the tubule. These cells regulate dentinogenesis (for-mation of dentin), ion, and protein content in the tubular fluid and within the dentin tissue. They also regulate the channeling of hydrokinetic forces and the secretion of sclerotic dentin upon carious attack or cell damage. Deposition of new dentin can occur even if the tissue, as for enamel, is avascularized. This is possible since the odontoblasts receive nutrition from the tubular fluid that originates from the blood vessels in the adjacent pulp tissue.

Dentin is composed of approximately 70 wt% mineralized material (cal-cium deficient hydroxyapatite, CDHA), 20 wt% organic components and the remaining fraction is water [12]. Apart from Ca2+ and PO4

3–, CDHA in dentin additionally contains CO4

3– (5.6 wt%), Mg2+ (1.23 wt%), Na+ (0.6 wt%), and small fractions of F–, K+, and Cl– [7]. The exact composition and the ratio between the inorganic and organic material varies across the tissue depending on location in relation to the pulp and the tubules.

The major part of dentin consists of intertubular dentin (ITD) that is made up of a framework of collagen fibrils (mainly collagen type I) with inclusions of non-collagenous proteins (e.g. proteoglycans and phosphoglycoproteins). The collagenous framework is mineralized by plate-like CDHA crystals (60 nm long and 2–5 nm thick) that are aligned along the length of the collagen fibrils [16–18]. Peritubular dentin (PTD), lining the dentin tubules, is hy-permineralized and denser. It can easily be distinguished from the ITD since it lacks a collagen framework [19]. The innermost surface of the PTD, on the tubule walls, resides the lamina limitans that lie in contact with the odontoblast process. It is hypocalcified and composed of a fibrous outer layer and a mem-branous inner layer.

16

2.1.1 Dentin hypersensitivity Dentin hypersensitivity is characterized by a sharp and sudden pain triggered by an external stimulus that can be either thermal, evaporative, osmotic, or tactile [20]. The condition arises when dentin is exposed to the oral environ-ment. This can occur as a result of erosion, abfraction (non-carious tissue loss caused by mechanical wear), attrition of the outer layer of the enamel, or gin-gival recession caused by excessive tooth brushing [21,22].

The underlying pain mechanism for dentin hypersensitivity has been dis-cussed and there are three suggested explanations; the direct intervention the-ory, the odontoblast theory, and the hydrodynamic theory. A schematic illus-tration is presented in Figure 2. The first two mechanisms are based on stim-ulation of nerves either by direct stimulation of nerves extending out in the tubules or by synaptic junctions between the odontoblasts and pulpal nerves [22]. The lack of evidence on the existence of such nerves or junctions makes the hydrodynamic theory the currently most accepted theory for the explana-tion of the underlying cause for pain. The theory was presented by Brännström in 1967, and it states that pain is evoked as a response to the movement of tubular fluid, causing mechanical deformation of the pulpal nerves [23].

The prevalence of dentin hypersensitivity is high, with up to 57 % of the adult population suffering from the condition, and the number is even higher for patients receiving periodontal treatment, reaching 84.5 % [2]. Thus, dentin hypersensitivity is widespread, which has led to a vast number of treatments for symptom relief that are available on the market. Treatments can be divided into two categories: in-office treatments and at-home treatments. In-office treatments include treatment with e.g. lasers, adhesive resins, and varnishes, that serves to induce or directly occlude (physically block) the exposed tubules [24–28]. To reduce the cost and simplify the treatments, much research and development have been focused on at-home treatments in the form of tooth-pastes, mousses, chewing gums, and mouthwashes, that with different mode of actions are reported to reduce pain [28].

Most desensitizing products intended for at-home use contain a potassium salt (e.g. nitrate or chloride) since potassium ions have been reported to depo-larize the interdental nerves through the changes in ion concentration in the extracellular fluid, and thereby reduce pain [29,30]. Since it is difficult to sus-tain potassium concentrations that are high enough to allow for long-lasting effects, potassium salts are seldom used alone for the treatment of dentin hy-persensitivity [31]. They can, however, offer fast pain relief and are therefore added as a complement to other types of desensitizing products.

To enable pain-relief that is sustained over a longer period, the addition of some kind of occluding agent is common in dental products intended for at-home treatment of dentin hypersensitivity. These are particles that can physi-cally block and/or induce the mineralization of a dentin-like material on the

17

dentin surface and inside the tubules. Formation of a mineralized material oc-curs if the release of calcium and phosphate ions is high enough, inducing the precipitation of calcium phosphate. Materials reported to have an occluding effect on exposed dentin tubules include Bioglass (calcium sodium phospho-silicate), arginine with calcium carbonate, stannous fluoride, and several cal-cium phosphate-based materials such as casein phosphopeptide amorphous calcium phosphate (CPP-ACP), hydroxyapatite (HA) and amorphous calcium phosphate (ACP) [32–40].

The ideal occluding agent should have a particle size that is small enough to penetrate the dentin tubules, and it should offer a fast release of ions to enable rapid mineralization. The mineralized material should additionally be resistant to a decrease in pH (acid attack) that can occur during the consump-tion of acidic beverages or food [41]. A possible increase in resistance towards acid attacks could be achieved by adding fluoride with the occluding agent. Fluoride is commonly used in regular toothpastes since it can prevent demin-eralization of dental tissues through the formation of fluorapatite (FA) that is less soluble compared to HA [42].

Figure 2. Schematic illustration of the three pain mechanisms suggested for dentin hypersensitivity including (a) the direct intervention theory (b) the odontoblast theory, and (c) the hydrodynamic theory. Adapted with modifications from [43].

18

3. Calcium phosphates in dentistry

Calcium phosphates are a category of ceramic materials that have gone through much development within the field of biomaterials over the latest dec-ades. Their resemblance to the mineral component in teeth and bone, along with their bioactive and osteoconductive properties, have made them interest-ing for hard tissue applications [4,5]. Within dentistry, calcium phosphates have been used in the form of granules, injectable cements, composites, coat-ings on implants, and as small micrometer to nanometer-sized particles in-cluded in toothpaste and other similar products [44]. This has allowed for treatment of, e.g. periodontal defects, augmentation of alveolar bone, tooth replacement, and treatment of dentin hypersensitivity.

There are a variety of different kinds of calcium phosphate materials that are distinguished by the constituent phosphate ion. These include the ortho-phosphates, metaphosphates, and pyrophosphates that are based on the PO4

3–, PO3

– and the P2O74– ions, respectively [7]. Within the field of dentistry, it is

mostly the calcium orthophosphates that are used, and thus the only group of calcium phosphates that will be considered in this thesis. Some of the common orthophosphates are listed in Table 1. In common for these is that the lower the Ca/P ratio is, the more water-soluble and acidic the calcium phosphate is [7]. These are important features for how the materials will behave in in vivo conditions in terms of degradation and interaction with surrounding tissues [45]. This thesis mainly focuses on calcium orthophosphates (from here on referred to as calcium phosphates) that can be prepared from aqueous solu-tions at neutral or basic pH.

Table 1. A summary of the most common calcium orthophosphates [7].

Ca/P Compound Abbreviation Chemical formula 0.5 Monocalcium phosphate monohydrate MCMP Ca(H2PO4)2·H2O 1.0 Monetite (dicalcium phosphate anhydrous) DCPA CaHPO4 1.0 Brushite (dicalcium phosphate dihydrate) DCPD CaHPO4·2H2O 1.33 Octacalcium phosphate OCP Ca8H2(PO4)6·5H2O 1.5 Alpha-tricalcium phosphate α-TCP Ca3(PO4)2 1.5 Beta-tricalcium phosphate β-TCP Ca3(PO4)2 1.2–2.2 Amorphous calcium phosphate ACP CaxHy(PO4)z·nH2O 1.67 Hydroxyapatite HA Ca10(PO4)6(OH)2

19

3.1 Ionic substitutes in calcium phosphates The calcium phosphates listed in Table 1 represent the stoichiometric forms of the materials. These seldom occur in nature where ionic substitution, both anionic and cationic, are common. HA occurring in nature is, for instance, always calcium deficient (CDHA, Ca10–x(HPO4)x(PO4)6–x) [5,7]. The vacan-cies of Ca2+ are compensated for by the protonation of OH– (inclusion of water in the structure) or substitution with other ions [7]. Ionic substitution can also be used in the preparation of synthetic calcium phosphates to control the prop-erties of the material, increase the bioactivity, or allow for the delivery of spe-cific ions with a therapeutic purpose [5,46].

3.1.1. Anionic substitutes Anions that can substitute for PO4

3– or OH– in calcium phosphates include among others, CO3

2–, SiO44–, SO2

4–, OH–, F–,Cl– and Br– [47]. Substitution with CO3

2– is common in calcified tissues and calcium phosphates prepared from aqueous solutions (due to dissolved atmospheric CO2). HA in teeth and bone are always substituted with CO3

2– by the replacement of PO43– (b-type

substitution) or OH– (a-type substitution) [12,48]. The b-type is more com-mon, and the a-type mostly occurs in synthetic apatites prepared at high tem-peratures. Incorporation of CO3

2– in the structure, accompanied by a distortion of the crystal structure, results in a solubility that is increasing with increasing degrees of substitution [48–50]. As for HA, naturally occurring ACP always contains CO3

2–, which is one of the ions reported to have a stabilizing effect on the otherwise metastable phase [46].

Another interesting anionic substitute is F– that naturally occurs in dental tissues (up to 0.01 wt% in enamel and 0.06 wt% in dentin) and bone [7]. In HA, F– substitutes for OH– at the center of the Ca(II) triangles (Figure 3), which results in the formation of a hydrogen bonding between the adjacent OH– group and the F– ion, accompanied by a reduction of the a-axis in the lattice [51]. This explains why F– substituted HA and FA (Ca5(PO4)3F2), are less soluble and can have an increased hardness in comparison to other apatites [52,53]. Looking at ACP, F– actually has the opposite effect in comparison to CO3

2–, since it promotes crystallization of HA [54].

3.1.2 Cationic substitutes Several cationic substituents can replace Ca2+ in calcium phosphates. Substi-tution of Ca2+ can occur with divalent ions such as e.g. Mg2+, Sr2+, Ba2+, Cd2+ and Pb2+ but other monovalent or trivalent ions such as Na+, K+ and Al3+ have also been reported as possible substituents [46,52]. Cationic substitution in HA occurs either by replacing Ca2+ at the Ca(I) or Ca(II) site in the structure (Figure 3). The difference between the two Ca2+ sites relates to the positions

20

of the surrounding PO43– and OH– groups. Due to this difference, the size of

the substituting ion in comparison to Ca2+ will determine the preferred site for substitution: smaller ions such as Mg2+ have a slight preference for the Ca(I) site and larger ions such as Sr2+ for the Ca(II) site [55,56].

Figure 3. Illustration of the crystal structure of HA seen from a view along the c-axis. Used with permission from [46].

Mg2+ is a well-studied and important substitute in calcium phosphates, and it is found naturally in both teeth and bone [7]. Due to its preferred site of sub-stitution, only a limited amount of Mg2+ can be incorporated in the HA struc-ture (up to 10 at%) without comprising the order in the crystal lattice [46]. Instead, at a higher degree of Mg+2 substitution, the formation of Mg2+ substi-tuted β-TCP or Whitlockite (WH) is favored. WH (Ca9Mg(PO4)6PO3OH) is an analog to β-TCP, but the two structures can be clearly distinguished from each other due to their different structural arrangement and the presence of HPO4

2– groups in WH [57]. A consequence of this is that Mg2+ efficiently can stabilize ACP. Investigations of the mechanism of stabilization have shown that this can occur in two ways: disruption of the order in the crystal structure and by adsorption on the material surface, resulting in a shielding effect (blocking active growth sites) towards the surrounding environment [58,59].

As for Mg2+, Sr2+ is also found in many biological calcified tissues [7]. In comparison to Mg2+, relatively large amounts of Sr2+ can be incorporated in the structure of HA, at the Ca(II) site (Figure 3), without comprising the order of the HA structure [46]. As a result of this, it is possible to incorporate Sr2+ in the HA structure over the whole range of composition, and it can therefore not stabilize ACP to the same extent as Mg2+.

21

3.2 Synthesis of nanostructured calcium phosphates The synthesis of calcium phosphates can be performed using both low and high-temperature processes. Synthesis at high temperature includes solid-state reactions and is mostly used for the preparation of high-temperature phases such as pure α-TCP and β-TCP, whereas low-temperature reactions include synthesis in aqueous media and preparation of hydraulic cements [5,7]. In low-temperature reactions, pH plays an important role, determining which type of calcium phosphate that forms [7]. Synthesis at low temperature, in aqueous media at neutral pH, is of particular interest in the synthesis of nanostructured calcium phosphates since it is possible to seek inspiration from biomineralization. Biomineralization is the process in nature from which com-plex hierarchical structures are produced from living organisms, e.g. the for-mation of calcified tissues containing calcium phosphates, such as teeth and bone [6]. Mineralization and self-assembly in these types of tissues are con-trolled by cellular processes and the presence of e.g. amino acids, proteins, or substituting ions (as described in the previous section), and/or by formation within a confined volume [6,60–63].

By the use of modulatory additives, similar to those found in natural pro-cesses, it is possible to control the nucleation, self-assembly, and crystalliza-tion of nanosized building blocks in the fabrication of synthetic nanostructured 3D-architectures [64]. Nanostructured synthetic calcium phosphates have shown to exhibit enhanced bioactivity in comparison to microscale materials, and the ultimate goal would be to achieve precise control over synthesis out-come to allow for tailoring of the material properties for a specific application [65]. Additives that have been studied in synthetic materials can offer possi-bilities to control the synthesis through templating, capping or internal modi-fication of crystal growth by the addition of substituting ions [64].

Precipitation reactions are among the simplest synthesis alternatives for nanostructured calcium phosphates. It is based on the formation of insoluble products as a result of supersaturation of dissolved salts, in some senses re-sembling the biomineralization processes. The simplicity of the method, how-ever, comes with the drawbacks that it can be difficult to control agglomera-tion and the size of particles. In this thesis, chemical precipitation has been used together with substituting ions as an approach for the formation of nanostructured core–shell particles, which will be described more in detail in the following section.

22

3.2.1 Synthesis of calcium phosphate core–shell particles Core–shell particles of calcium phosphates are interesting materials within bi-omedicine due to their potential use as delivery vehicles for therapeutic agents (drugs, genes, and proteins) and use as building blocks or ion reservoirs in hard tissue applications [5]. Synthesis of hollow core–shell particles from aqueous solutions often includes the use of additives with templating func-tions. These can be either hard (solid materials) or soft (lacking a rigid struc-ture) forming during the reaction, but common for them is that they have to be removed at the end of the synthesis to obtain a hollow core [66]. This can be achieved either by heating or dissolution, but it induces the risk of damag-ing the core–shell structures. As a result of this, much effort has been put into the development of self-templating or template-free methods based on differ-ent principles. This includes e.g. Ostwald ripening that can result in hollow particles if the reaction time is long enough to allow for dissolution, diffusion, and reprecipitation of material [67]. This is due to differences in solubility between small and large particles. Another strategy, that could be considered template-free or self-templating, is the use of gas bubbles that can guide the formation of core–shell particles. In that case, the surface of the bubbles could function as a soft template, without the need for removal at the end of the reaction.

3.2.2. Gas bubbles as soft templates The use of gas bubbles as soft templates have been reported in the synthesis of hollow core–shell particles of several different material categories includ-ing phosphates, carbonates, oxides, sulfides, and pure metals [68–77]. The formation of bubbles in the reaction is commonly explained as a result of the decomposition of precursors occurring upon heating. Removal of such precur-sors resulting in the lack of formation of core–shell particles have been used as a confirmation of the templating function of the gas bubbles, but there is yet no other empirical evidence of their function as soft templates [72,74,77].

The size of particles reported to be synthesized using gas bubbles has di-ameters ranging from 80 nm–10 µm [73,75]. For particles in the smaller size range, the templating bubbles should be what is referred to as nanobubbles, i.e. stable gas bubbles having diameters smaller than 1 µm. Their existence, and especially their stability, are highly debated due to their extremely high internal pressure (caused by the curvature and surface tension of the bubble) that should cause them to disappear within microseconds [78]. Among the proposed explanations for their stability are a special arrangement of the mol-ecules in water and/or selective adsorption of inorganic salts that could coun-teract the internal pressure, and the existence of organic or surface-active con-taminants that could prevent the outward flow of gas [79–86].

23

The suggested mechanisms of formation of the core–shell particles using gas bubbles as soft templates are similar when comparing several studies, and a schematic illustration is shown in Figure 4. The first step is the simultaneous formation of primary nanoparticles (NPs) and gas bubbles in the solution. The NPs adsorb on the bubble surface, driven by the minimization of interfacial energy, which could be compared to the mechanism of stabilization in Pick-ering emulsions [87]. With time, more and more particles will adsorb and ag-gregate on the bubble surface, in multiple layers, resulting in a complete core–shell particle.

Figure 4. Schematic illustration of the mechanism of formation of core–shell parti-cles using gas bubbles as soft templates.

24

4. Summary of aims and objectives

The overall aim of this thesis was to gain increased knowledge about the syn-thesis of ceramic core–shell particles, in particular of calcium phosphates, and the use of those within dentistry. The thesis builds on investigations of using precipitation reactions in the synthesis of ceramic core–shell particles, where the mechanism of formation and the role of the substituting ions were studied by morphological, structural, and compositional evaluation. The use of core–shell particles of amorphous calcium magnesium phosphate (ACMP) as an occluding/mineralization agent, for the treatment of dentin hypersensitivity, was also investigated in a series of in vitro studies. The specific objectives of the appended papers were:

I. Primarily, to assess the mechanism of formation of calcium phosphate core–shell particles and the potential influence from gas bubbles. Sec-ondarily, to evaluate the role of substituting ions in terms of ionic ra-dius and concentration.

II. To determine if the synthesis approach from Paper I could be ex-

tended to other materials and to evaluate if the characteristics of the formation of the core–shell particles followed the same pattern.

III. To determine the mode of action when using ACMP particles as an

occluding/mineralization agent. IV. First, to study the mineralization on the PTD after the use of ACMP

particles as an occluding/mineralization agent. Second, to determine the effect of additional use of a fluoride toothpaste.

V. To compare the ACMP particles with six commercially available de-

sensitizing products regarding their occluding/mineralization perfor-mance and resistance towards acid attacks.

25

5. Preparation and characterization methods

This section describes the methods used in the thesis in terms of the synthesis of ceramic core–shell particles and the use of these particles as an occlud-ing/mineralization agent. The procedures used in each study are described briefly, while detailed information about the methods used in synthesis, in vitro studies, and characterization can be found in the respective papers.

5.1 Synthesis of ceramic core–shell particles Investigations of the mechanism of formation of the core–shell particles and the role of substituting ions were performed using an in-house developed method as the base-line [88]. This method used Sr2+ and Mg2+ as substituting ions in a precipitation reaction in aqueous solutions, allowing for the for-mation of core–shell particles by structural regulation of the precipitated ma-terial. Slight modifications of this method were used in Paper I and II where solutions containing the anions (Na2HPO4 and KH2PO4) and the cations (CaCl2, MgCl2·6H2O, Sr(NO3)2 and/or BaCl2·2H2O) were prepared sepa-rately, and appropriate volumes were mixed to achieve the desired concentra-tions (Table 2). The salt solutions were mixed at room temperature, forming a clear solution, which were heated (60–100 °C) to induce precipitation. Parti-cles were collected by either filtration or centrifugation followed by washing with deionized water and ethanol to remove any salt residues.

Table 2. Summary of the salt concentrations that were used in the synthesis of the core–shell particles in Paper I and II.

Material Study Concentrations (mM)

Mg2+ Ca2+ Sr2+ Ba2+ PO43– Calcium phosphate Paper I 0-0.9 0.9 0-0.6 - 10 Strontium phosphate Paper II 0-0.9 0-0.9 0.9 - 10 Barium phosphate Paper II 0-0.9 0-0.9 0-0.5 0.9 10

In Paper I, the synthesis of nanostructured calcium phosphate core–shell par-ticles was investigated. The incorporation of substituting ions in calcium phos-phates is well studied, but not specifically in the context of the formation of core–shell particles. The effect of using Sr2+ and Mg2+, together and sepa-rately, were compared in terms of self-assembly patterns, morphology, size,

26

and crystal structure. Furthermore, the effect of reaction temperature and con-centration of the ion was evaluated to pin down which role of the substituting ion and the reaction conditions have in the formation of the core–shell parti-cles.

To evaluate if substituting ions could be used as a general approach for the formation of core–shell particles, Paper II extended the synthesis procedure to other alkaline earth phosphates (strontium and barium). The effect of the ionic radius and concentration of the substituting ions (Mg2+, Ca2+, and Sr2+) were evaluated to investigate how they influenced the outcome of the synthe-sis and as a proof of concept.

5.2 Dentin occlusion/mineralization The ideal desensitizing agent should result in fast and long-lasting occlu-sion/mineralization of exposed dentin, hindering the movement of fluid inside the dentin tubules. Limitations with the available technologies are related to non-ideal solubility of the occluding material, large particle sizes, and poor resistance to acid attacks. To overcome these issues, the occluding agent should have a particle size that allows for intratubular penetration as well as suitable degradation properties, i.e. a continuous release of ions, at a level that is high enough to induce mineralization. Considering this, submicron particles of ACP were regarded to be a promising candidate due to the excellent bioac-tivity and adjustable degradation rates of the amorphous material [89].

Using the synthesis method in Paper I, it was shown possible to synthesize calcium phosphate core–shell particles with an amorphous structure by the incorporation of Mg2+. The addition of the substituting ion did not only allow for the synthesis of core–shell particles, but it could also improve the handling properties and shelf-life of the otherwise metastable material [90]. Based on the same principle as used in Paper I, a synthesis approach for the fabrication of submicron particles was developed by collaborators (Psilox AB, Uppsala, Sweden) to fabricate particles of ACMP at a large scale. The salt concentra-tions during the synthesis were generally higher than in Paper I, and mixing of the solutions was conducted at elevated temperatures. This generated amor-phous core–shell particles with diameters between 180–440 nm, with a com-position of 22 ± 2 wt% Ca, 6 ± 2 wt% Mg, 58 ± 2 wt% PO4, and 14 ± 2wt% H2O. The use of the ACMP particles as an occluding agent was investigated in Paper III, IV, and V. In vitro evaluation of materials intended for dentin occlusion/mineralization will differ from in vivo evaluation due to mechanical stress arising from tooth brushing, food debris, salivary content, and varying pH-conditions, but it can be a good starting point to predict the behavior of the material in clinical use.

27

5.2.1 Degradation properties The degradation properties and ion release from an occluding agent are of great importance since they determine the character and rate of mineralization. Calcium phosphates are interesting alternatives as occluding agents since they are composed of ions that, upon being dissolved, could form a dentin-like ma-terial. The solubility of calcium phosphates is dependent on the Ca/P ratio, as described previously (in section 3). At neutral pH, HA that has a high Ca/P ratio is stable and the ion release is therefore slow [52]. ACP can offer a much faster release of ions, but since it is a metastable phase that easily dissolves or transforms into other more stable calcium phosphate phases, clinical use is challenging in terms of handling properties and shelf life [7,90]. To overcome these issues there are several possible alternatives for stabilization of ACP enabling use within dentistry. One example of this is stabilization using casein phosphopeptides (CPP) [36]. The milk-derived phosphopeptide can stabilize ACP by complexing with Ca2+, but the drawback is that it cannot be used by individuals with milk-based allergies. Another option for stabilization of ACP is to introduce other ions, such as Mg2+ in ACMP, which could inhibit spon-taneous transformation and potentially extend the ion release from the amor-phous material [59,90,91]. Thus, the degradation properties of the ACMP par-ticles were investigated in Paper III to determine the effects of Mg2+ and the mode of action of the ACMP particles.

When performing a degradation study, several things can affect the out-come of the study. Choice of media, pH, and temperature are among the most important factors to consider to obtain relevant data. Ion release and transfor-mation of ACP have previously been studied in several different media in-cluding phosphate-buffered saline (PBS), artificial saliva, and tris(hy-droxymethyl)aminomethane (Tris) buffer [92–95]. In Paper III, the degrada-tion properties of the ACMP particles were evaluated in Tris-HCl buffer, at pH 7.4, at 37 °C, and over a period of 30 minutes to 8 weeks. The pH and temperature were chosen to mimic the in vivo conditions. Tris-HCl was cho-sen as the media even if the aforementioned buffers might have been more representative for intra-oral conditions. This was done to avoid the high Na+ concentrations in the other buffers that can cause chemical interferences (ion-ization) in emission spectroscopy during the quantification of ion release. The degradation properties were evaluated in terms of ion concentrations in the supernatants and evolution of morphology, phase composition, and atomic composition of the particles over time.

28

5.2.2 In vitro evaluation of dentin occlusion/mineralization In the development of dental products, there is a need for a representative and reproducible platform that can be used for in vitro evaluation of the products before clinical evaluation. The dentin-disc model is such a platform that has been used extensively in the evaluation of products intended for sensitivity relief [3,96]. Dentin specimens are prepared from extracted teeth by section-ing thin discs in the transverse plane in the coronal region of the tooth. Fol-lowing removal of the smear layer (grinding debris from cutting), by etching in acid, creates samples that can be used for testing in intra-oral conditions. Dentin-disc specimens can be used for in situ and ex situ evaluation of occlu-sion, both in terms of visualization in a microscope and for evaluation of the permeability, e.g. by measuring the hydraulic conductance [97,98]. It is, how-ever, important to keep in mind that the natural variation in the dentin tubule appearance across the tooth, and the effects from etching, can affect the out-come of the use of the model [96].

The dentin-disc model was used in Paper III, IV, and V for in vitro eval-uation of the ACMP particles for dentin occlusion/remineralization. The ACMP particles were included in a sticky gel composed of glycerol, water, potassium nitrate, xanthan gum, potassium hydroxide, Carbopol 980, mint flavor, sodium benzoate, monosodium phosphate, and calcium chloride. Ap-plication of the gel on the dentin specimens was performed by manually brushing the specimens using a soft-bristled toothbrush, storing the samples in complete artificial saliva at 37 °C between treatments (treatment fre-quency and duration varied between studies).

As a part of determining the mode of action of the ACMP particles in Paper III, the morphological evolution of the particles over time was evaluated after application of the gel four times during one day and following incubation in saliva. The morphology of the particles and mineralized material on the treated dentin specimens were observed between 12 hours and 7 days, both on the surface and inside the tubules after preparing longitudinal cross-sections.

To gain further knowledge about the intratubular mineralization, and the interface between the PTD and the mineralized material, transverse cross-sec-tions of the dentin discs were investigated in Paper IV. This was done both in terms of visual observation of the specimens and evaluation of the elemental composition of the specimen and the mineralized material inside the tubules. The effect from combining the application of the ACMP particles with a flu-oride toothpaste (Pepsodent Super Fluor, 1450 ppm F–) was also investigated since F– is known to have several benefits when included in dental products (e.g. anti-caries properties, inhibition of demineralization, and promotion of remineralization of FA or F– substituted HA) [42,99].

There are numerous desensitizing products available on the market, but there are few comprehensive studies that compare the effect of those in terms of occlusion/mineralization efficiency and resistance towards acid attacks.

29

There are especially no recent studies that are up to date. Hence, in Paper V the effect of using the ACMP particles was compared to six other desensitiz-ing products available on the market (listed in Table 3). Specimens were treated with the same protocol, and the products were applied as instructed by the manufacturers. One set of specimens were observed directly after the fin-ished treatment sequence, and another set was observed after exposure to 2 wt% citric acid, to mimic the pH drop resulting after drinking e.g. an acidic beverage.

Table 3. Desensitizing products compared in Paper V. Product Manufacturer Technology

PSF + ACMP gel ACMP Sensodyne Repair & Protect GlaxoSmithKline Bioglass (NovaMin®) Colgate Sensitive PRO-Relief Colgate-Palmolive Arginine (Pro-Argin®) Oral-B Pro-Expert Procter&Gamble Stannous fluoride MI Paste Plus GC Corporation CPP-ACP (RECALDENT™) GUM SensiVital+ Sunstar HA Enamelon Preventive Treatment Gel Premier ACP

5.3 Characterization Multiple techniques were used for the characterization of synthesized samples and for the evaluation of specimens in the in vitro studies. The most important of the techniques used for imaging, determination of elemental composition and structure are described in the following sections in terms of underlying theories, and which information that can be retrieved from respective tech-nique.

5.3.1 Imaging High-resolution imaging using electron microscopy has been used extensively throughout the work in this thesis (Paper I, II, III, IV, and V). Electron micros-copy uses a focused electron beam as the source of illuminating radiation, and it is the short wavelength of the electrons that allows for imaging with high resolution.

Scanning electron microscopy (SEM) SEM can be used for surface analysis of samples. Imaging is enabled by scan-ning a focused electron beam in a raster pattern, where scattered electrons are used to create an image. Depending on the character of the interaction between the sample and the scattered electrons, various types of signals can be used to retrieve information about the sample. Secondary electrons (resulting from in-elastic scattering) are useful for imaging of surface structures and topology,

30

whereas backscattered electrons (resulting from elastic scattering) results in atomic contrast that can be used for studying compositional variations in ma-terials. SEM requires conductive samples to avoid charging that can disturb imaging and create artifacts. All samples analyzed in this thesis have been non-conductive, resulting in the need for coating with a conductive layer of Au/Pd prior to analysis.

Transmission electron microscopy (TEM) TEM can be used when the resolution in SEM is not high enough, and when information about the internal structure of the material is needed. As the name implies, TEM uses transmitted electrons to create an image. Using either elas-tically or inelastically scattered electrons, samples can be imaged either in bright field (BF) or dark field (DF) mode, respectively. These modes have different advantages, and preferred use depends on which sample characteris-tics that are of interest (e.g. morphology, crystal lattices, defects, and grain boundaries). TEM has successfully been used for the examination of the ul-trastructure of biological tissues such as bone and teeth [100–104]. BF-TEM was therefore used in Paper IV for the analysis of transverse cross-sections of the dentin specimens, with a focus on the mineralized material inside the tu-bules and the interface towards the PTD. TEM requires electron transparent samples (< 100 nm), which in Paper IV was achieved using focused ion beam (FIB). Samples were mounted on a Cu-lift out grid, and a focused beam of Ga+ was used for thinning of the samples (sputtering material off the surface), first with an acceleration voltage of 30 kV followed by a final polishing step using only 5 kV.

Scanning transmission electron microscopy (STEM) STEM combines the features from SEM and TEM. Imaging is performed in-side a TEM and images are created by transmitted electrons. In contrast to TEM, the electron beam is focused to a fine spot that is scanned in a raster pattern, as in SEM. STEM can be used as a separate imaging mode in TEM, and in combination with elemental analysis such as energy-dispersive X-ray spectroscopy (EDX), which was done in Paper IV.

31

5.3.2 Elemental composition Several different techniques can be used for elemental analysis of materials and solutions. Many of these are spectroscopic techniques based on the inter-action between photons or electrons and the sample, resulting in a signal that can be used for qualitative and/or quantitative analysis.

Inductively coupled plasma optical emission spectroscopy (ICP-OES) Adsorption and emission spectroscopy in the UV-visible wavelength range (190–900 nm) can favorably be used for elemental analysis when high sensi-tivity is needed. Among different techniques, ICP-OES is a powerful method for multi-elemental analysis of both solutions and solids. The technique was used in Paper I, II, and III to analyze elemental composition in the studied materials as well as ion concentrations in solution, i.e. in the degradation study in Paper III. ICP-OES requires atomization of samples, meaning that they need to be in solution. Solids, therefore, have to be digested (e.g. in acid) be-fore analysis. The setup of a typical ICP-OES is illustrated in Figure 5, but different samples may require different configurations of the instrument (e.g. radial or axial view of the plasma). The first step in the analysis procedure is that the sample passes through the nebulizer where an aerosol of fine droplets of the sample solution is created. Droplets that are small enough (sorted out in the spray chamber) are transported with a carrier gas (commonly Ar) to a plasma where the sample is atomized, sometimes also ionized, and excited. When relaxation occurs, light corresponding to the specific energy levels of the atoms and ions will be emitted, and the intensity of the light can be used for quantitative determination of the concentration of one or several specified elements.

Figure 5. Illustration of a typical ICP-OES setup with a radial plasma view (from the side). Used with permission from [105].

32

Energy-dispersive X-ray spectroscopy (EDX) The elemental composition of solid samples can also be determined using EDX. This technique is used within a SEM or STEM (Paper III and IV) and allows for elemental analysis of an area that can be imaged simultaneously. The technique is based on the emission of characteristic X-rays from a mate-rial arising as a result of exposure to an electron beam. The number and energy of the emitted X-rays are determined by an energy-dispersive spectrometer, and it is possible to achieve both qualitative and quantitative information from the sample. The sensitivity of EDX is generally relatively low, but the spatial resolution is higher in STEM in comparison to in SEM. Elemental mapping in STEM-EDX can therefore be performed with higher resolution.

5.3.3 Structural characterization

X-ray diffraction (XRD) Characterization of the crystal structure is of great importance to understand the origin of other material properties in a sample, such as morphology and degradation properties. Structural information of solids can be retrieved using XRD, which is based on the diffraction of light caused by the long-range order (periodic arrangement of atoms) in a crystalline material. When monochro-matic X-rays are incident on a crystalline material, the atomic planes in the crystal structure will cause elastic scattering of the photons. If the spacing be-tween the atomic planes in the structure is comparable to the wavelength of the X-rays, diffraction (constructive interference) will occur at certain angles (according to Bragg’s law). Diffracted light can be collected over a range of angles, and by using the intensity and scattering angle of the resulting diffrac-tion peaks, the crystal structure of the material can be determined. XRD was used in Paper I, II, and III to determine the crystal structure of synthesized materials and to follow the phase evolution in the degradation study (Paper III).

Small-angle X-ray scattering (SAXS) SAXS is based on the analysis of the elastic scattering behavior of X-rays when traveling through a material. The scattering is, as the name implies, rec-orded at low angles (0.1-10°), resulting in a 2D-scattering pattern that can be used for the characterization of materials at the nanoscale. Measurements can be made on many different types of samples including colloidal dispersions, gels, solids, etc. with the possibility of retrieving information such as nano-particle size distributions, particle shape, and structure, pore-size distribution, agglomeration, and self-assembly patterns, among others. SAXS was used in Paper I to study the formation of calcium phosphate core–shell particles.

33

6. Synthesis of core–shell particles

The following section summarizes the key results from the studies investigat-ing the synthesis of core–shell particles of calcium phosphate and other alka-line earth phosphates from aqueous solutions. The results are presented in terms of the mechanism of formation, and the role of the substituting ion (ionic radius and concentration) in the synthesis of the core–shell particles.

6.1 Mechanism of formation The results from Paper I and II showed that it was possible to synthesize core–shell particles of both calcium phosphate and other alkaline earth phosphates, given the use of appropriate salt concentrations and substituting ions. It ap-peared like the formation of the core–shell particles was not material specific, suggesting that the synthesis approach may be extended to other types of ma-terials if modified according to the solubility of the product and desired char-acteristics of the particles.

It was possible to follow the formation and morphological transformation of the core–shell particles by collecting precipitates at different time points during the reaction. Observation in SEM revealed that the formation of the particles in Paper I was initiated by the precipitation and self-assembly of pri-mary NPs around hollow cores, see Figure 6. The NPs had diameters of ~ 20–40 nm and morphologies resembling that of ACP (Figure 6a-b) [90,106]. With increasing reaction time, more NPs assembled and aggregated around the cores, forming complete shells after 2 hours (Figure 6c-f). The particles formed in Paper II showed the same characteristics and followed the same pattern of formation, with the exception that it was faster.

Observation of cross-sections of the core–shell particles in Paper I, con-firmed that they remained hollow after 24 hours, see Figure 7. The shells were composed of several layers of aggregated NPs, resulting in a shell thickness of ~ 250 nm. The cross-sections furthermore revealed that the particles could be connected in three different ways. Some were connected by the shells as a result of aggregation of particles after the adsorption of primary NPs (Figure 7a). Other particles had cores that were connected with the cores of adjacent particles (Figure 7b). Another interesting feature that was observed was in some particles whose hollow cores were connected by a “bridge”, constructed in the same way as the shells of the particles (Figure 7c).

34

Figure 6. SEM-micrographs of core–shell particles (calcium phosphate with Mg2+ and Sr2+) collected at (a-b) 5 min, (c) 40 min, (d) 75 min, (e) 2 hours, and (f) 24 hours after precipitation.

Figure 7. Cross-sections of core–shell particles (calcium phosphate with Mg2+ and Sr2+) embedded in resin. The SEM-micrographs show (a) particles connected by the shells, (b) particles with cores in direct connection with the cores of adjacent particles, and (c) particles connected by a “bridge”.

35

The characteristics of the formation of the core–shell particles and their mor-phological evolution were very similar to those in studies suggesting that gas bubbles in the reaction solution could have a templating function, as illustrated in Figure 4 [69,73,75]. Suggesting that this mechanism also applies to the for-mation of the core–shell particles in Paper I and II is somewhat speculative, but no other explanation is found applicable.

Common approaches presented for template-free or self-templating synthe-ses, such as Ostwald ripening or the Kirkendall effect, have been considered as explanations but have been rejected. Ostwald ripening is dependent on dis-solution, diffusion, and reprecipitation of material to form hollow particles [67,107]. It can therefore not explain the self-assembly of primary NPs that was observed early in the synthesis, shortly after precipitation (Figure 6a-b). The Kirkendall effect does not apply to the formation of particles with sizes observed in Paper I and II. Synthesis in aqueous solutions is furthermore most commonly dependent on regular diffusion or surface reactions rather than the Kirkendall effect [108].

Since no gas-forming precursor was used in the synthesis used in Paper I and II, the formation of gas bubbles could instead be explained by decreasing solubility of dissolved O2, N2, and/or CO2 upon heating (according to Henry’s law) [109]. Bubbles forming in the solution would be unstable due to their buoyancy and high surface tension [110]. Other studies reporting on the use of gas bubbles as soft templates claim that particle adsorption at the bubble surfaces is a result of minimization of interfacial energy [69,73,75]. This is similar to what has been reported for solid particles in Pickering emulsions and in foams [87,111,112]. Stabilization in these systems is explained by high adsorption energies of the solid particles that result in irreversible adsorption. The adsorption energy (∆Gads) of a particle at a gas–water interface can be described by: ∆ = / (1 − )

, where γg/w is the surface tension at the interface, r is the radius of the particle, and θ is the contact angle. This indicates that the potential adsorption of solid particles at small bubbles (with high surface tension) could reach very high adsorption energies, increasing the stability of the bubbles [112]. Supporting this is studies performed by Mohamedi et al. and Du et al. who showed that micrometer-sized bubbles could be stabilized by gold and silica NPs, respec-tively [113,114]. The formation of core–shell particles in Paper I and II was therefore interpreted as the simultaneous formation of gas bubbles and pri-mary NPs, where the templating function of the gas bubbles stems from their instability. The appearance of the cross-sections in Figure 7b-c indicates that aggregates/clusters of bubbles also could have a similar templating function, resulting in the formation of agglomerated core–shell particles. Adsorption of solid particles at curved interfaces has shown to be dependent on particle size,

36

shape, and concentration [115]. The spherical NPs that were observed in Pa-per I and II appeared to result in complete surface coverage with high packing density, which further highlights the importance of preventing rapid crystalli-zation.

Attempts to characterize the formation and function of the gas bubbles have been performed in work outside this thesis. These attempts have so far not allowed for characterization of the bubbles in situ, during the reaction. Fur-thermore, the greatest obstacle remaining is to find a characterization tech-nique that can distinguish between solid particles and bubbles in this type of bulk reaction.

6.2 The role of the substituting ion The formation of the core–shell particles in Paper I and II was shown to be highly dependent on the use of substituting ions. The synthesis approach al-lowed for the synthesis of not only calcium phosphates, but also strontium and barium phosphates. Having this in mind, together with the mechanism of for-mation described in the previous section, it appears like the key role of the substituting ions is to prevent rapid crystallization as well as prolong the life-time of the primary NPs of the amorphous phase. For calcium phosphates, it has been shown that substituting ions as a broad term can be used to inhibit crystal growth in HA and to stabilize ACP [7,46]. This is achieved by the replacement of Ca2+ ions in the crystal structure, or by adsorption of ions on the material surface (causing a shielding effect) [58,59]. Replacement of the Ca2+ depends on the radius and the concentration of a specific ion, i.e. the effect from different ions and their concentration in the synthesis could affect the formation of core–shell particles [46]. Since the formation of strontium and barium phosphates appeared to follow the same formation pattern as the calcium phosphates, the effect of the substituting ions likely plays a similar role in these materials.

6.2.1 Effect of ionic radius In Paper I, the effect of using Sr2+ and Mg2+ together and separately was com-pared in the synthesis of calcium phosphate. Combined use resulted in the formation of the particles shown in Figure 6, with smooth surfaces and diam-eters of 700 nm–1.5 µm. As can be seen in Figure 8, using only Sr2+ did not result in the formation of any core–shell particles, whereas comparable con-centrations of Mg2+ did. This indicates that the ionic radius indeed affects the stabilization of ACP, prevention of rapid crystal growth, and the ability to in-duce the formation of core–shell particles. Sr2+ that prefers substitution at the Ca(II) site can substitute for Ca2+ over the whole range of composition in HA without comprising the structure [46]. As a result of this, it cannot efficiently

37

stabilize ACP, which was reflected in the XRD results. Formation of HA was noted, but the peaks were shifted towards lower angles as a result of the ex-pansion of lattice parameters [116]. Following this was the lack of formation of core–shell particles in the synthesis where flake-like structures formed in-stead (Figure 8b). Mg2+, on the other hand, that instead favor substitution at the Ca(I) site, cannot be incorporated in the HA structure to the same extent. This results in stabilization of ACP, which allowed for the formation of core–shell particles with diameters between 400–800 nm (Figure 8c) and the for-mation of a WH structure upon crystallization [57].

Figure 8. SEM-micrographs showing the difference between synthesis outcome in reaction solutions (a) without substituting ions, (b) with 0.6 mM Sr2+, and (c) with 0.5 mM Mg2+.

In Paper II, the effect of different substituting ions was evaluated in strontium and barium phosphates. In contrast to Paper I, all substituting ions (Mg2+, Ca2+, and Sr2+) were smaller than the main constituent ion (i.e. Sr2+ or Ba2+). The difference in ionic radius was nonetheless reflected in the morphology, the critical concentration of the substituting ion to achieve core–shell particles, and the degree of crystallinity of the material. A greater difference in the ionic radius resulted in a lower concentration required to induce the formation of core–shell particles. This is illustrated in Figure 9, comparing the effect of Mg2+ and Ca2+ substitution in strontium phosphate. In this case, the lowest evaluated concentration of Mg2+ resulted in the formation of core–shell parti-cles, whereas the highest concentration was needed for Ca2+. Similarly, the difference in ionic radius was also reflected in the crystallinity of the synthe-sized materials. A small difference in radius resulted in crystallization, whereas the same concentration of the substituting ion, but with a large radius difference, promoted the amorphous phase to be sustained throughout the re-action. As for calcium phosphates, this is most likely a result of the distortion of the crystal structure and stabilization of the amorphous phase. The same trend was noted for the barium phosphates. The crystal structure of strontium and barium phosphates is, however, not as well explored as the calcium phos-phates. Therefore, preferred sites of substitution of different ions are not known, suggesting that further investigations are needed.

38

Figure 9. SEM-micrographs showing the morphology of strontium phosphates syn-thesized with with (a) 0.25 mM Mg2+, (b) 0.5 mM Mg+2 (inset showing a hollow par-ticle), (c) 0.9 mM Mg2+, and (c) 0.9 mM Ca2+.

6.2.2 Effect of concentration In previous work, it was shown that it was possible to alter the characteristics of the core–shell particles by altering the concentration of the substituting ion, in that case by changing the concentration of Sr2+ [88]. It was, however, not determined how the reaction conditions affected the actual composition in the formed material and the crystal structure.

By altering the concentration of Mg2+ in the synthesis of calcium phosphate in Paper I, the amount of Mg2+ substitution increased linearly with the con-centration (Figure 10a). Since the Ca2+ content decreased similarly, while the (Ca+Mg)/P ratio was kept more or less constant, it could be confirmed that it indeed was substitution of ions in the structure. It was, however, not possible to exclude the possibility that some of the detected Mg2+ potentially could have been surface adsorbed ions. As a result of the increasing degree of sub-stitution, the crystallinity of the material changed as well. As can be seen in Figure 10b, the samples with the two lowest Mg2+ concentrations were com-posed of WH, whose crystallinity decreased with increasing concentrations of Mg2+. When increasing the concentration further, the material became amor-phous. Similar observations have been made in other studies where Mg2+ has been shown to efficiently stabilize ACP as well as favored the crystallization of WH over HA when the substitution has been exceeding 10 at% [46,92].

39

Comparable observations were also made in Paper II when evaluating the ef-fect of different Mg2+ concentrations in the synthesis of strontium and barium phosphates (Figure 9).

As a result of the difference in crystallinity, the amount of incorporated water also varied among synthesized materials. The water content was deter-mined using thermogravimetric analysis (TGA) in Paper I. The analysis showed that the amorphous materials contained 18-19 wt% of water, which lies within the range of what previously has been reported for ACP [7,117]. The water loss was occurring in two steps corresponding to surface adsorbed water up to 125 °C, and chemically bound water between 225–450 °C (Figure 10c) [118,119]. The same trend was noted for the crystalline samples but the total amount of water was smaller, 11, and 14 wt% respectively.

Figure 10. Effects of varying Mg2+ concentrations in the reaction solutions showing (a) the atomic ratios (determined with ICP-OES), (b) XRD patterns, and (c) TGAcurves for the samples.

40

SAXS was used in Paper I to study the effect of different Mg2+ concen-trations on the formation of primary NPs before aggregation. Precipi-tates were analyzed in dilute particle-ethanol solutions to avoid poten-tial crystallization or aggregation caused by the drying of the particles. The results, in terms of size and size distribution, are summarized in Figure 11. Assuming that the primary NPs had a spherical shape (from the isotropic nature of ACP), employing a lognormal size distribution due to the lack of a clear modulation in the intensity of the SAXS data, the fitted results match the experimental data reasonably well (Figure 11a) [90]. The sizes of the particles varied between 39.7 ± 0.25 to 41.7 ± 4.3 nm in the analyzed samples (Figure 11b). This was in the same size-range as observed in SEM in Figure 6. Since all evaluated Mg2+ concentrations resulted in primary NPs with similar sizes, it can be as-sumed that the morphologies at a larger scale are a result of crystalliza-tion and crystal growth occurring after the formation of the shells. This suggests that the morphology, e.g. the smoothness of the particle sur-faces, does not stem from the size or shape of the primary NPs of ACP, but rather their composition.

Figure 11. Results from SAXS measurements of calcium phosphate samples with var-ying Mg2+ concentrations showing (a) experimental scattering data (void circles) and fitting results (solid lines) obtained by using the form factor of a sphere and a lognor-mal polydispersion assumption and (b) the polydispersity of the NPs modelled using lognormal size distribution functions.

41

7. Dentin occlusion and mineralization usingACMP particles

In this thesis, ACMP particles have been used as an occluding/mineralization agent to evaluate the possibility of using the material in the treatment of dentin hypersensitivity. This section of the thesis presents the results obtained in these studies.

7.1 Mode of action The mode of action of an occluding agent is the physical and/or chemical mechanism by which the material induces occlusion or mineralization. It was determined for the ACMP particles in Paper III to predict the in vivo behavior of the material and to estimate its efficiency for clinical use.

Ion release from the ACMP particles and the particle morphology, atomic composition, and phase evolution over time were evaluated up to 8 weeks of degradation in Tris-HCl buffer (pH 7.4 at 37 °C). Initially, all element con-centrations increased in the solution, see Figure 12. After 6 hours, the Ca con-centration started to decrease, whilst the Mg and P concentration continued to increase during 14 days. The release patterns and the atomic composition of the particles, where the Ca/P ratio was increasing and the Mg/Ca ratio was decreasing, indicated that the amorphous material initially crystallized with lower Mg content compared to the ACMP particles. The formation of HA was confirmed with XRD, where characteristic HA peaks appeared after 14 days (Figure 13). The continuous decrease in the concentration of all elements after 14 days, accompanied by increasing intensities of the diffraction peaks, indi-cated continuous formation and crystallization of HA. Observation of the transformation of the ACMP particles in SEM revealed that it followed a typ-ical route of transformation from ACP to HA [120]. It also showed that the formed HA had a nanocrystalline character with poor crystal definition throughout the study, which together with e.g. CO3

2– substitution could ex-plain the low degree of crystallinity noted in XRD even after 8 weeks (Figure 13).

42

Figure 12. Ion release and atomic composition of the ACMP particles determined with ICP-OES. Concentrations of Ca, Mg, and P in the solutions between (a) 30 min to 8 weeks and (b) 30 min to 24 h in detail. Atomic ratios in terms of (c) Ca/P and (d) Mg/Ca in the ACMP particles from 24 h to 8 weeks.

Figure 13. Diffraction patterns of the ACMP particles from the degradation study in Tris-HCl over 24 hours to 8 weeks.

The morphological evolution of the ACMP particles in vitro was evaluated after application of the gel containing the particles on the dentin-discs, 4 times during one day, followed by incubation in artificial saliva at 37 °C for up to 7

43

days. SEM observation of the dentin specimens showed that the particles ad-hered to the dentin surface as well as penetrated the tubules down to a depth of 100 µm (Figure 14a). The formation of nanocrystalline HA (nanoHA) was rapid on the dentin surface due to the supersaturated nature of saliva [121]. Complete coverage of the surface was noted already after 24 hours. Transfor-mation of the ACMP particles and mineralization appeared to follow the same route as in the degradation study in terms of morphology, but it was faster.

SEM observations of the longitudinal cross-sections of the specimens re-vealed that the transformation of the particles, and mineralization, were slower inside the tubules in comparison to on the surface. This was most likely caused by a gradient in ion concentrations, which was dependent on the amount of particles (being greater close to the dentin surface). Figure 14 illustrates the transformation of the ACMP particles and the formation of mineralized mate-rial inside the tubules, and clearly shows the different steps in the process (100–400 µm from the surface). After 12 hours, the ACMP particles lodged deeper down in the tubules remained unaffected in terms of morphology (Fig-ure 14b). After 24 hours, degradation was indicated by the roughening of the particle surfaces (Figure 14c). Mineralized material appeared on the tubule walls after 3 days (Figure 14d), and was complemented by mineralized mate-rial in the tubule volume after 5 days (Figure 14e-f). Complete intratubular occlusion of the tubules was noted after 7 days, with nanoHA filling the entire tubule volume (Figure 14g). Even if the ACMP particles were found to pene-trate the tubules down to a depth of 100 µm (Figure 9a-b), the intratubular mineralization was most likely also dependent on diffusion of ions and/or Pos-ner clusters that could induce in heterogeneous nucleation of HA [90].

Figure 14. Longitudinal cross-sections of dentin specimens treated with the ACMP particles. The micrographs are showing the evolution of the particles inside the tu-bules. Specimens were incubated in artificial saliva for (a-b) 12 h, (c) 24h, (d) 3 days, (e-f) 5 days, and (g) 7 days.

44

In summary, the mode of action of the ACMP particles can be assigned to two separate mechanisms. The mechanism considered to be the main contribution to the occlusion is the interaction between the saliva and the particles, result-ing in rapid release of Ca2+, Mg2+, and phosphate ions. The incorporation of Mg2+ in the ACMP particles appeared to have a stabilizing effect on the ACMP particles, similar to CPP stabilized ACP [36]. This allows for a con-tinuous release of ions that can trigger the nucleation and crystal growth of HA, which efficiently can contribute to both surface, and intratubular miner-alization. Additionally, just after application of the gel, the ACMP particles can physically block the exposed tubules, and thanks to their small particle size penetrate the tubules, resulting in intratubular occlusion as well.

7.2 Mineralization on peritubular dentin Most studies related to the occlusion and mineralization of dentin are re-stricted to observation of the dentin surface and longitudinal cross-sections using SEM or optical microscopy. To allow for observation of the ultrastruc-ture of dentin together with the interface between the mineralized material inside the tubules and the PTD, the crystal orientation of the mineralized ma-terial, and elemental composition, TEM and STEM-EDX was used in Paper IV as a complement to SEM.

Observation of an untreated sample revealed open tubules, both at the sur-face and in the longitudinal cross-sections (Figure 15a-c) as well as the trans-verse cross-sections (Figure 16a-c). The characteristic features of dentin were observed as well, with its collagenous framework in the ITD (appearing bright), and the more dense PTD lining the tubule openings (appearing darker). STEM-EDX spectral imaging revealed the compositional difference between the PTD and the ITD. The collagenous ITD showed higher intensities of C and N, whereas the PTD had higher intensities of Ca, P, and O, which indicated a higher mineral content (Figure 17). This was similar to what pre-viously has been found when examining dentin in SEM/STEM-EDX [122,123]. Despite the difference in elemental composition between the ITD and the PTD, the structures had similar Ca/P ratios ranging between 1.6–1.7, which is comparable to HA [7]. The differences between the ITD and the PTD should therefore be assigned to structural parameters such as grain size, the packing density of HA crystals, and differences in the volume fraction of the mineral phase [123].

The brighter regions observed between the tubule lumen and the PTD in the reference specimen are most likely sclerotic dentin forming as a result of aging or trauma, gradually closing the tubule lumens [124]. It usually has a lower density compared to normal dentin, explaining why it appeared brighter in TEM [125]. Unfortunately, the dentin discs used in Paper IV were taken from different molars, from different individuals, without any knowledge

45

about factors such as age and oral health. This could explain some of the dif-ferences noted among the used specimens that could not be assigned to the application of the ACMP particles.

The sample treated with the ACMP particles exhibited a completely oc-cluded surface with no trace of the underlying tubules, and intratubular min-eralization along the tubule length (Figure 15d-f). The appearance of the min-eralized material was similar to the morphology that was observed in Paper III, i.e. resembling poorly crystalline HA (Figure 16d-f). The STEM-EDX mapping revealed a high C content, which indicated CO3

2– substitution that partly could be explained by the high bicarbonate concentration in saliva (Fig-ure 17) [126]. Most of the tubules analyzed exhibited complete occlusion with mineralized material filling the entire tubule volume. Some tubules had an inner region of the tubule that remained open, showing that the mineralization was initiated on the PTD with crystals extending out in the tubule volume (Figure 16e). Continued mineralization resulted in densification of the mate-rial and the addition of material until the tubule was completely occluded (Fig-ure 16f). The fact that the intratubular mineralization appeared to be initiated on the tubule walls, resulted in good adherence of the mineralized material to the PTD, which could be of benefit for long-lasting pain relief when used in vivo.

Figure 15. SEM-micrographs of the surface and the longitudinal cross-sections of the dentin specimens. Images show the (a-c) untreated specimen, (d-f) the specimen treated with the ACMP particles, and (g-i) the specimen treated with PSF and the ACMP particles.

46

Figure 16. TEM-micrographs from the transverse cross-sections of dentin specimens. The left column shows images at low magnification as an overview of the specimens and the right columns show images with higher magnification of single tubules in the specimens of the (a-c) untreated specimen, (d-f) the specimen treated with the ACMP particles, and (g-i) the specimen treated with PSF and the ACMP particles.

7.3 Effect of fluoride treatment Due to the positive effects of fluoride use in dentistry, additional application of fluoride toothpaste was evaluated together with the application of the ACMP particles in Paper IV. Evaluation was performed in terms of composi-tion and character of the mineralization.

The effect of fluoride treatment was not apparent when examining the den-tin surface in SEM. However, observations of the longitudinal cross-sections in SEM (Figure 15h-i), and the transverse cross-sections in the TEM (Figure 16g-h), showed that it affected the mineralization inside the tubules. The min-eralized material exhibited increased crystal definition and aspect ratios in comparison to pure HA, and the crystal growth appeared to be directed to-wards the center of the tubule lumen. This could be explained by the incorpo-ration of F– in the mineralized material that was confirmed with STEM-EDX (Figure 17). The element map showed that the F content was concentrated to the center of the tubule lumen. Eanes and Meyer reported that F– substitution

47

in HA (prepared in aqueous solution) resulted in the formation of needle-like structures as a result of reduced growth of the (100) plane in the crystal struc-ture [127]. It appeared like the use of a fluoride toothpaste had such an effect on the intratubular mineralization even at depths down to 50 µm. The for-mation of the needle-like structures inside the tubules can therefore be inter-preted as preferential growth along the c-axis caused by F– substitution.

Incorporation of F– in the mineralized material furthermore lead to a densi-fication of the mineralized material, and it was not possible to distinguish the interface to the PTD (Figure 15h-i and Figure 16g-i). These aspects, together with the fact that F– substituted HA is known to have a lower solubility, could be beneficial for use in vivo [7].

Figure 17. EDX in STEM showing the elemental composition of the area closest to the tubule lumens. (a) STEM images indicating the analyzed regions, (b) elemental maps, and (c) EDX spectra (normalized against the P peak) for the untreated specimen (black), the specimen treated with ACMP (red), and the specimen treated with PSF and ACMP (blue).

48

7.4 Comparison to similar products available on the market To evaluate the occlusion performance and resistance of the mineralized ma-terial against acid attacks, treatment with the ACMP particles was compared with six other products available on the market (Table 3). Despite the number of available technologies and treatment options, few comprehensive studies are up to date.

The occluding performance of the ACMP particles was compared by ob-servation in SEM after finished treatment sequences. As can be seen in Figure 18, it was only the specimen treated with PSF and the ACMP particles that exhibited a complete surface occlusion with a mineralized layer covering the entire dentin surface. Treatment with the other products only resulted in oc-clusion that was dominated by particle deposition on the dentin surface. Sim-ilarly, it was only the same specimen that exhibited intratubular mineralization with good integration with the PTD. The key factor for pain-relief through reduction of the hydraulic conductance (movement of tubular fluid), is to re-duce the anatomic tubule radius [128]. This indicates that the occlusion and mineralization after treatment with PSF and the ACMP particles potentially could offer a higher degree of pain-relief compared to the other products.

The resistance towards acid attacks was evaluated by exposing one set of specimens to 2 wt% citric acid before observation in SEM. Acid resistance of the occluding material is of great importance since it can simulate the outcome of in vivo chemical erosion. Observation of the specimens, and comparison to the images collected prior to acid exposure, revealed differences in acid re-sistance of the evaluated products. The specimens treated with the Sensodyne Repair & Protect and Oral-B Pro-Expert toothpastes were least affected by a lowered pH, owing to the low solubility of the occluding agents in these prod-ucts, i.e. Bioglass and stannous fluoride (SnF2). Similar observations have been made in other studies, but it should be noted that comparisons between different studies with varying study designs (treatment sequence, treatment duration, approach evaluation of acid resistance, etc.) should be treated with caution [32,129]. Surface evaluation of the specimen treated with the PSF and the ACMP particles revealed that the acid exposure affected the mineralized material on the dentin surface since the tubule openings reappeared. The in-tratubular mineralization observed after this treatment could, however, miti-gate the effect of acid solubilization since the material lodged inside the tu-bules remained relatively unaffected for all evaluated products.

In summary, this comparison between desensitizing products reveals great variability in terms of the degree of occlusion and acid resistance among the evaluated products. Given the differences noted in this study, it would be of interest to complement the qualitative observations with quantitative studies to support the predictions regarding the efficiency for in vivo use.

49

Figure 18. Representative SEM micrographs of specimens treated with different oc-cluding agents. (a-b) PSF + ACMP gel, (c-d) Sensodyne Repair & Protect, (e-f) Col-gate PRO-Relief, (g-h) Oral-B Pro-Expert, (i-j) MI Paste Plus, (k-l) GUM SensiVital+ and (m-n) Enamelon Gel. The left panel shows images at low magnification and the right panel shows images taken at higher magnification.

50

8. Concluding remarks

The work presented in this thesis is based on five separate studies about the synthesis of ceramic core–shell particles and the use of those as an occluding agent in the treatment of dentin hypersensitivity. It contributes to an increased understanding of the synthesis approach as well as functional aspects of the use of calcium phosphate core–shell particles within dentistry.

By using the synthesis strategy presented in Paper I and II, it was shown to be possible to synthesize core–shell particles with diameters between 400 nm–1.5 µm (depending on reaction conditions). The formation was a result of the self-assembly of primary NPs around hollow cores, presumed to be gas bubbles that formed simultaneously in the reaction, functioning as soft tem-plates. The inherent instability of gas bubbles in solution drives the formation of core–shell particles since adsorption of primary NPs on the bubble surfaces lowers the interfacial energy and stabilizes the bubbles. Another key factor in the synthesis, allowing for a controlled self-assembly process, was the preven-tion of rapid crystal growth and prolonged lifetime of the amorphous primary NPs. This was achieved by including substituting ions in the precipitation re-actions. The radius of the substituting ion, together with its concentration was shown to be important factors affecting the formation of the core–shell parti-cles. Furthermore, it was demonstrated that these synthesis parameters could be altered to consciously modify the morphology, crystal structure, and com-position of the resulting core–shell particles. This could be of benefit when synthesizing these types of particles when targeting a specific application.

The synthesis approach was shown to apply, not only for calcium phos-phates but also to other alkaline earth phosphates, suggesting that it may be extended to other material categories as well. Modifications of the synthesis parameters related to the solubility of the product and desired properties of the particles should in that case be taken into account to achieve a successful out-come.

The in vitro studies that were performed to evaluate the use of the ACMP particles as an occluding/mineralization agent in Paper III, IV, and V, indi-cated that the material is a promising candidate for clinical use. In comparison to similar desensitizing products available on the market, the application of the ACMP particles resulted in complete occlusion of exposed tubules and intratubular mineralization could further enhance the resistance towards acid attacks. The occluding efficiency was assigned to the mode of action of the

51

material, i.e. interactions with saliva resulting in rapid and continuous release of Ca2+, Mg2+ and phosphate, inducing the crystallization of HA.

Investigations of the mineralization in relation to the ultrastructure of den-tin, revealed that the mineralized material was composed of poorly crystalline HA substituted with CO3

2–. The mineralized material was adhering well to the PTD, being a result of that the mineralization was initiated on the tubule walls, gradually closing the tubules. The degree of occlusion and the adherence of the mineralized material is important for the prevention of flow of tubular flu-ids and to achieve a long-lasting effect that can withstand the erosive, abrasive and mechanical challenges that can arise in clinical use.

The additional use of a fluoride toothpaste applied before the ACMP parti-cles was shown to result in the incorporation of F– in the mineralized material. This was expressed both in terms of morphology and elemental composition. Apart from the known benefits from F– incorporation in dental tissues (e.g. lowered solubility), these results show that the mechanism behind the intra-tubular occlusion i.e. that it is a result of both particle penetration and the dif-fusion of ion/Posner clusters with resulting heterogeneous nucleation.

52

9. Future outlooks

There is more to explore in the future when it comes to the synthesis of the core–shell particles. In situ analysis of the precipitation, self-assembly, and transformation of the particles would be of great interest to extend the knowledge about the synthesis approach. This could, for instance, be per-formed using synchrotron radiation (for structural analysis or imaging), allow-ing for fast acquisition time and high-resolution analysis within the short time-frame between initial precipitation and formation of the core–shell particles. Such analysis, especially if extended to later stages in the reaction as well, would allow for a better understanding of the relationship between the self-assembly patterns, structure and morphology.

Furthermore, the presence and function of the gas bubbles in the reaction need to be confirmed using a suitable characterization technique. This tech-nique not only has to capture the fast process of particle and bubble formation, but it also has to be able to distinguish between solid particles and gas bubbles to generate reliable results. If the existence of the bubbles cannot be confirmed or if it is rebutted, another reasonable explanation for the mechanism of for-mation of the core–shell particles should be suggested.

The in vitro studies of the occluding/mineralization performance of the ACMP particles indicated its potential clinical efficiency. These studies, how-ever, were restricted to qualitative evaluation, which manifests the need for additional quantitative analysis and comparisons. This could be performed e.g. by measuring and comparing the hydraulic conductance [98]. It would also be of interest to compare the outcome of using the ACMP particles with other types of treatment options. This could include e.g. laser treatments or the use of adhesive resins that have proven to be efficient in terms of pain-relief, but complicated and expensive due to the restricted use at dental prac-tices.

There is a need for standardization of test methods for in vitro evaluation to ensure accurate comparisons between different qualitative or quantitative studies. Today there is no consensus and specification on how e.g. the dentin-disc model should be used. As a result of this, there is great variability between studies regarding how the samples are treated before the application of the occluding agent, how the application of the products is performed, which treatment sequence that is used, and for how long the studies last. Similarly,

53

there is no standard set for testing the acid resistance. This makes the compar-ison between different studies complicated, and there is a risk of bias when comparing results between different studies.

As a follow-up on additional in vitro testing, clinical evaluation of using the ACMP particles would be important to prove the “real” efficiency of the occluding agent. The clinical efficiency of a desensitizing product can be de-termined by applying an artificial stimulus that can induce pain in sensitive teeth. The degree of pain is subsequently estimated based on a specific set scale. Tactile tooth sensitivity is evaluated based on the Yeaple probe index (using an electronic pressure-sensitive device), and the thermal/evaporative tooth sensitivity is evaluated based on the Schiff air index (using a blast of air) [130,131]. Comparison of the pain experienced before and after the finished treatment can be used to estimate the efficiency of the treatment.

54

Svensk sammanfattning

Munhälsa är bland de största problemen kopplade till folkhälsa världen över. En äldre befolkning, ändrad livsstil, med ökad konsumtion av socker, tobak och alkohol, har gjort att efterfrågan på dentalmaterial ökat. De används för behandling och lindring av en rad olika sjukdomar och tillstånd. Bland dessa är hypersensitivitet (ilningar i tänderna) ett utav de vanligast förekommande problemen. En stor del av den vuxna befolkningen i världen lider av detta i varierande grad.

Hypersensitivitet kan beskrivas som en kort och skarp smärta som uppstår när en tand utsätts för ett yttre stimulus som kan vara antingen taktilt, termiskt, osmotiskt eller evaporativt. Smärtan uppkommer som ett resultat av att dentin exponeras mot munhålan. Dentin är den hårda tandvävnad som omsluter tand-pulpan och som täcks av emaljen på kronan och av tandcementet på roten. Vävnaden består av en organisk del som till största del utgörs av kollagen och en mineraldel som består av kalciumfosfat. Det som gör dentin lätt att särskilja från andra hårdvävnader i tanden, är att det är uppbyggt av mikrometerbreda kanaler. Dessa sträcker sig från pulpan (den innersta delen av tanden) ända ut till emaljen. Exponering av dentin kan ske om emaljen löses upp eller nöts bort, eller som följd av tandköttsretraktion. När den vätska som finns inne i dentinkanalerna börjar röra på sig till följd av något av de stimuli som beskri-vits ovan, så stimuleras nerver i pulpan och smärta uppstår.

De tidiga behandlingsmetoderna för hypersensitivitet var baserade på an-vändandet av laserbehandlingar och syntetiska hartser (plaster). Dessa typer av behandlingsmetoder måste utföras på en tandvårdsklinik, vilket gör dem både dyra och komplicerade. Ett resultat av detta är att det under senare tid lagts mycket möda på att utveckla behandlingsalternativ som kan användas hemma och som då är både enklare och billigare. Intressanta alternativ för sådana behandlingar är att använda sig av partiklar som kan ockludera (täppa till) exponerade dentinkanaler och därmed förhindra flödet av vätska i dessa. Idag finns det flera typer av sådana produkter tillgängliga på marknaden i form av tandkrämer, geler, munskölj, etc. Effektiviteten hos dessa behandlingsme-toder är dock ofta låg. Detta beror på att partiklarna är för stora för att ta sig in i dentinkanalerna och/eller att de inte har egenskapen att ombildas till ett dentinliknande material. Ett intressant alternativ är därför att använda sig par-tiklar av kalciumfosfat som är tillräckligt små för att ta sig in i dentinakana-lerna och som vidare liknar mineraldelen i dentin.

55

Första delen av denna avhandling fokuserade på att undersöka och utveckla en syntesmetod för framställande av små ihåliga kalciumfosfatpartiklar där partiklarnas storlek, struktur och sammansättning kunde kontrolleras. Detta gjordes genom att använda sig av jonsubstitution vilket är ett naturligt före-kommande fenomen för kontroll av kristalltillväxt i flertalet mineraler, till ex-empel i kalciumfosfat i tänder och ben. Jonsubstitution innebär att man byter ut några joner i en ordnad kristallin struktur. Detta kan leda till att man kan styra hur materialet ser ut både på mikro- och nanometernivå. I kalciumfosfa-ter, som är sammansatta av kalciumjoner (Ca2+), fosfatjoner (PO4

3–), och ibland hydroxidjoner (OH–), kan man i stort sett byta ut andelar av alla typer av joner som ingår i materialet.

Tidigare arbete vid Uppsala universitet visade att tillsats av strontium- (Sr2+) och magnesiumjoner (Mg2+) i en utfällningsreaktion, kan möjliggöra för bildandet av små ihåliga sfäriska partiklar av kalciumfosfat. Syntes av kalci-umfosfater i vattenlösning inleds normalt av bildande av ett amorft (oordnat) material i form av kluster av nanopartiklar. Vid ett neutralt pH bildas hydroxy-apatit när det amorfa materialet kristalliserar. Denna process är vanligtvis re-lativt snabb. Genom att tillsätta substituerande joner är det dock möjligt att störa kristalliseringen och istället möjliggöra för bildandet av ihåliga sfäriska partiklar. För att undersöka vad det är som bestämmer egenskaperna hos par-tiklarna och mekanismen bakom hur de bildas, studerades effekterna av kon-centrationen av de substituerande jonerna och deras jonradie.

Bildandet av de ihåliga partiklarna visade sig vara ett resultat av ansamling av amorfa nanopartiklar, ~ 20–40 nm i diameter, runt ihåliga kärnor (se Figur 19). Nanopartiklarna växte med tiden samman och bildade kompletta skal runt kärnan som antogs vara bubblor av gas som bildats under reaktionen. I denna process visade sig både jonradien och koncentrationen vara viktiga för partik-larnas egenskaper. Koncentrationen av olika joner kunde användas för att kon-trollera egenskaper så som exempelvis ytstruktur, sammansättning och kristallinitet. Förutom att metoden möjliggjorde syntes av partiklar av kalci-umfosfat så visade det sig att även framställning av andra fosfater av de alka-liska jordartsmetallerna (strontium och barium) var möjlig. Detta indikerar att syntesmetoden skulle kunna fungera som en generell metod för framställning av liknande partiklar av andra materialtyper.

I den andra delen av avhandlingen undersöktes användandet av kalcium-fosfatpartiklar som ett alternativ för behandling av hypersensitivitet. Fram-ställningen av dessa liknade i stora drag den metod som presenterats ovan och partiklarna hade diametrar mellan 180–440 nm, samt en amorf struktur. För att undersöka partiklarnas förmåga att ockludera exponerade dentinkanaler så studerades de in vitro, det vill säga i en miljö som efterliknar den i munnen. Partiklarna applicerades i form av en gel på provskivor kapade från mänskliga tänder. Proverna studerades sedan i elektronmikroskop, både på ytan och i genomskärning, för att kunna observera strukturer ner på nanometernivå.

56

Partiklarna fäste väl på dentinytan och de kunde även ta sig djupt ner i dentin-kanalerna. En annan fördel var att de snabbt ombildades till ett lager av mine-raliserad hydroxyapatit som täckte hela dentinytan och som fyllde ut dentin-kanalerna, även djupt ner under ytan. Detta indikerade att partiklarna och de-ras omvandling, mest troligt skulle kunna förhindra flödet av vätska i dentin-kanelaerna på ett effektivt sätt. Vidare studier visade även att en kompletterande behandling med en fluortandkräm skulle kunna förbättra ut-fallet ytterligare. Fluorjoner (F–) från tandkrämen inkorporerades i det mine-raliserade materialet, vilket kan ha en rad positiva effekter, bland annat mins-kad löslighet.

Effektiviteten hos partiklarna jämfördes slutligen med sex andra liknande produkter som finns tillgängliga på marknaden. Detta för att kvalitativt jäm-föra deras effekt och väl hur de kan motstå syraattacker som uppstår vid kon-sumtion av exempelvis sura drycker (så som juice eller kaffe). Undersök-ningen visade att det endast var kalciumfosfatpartiklarna som resulterade i en fullständig ockludering av dentinytan. Det mineraliserade materialet som bil-dats i dentinkanalerna klarade dessutom av att motstå en artificiell syraattack vilket indikerar en möjlig långvarig effekt.

Sammanfattningsvis så har arbetet i denna avhandling lett till en ökad för-ståelse för framställningen av ihåliga sfäriska partiklar av kalciumfosfat och möjligheterna att kontrollera deras egenskaper under syntesen. Kalciumfos-fatpartiklar framställda baserat på denna metod, visade sig dessutom vara ett möjligt effektivt alternativ för behandling av hypersensitivitet.

Figur 19. En bild tagen med ett svepelektronmikroskop som visar bildandet av ihåliga kalciumfosfatpartiklar.

57

Acknowledgements

First and foremost, I would like to thank my supervisors Wei and Håkan. Wei, thank you for introducing me to the world of biomaterials and for all your support throughout these years. I am very impressed with your drive and es-pecially your curiosity for research. Whenever I have been in doubt about my results or failing experiments you have always replied with “this is interest-ing!”. I should learn from you. Håkan, thank you for inspiring talks and for pushing me in the right direction when needed. I must admit that I was a little bit afraid of you in the beginning, but that is now long gone and replaced with admiration for your way of turning research into reality.

Erik, thank you for all the help and encouragement I have received from you. Thanks for commenting on papers, providing me with samples, and for being calm and rational whenever I have been stressed or maybe a bit too emotional. Thanks to Lars Riekehr for all the help with the TEM analysis, and the discussions about all the “fun” things one can explore within the world of biological samples. Thanks to Shun Yu at RISE for the help with data analysis and Andreas Thor for a nice collaboration regarding teeth and cakes.

At Ångström, there are many people I would like to express my gratitude to. Amina and Susanne, I am so happy that I have had your company and sup-port during these years. Things would not have been the same without you. Amina, thank you for being an excellent office companion (alla är dumma), cleaning police, and company during outdoor adventures (sleeping outside is never a bad idea). It is difficult to find someone more caring than you. Su-sanne, thank you for great travelling company (Schiphol is a decent place), run-day-fun-days (I will learn how to run one day), and for your endless amount of encouragement and help. Seeing you finish your PhD was truly inspirational and I am so impressed with all your hard work. Thanks to Tor-björn for nice fika-times and walks. It has been nice to have someone that can be equally excited about a sleeping bag or anything else belonging to the out-doors. Alejandro, thank you for keeping everything in order in the lab and for answering one million questions. May the ICP live on without our love! Thanks to Giannis and Salim for giving us something to look forward to. You did not disappoint me on your arrival and I have really appreciated your com-pany and updates on the latest observations (gossip). Hanna, thank you for spreading positive vibes and for being so nice and friendly. Thanks to Char-lotte for nice chats and for bringing interesting topics of conversation to lunches and fika. Many thanks to Céline A for making my first year as a PhD

58

student eventful and fun. Zürich and Uppsala is too far apart. To all the other present and former members of the MiM and BMS groups: Cecilia, Caroline, Estefanía, Ana, Lisa, Andrea, Alexandra, Yijun, Huasi, Michael, Marjam, Lui-mar, Lee, Anna, Elin, Magali, Roger, Le, Jun, Céline R, Dan, Oscar, and the rest, thank you for making Ångström a nice workplace. I have really appreci-ated your company in the lab and outside work. Thanks to all the people in the Tribo group and at Chemistry and MST for fun times both at work and outside Ångström. Thanks to Anders and Carl Johan for good company when sharing office (not trash bins) and for helping out with all sorts of things. A special thanks to Gabriel, my favorite German person, for all the nice coffee breaks and for bringing some proper engineering to the table when needed. Thanks to Jonatan for all the help with computers and things that I know nothing about and thanks to Sara and Ingrid R for help with administrative stuff.

Ett stort tack till Johanna R för hjälpen med den fina framsidan till den här avhandlingen. Tur att det finns folk som har bra mycket mer sinne för estetik än oss fyrkantiga ingenjörer!

Ett stort tack till alla mina vänner för trevligt sällskap, upptåg och upp-muntran. Det hade sannerligen inte gått lika bra utan er och jag hoppas verk-ligen vi snart kan umgås lite mer än den senaste tiden. Tack till Johanna och Micke för att er dörr alltid står öppen och för att ni alltid ställer upp, vare sig det gäller praktikaliteter eller själavård. BengtHans kan man alltid lita på! Tack Ida, Amanda och Liora för all pepp, trevliga stunder och strapatser. Det är skönt att veta att ni finns där, både på telefon men också live när tillfälle ges. Uppsala är inte detsamma utan er. Voltigen, både människor och hästar, ska också ha ett stort tack för att ha skänkt mig mycket glädje under alla år och för att jag har haft något roligt att hänga upp livet på utanför jobbet.

Ett stort tack till min familj som ställer upp i vått och torrt och som kan få de allra gråaste dagarna att kännas soliga. Mamma, jag önskar att du hade kunnat vara med idag och alla andra dagar. Jag tänker ändå att det var tur att du visade med mig hur man navigerar i universitetsvärlden. Diverse sångsnut-tar, placering av papperslappar och sättet att ta sig an problem har fått det hela att kännas lite mindre som en svårforcerad djungel. Det väger såklart inte upp till hur mycket jag saknar dig. Tack till Mormor för att du alltid är intresserad av vad jag gör, hur det går och hur jag mår. Framförallt så tycker jag att du ger mig perspektiv på vad som är viktigt i livet (eller jag kanske inte alltid tycker fönsterputs är superviktigt). Jag säger som jag brukar: det är tur att vi har varandra! Ett stort tack till Pappa som tålmodigt stått ut med att läsa ige-nom allt som jag någonsin skrivit, från första klass med uppochnedvända bok-stäver, till det sista ordet i kappan. Tack för ditt stöd och fix med mat, fika, och naturkul i allmänhet, men framförallt de gånger när det verkligen behövts. I lav you! Ett minst lika stort tack till min storasyster Hannah (Dalin!). Vad livet skulle vara utan dig vet jag inte. Förmodligen tomt, tråkigt och alldeles värdelöst. Tack för att du aldrig är längre bort än ett telefonsamtal (fast borde vi inte bo närmare varandra?) och för att du förstår dig på mig till och med de

59

gånger när jag inte ens gör det själv. August, tack för att du har stått ut med mig den senaste tiden och för att du peppat och styrt upp tillvaron. Din optim-ism, påhittighet och kärlek har gjort livet så mycket bättre och jag är så glad att jag får vara med just dig. Nu väntar ljusare tider!

60

References

[1] P.E. Petersen, Challenges to improvement of oral health in the 21st century - The approach of the WHO Global Oral Health Programme, Int. Dent. J. 54 (2004) 329–343.

[2] C.H. Splieth, A. Tachou, Epidemiology of dentin hypersensitivity, Clin. Oral Investig. 17 (2013) 3–8.

[3] R. Orchardson, Managing dentin hypersensitivity, J. Am. Dent. Assoc. 137 (2006) 990–999.

[4] M. Bongio, J.J.J.P. Van Den Beucken, S.C.G. Leeuwenburgh, J.A. Jansen, Development of bone substitute materials: From “biocompatible” to “instructive,” J. Mater. Chem. 20 (2010) 8747–8759.

[5] W. Habraken, P. Habibovic, M. Epple, M. Bohner, Calcium phosphates in biomedical applications: Materials for the future?, Mater. Today. 19 (2016) 69–87.

[6] Y. Cai, R. Tang, Calcium phosphate nanoparticles in biomineralization and biomaterials, J. Mater. Chem. 18 (2008) 3775–3787.

[7] S. V Dorozhkin, Calcium orthophosphates: Occurrence, properties, biomineralization, pathological calcification and biomimetic applications, Biomatter. 1 (2011) 121–164.

[8] D.F. Kinane, P.G. Stathopoulou, P.N. Papapanou, Periodontal diseases, Nat. Rev. Dis. Prim. 3 (2017) 1–14.

[9] C. Dawes, What is the critical pH and why does a tooth dissolve in acid?, J. Can. Dent. Assoc. 69 (2003) 722–724.

[10] J.L. Cuy, A.B. Mann, K.J. Livi, M.F. Teaford, T.P. Weihs, Nanoindentation mapping of the mechanical properties of human molar tooth enamel, Arch. Oral Biol. 47 (2002) 281–291.

[11] SMART - Servier Medical Art, (n.d.). [12] A. Nanci, Ten Cate’s Oral Histology: Development, Structure and Function,

8th ed., 2012. [13] A. Lussi, N. Schlueter, E. Rakhmatullina, C. Ganss, Dental erosion - An

overview with emphasis on chemical and histopathological aspects, Caries Res. 45 (2011) 2–12.

[14] R. Schilke, J.A. Lisson, O. Bauß, W. Geurtsen, Comparison of the number and diameter of dentinal tubules in human and bovine dentine by scanning electron microscopic investigation, Arch. Oral Biol. 45 (2000) 355–361.

[15] T.L. Lenzi, C.D.A.B. Guglielmi, V.E. Arana-chavez, D.P. Raggio, M icroscopy M icroanalysis Tubule Density and Diameter in Coronal Dentin from Primary and Permanent Human Teeth, (2006) 1–5.

[16] M. Goldberg, A.B. Kulkarni, M. Young, A. Boskey, Dentin: structure, composition and mineralization, Front. Biosci. (Elite Ed). 3 (2011) 711–35.

61

[17] J.C. Voegel, R.M. Frank, Ultrastructural study of apatite crystal dissolution in human dentine and bone, J. Biol. Buccale. 5 (1977) 181–194.

[18] E. Johansen, R.F. Parks, Electron-microscopic observations on sound human dentin stance of dentine, Arch. Oral Biol. 7 (1962) 185–193.

[19] L. Tjäderhane, M.R. Carrilho, L. Breschi, F.R. Tay, D.H. Pashley, Dentin basic structure and composition-an overview, Endod. Top. 20 (2009) 3–29.

[20] C. Recommendations, D. Hypersensitivity, Consensus-based recommendations for the diagnosis and management of dentin hypersensitivity., J. Can. Dent. Assoc. (Tor). 69 (2003) 221–226.

[21] B.G. Smith, Toothwear: aetiology and diagnosis, Dent. Update. 16 (1989) 204–212.

[22] N.X. West, A. Lussi, J. Seong, E. Hellwig, Dentin hypersensitivity: Pain mechanisms and aetiology of exposed cervical dentin, Clin. Oral Investig. 17 (2013) 9–19.

[23] M. Brännström, The hydrodynamics of the dental tubule and of pulp fluid. A discussion of its significance in relation to dentinal sensitivity, Caries Res. 1 (1967) 310–317.

[24] Y. Kimura, P. Wilder-Smith, K. Yonaga, K. Matsumoto, Treatment of dentine hypersensitivity by lasers: A review, J. Clin. Periodontol. 27 (2000) 715–721.

[25] A. V. Ritter, W.D.L. Dias, P. Miguez, D.J. Caplan, E.J. Swift, Treating cervical dentin hypersensitivity with fluoride varnish: A randomized clinical study, J. Am. Dent. Assoc. 137 (2006) 1013–1020.

[26] A. Gaffar, Treating hypersensitivity with fluoride varnish., Compend. Contin. Educ. Dent. 20 (1999) 27–33; quiz 35.

[27] G.D. Canali, S.A. Ignacio, R.N. Rached, E.M. Souza, Clinical efficacy of resin-based materials for dentin hypersensitivity treatment., Am. J. Dent. 30 (2017) 201–204.

[28] R. Orchardson, D.G. Gillam, Managing dentin hypersensitivity, J. Amarican Dent. Assiciation. 137 (2014) 990–998.

[29] K. Markowitz, G. Bilotto, S. Kim, Decreasing intradental nerve activity in the cat with potassium and divalent cations, Arch. Oral Biol. 36 (1991) 1–7.

[30] J.M. Peacock, R. Orchardson, Action potential conduction block of nerves in vitro by potassium citrate, potassium tartrate and potassium oxalate, J. Clin. Periodontol. 26 (1999) 33–37.

[31] D.H. Pashley, F.R. Tay, Dentine Hypersensitivity: Current State of the Art and Science, Dentine Hypersensitivity Consens. Monogr. 4 (2008) 1–12.

[32] A. Burwell, D. Jennings, D. Muscle, D.C. Greenspan, NovaMin and dentin hypersensitivity-in vitro evidence of efficacy., J. Clin. Dent. 21 (2010) 66–71.

[33] S.A. Lavender, I. Petrou, R. Heu, M.A. Stranick, D. Cummins, L. Kilpatrick-Liverman, R.J. Sullivan, R.P. 3rd Santarpia, Mode of action studies on a new desensitizing dentifrice containing 8.0% arginine, a high cleaning calcium carbonate system and 1450 ppm fluoride, Am. J. Dent. 23 Spec No (2010) 14A-19A.

62

[34] I. Petrou, R. Heu, M. Stranick, S. Lavender, L. Zaidel, D. Cummins, R.J. Sullivan, C. Hsueh, J.K. Gimzewski, A breakthrough therapy for dentin hypersensitivity: how dental products containing 8% arginine and calcium carbonate work to deliver effective relief of sensitive teeth, J. Clin. Dent. 20 (2009) 23–31.

[35] S. Miller, T. Truong, R. Heu, M. Stranick, D. Bouchard, A. Gaffar, Recent advances in stannous fluoride technology: antibacterial efficacy and mechanism of action towards hypersensitivity, Int. Dent. J. 44 (1994) 83–98.

[36] E.C. Reynolds, Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions, J. Dent. Res. 76 (1997) 1587–1595.

[37] N.J. Cochrane, E.C. Reynolds, Advances in Dental Research Calcium Phosphopeptides — Mechanisms of Action and Evidence for Clinical Efficacy, Adv. Dent. Res. 24 (2012) 41–47.

[38] B.T. Amaechi, S.M. Mathews, K. Ramalingam, P.K. Mensinkai, Evaluation of nanohydroxyapatite-containing toothpaste for occluding dentin tubules, Am. J. Dent. 28 (2015) 33–39.

[39] S. Kardos, B. Shi, T. Sipos, The in vitro demineralization potential of a sodium fluoride, calcium and phosphate ion-containing dentifrice under various experimental conditions, J. Clin. Dent. 10 (1999) 22–25.

[40] A. Winston, N. Usen, Processes for the remineralization and mineralization of teeth, US6036944, 2000.

[41] T. Imfeld, Dental erosion. Definition, classification and links, European. 104 (1996) 151–155.

[42] J.M. Ten Cate, Contemporary perspective on the use of fluoride products in caries prevention, Br. Dent. J. 214 (2013) 161–167.

[43] N. Garg, A. Garg, Textbook of operative dentistry, in: Third, Jaypee/The Health Sciences Publisher, New Delhi, 2015.

[44] S. V. Dorozhkin, Calcium orthophosphates in dentistry, J. Mater. Sci. Mater. Med. 24 (2013) 1335–1363.

[45] M. Bohner, Calcium orthophosphates in medicine: From ceramics to calcium phosphate cements, Injury. 31 (2000).

[46] E. Boanini, M. Gazzano, A. Bigi, Ionic substitutions in calcium phosphates synthesized at low temperature, Acta Biomater. 6 (2010) 1882–1894.

[47] J.C. Elliot, Structure and Chemistry of the Apatites and Other Calcium Orthophosphates, J. Am. Chem. Soc. 118 (1996) 3072.

[48] R.Z. LeGeros, O.R. Trautz, E. Klein, J.P. LeGeros, Two types of carbonate substitution in the apatite structure, Experientia. 25 (1969) 5–7.

[49] H. Pan, B.W. Darvell, Effect of carbonate on hydroxyapatite Solubility, Cryst. Growth Des. 10 (2010) 845–850.

[50] R. Zapanta-LeGeros, Effect of Carbonate on the Lattice Parameters of Apatite, Nature. 006 (1965) 403–404.

[51] L.M. Rodríguez-Lorenzo, J.N. Hart, K.A. Gross, Structural and chemical analysis of well-crystallized hydroxyfluorapatites, J. Phys. Chem. B. 107 (2003) 8316–8320.

63

[52] S. V. Dorozhkin, Calcium orthophosphates: occurrence, properties, biomineralization, pathological calcification and biomimetic applications, Biomatter. 1 (2011) 121–164.

[53] E.C. Moreno, M. Kresak, R.T. Zahradnik, Fluoridated hydroxyapatite solubility and caries formation, Nature. 247 (1974) 64–65.

[54] T. Aoba, The effect of fluoride on apatite structure and growth, Crit. Rev. Oral Biol. Med. 8 (1997) 136–153.

[55] A. Bigi, G. Falini, E. Foresti, M. Gazzano, A. Ripamonti, N. Roveri, Rietveld Structure Refinements of Calcium Hydroxylapatite Containing Magnesium, Acta Crystallogr. Sect. B Struct. Sci. 52 (1996) 87–92.

[56] A. Bigi, E. Boanini, C. Capuccini, M. Gazzano, Strontium-substituted hydroxyapatite nanocrystals, Inorganica Chim. Acta. 360 (2007) 1009–1016.

[57] H.L. Jang, K. Jin, J. Lee, Y. Kim, S.H. Nahm, K.S. Hong, K.T. Nam, Revisiting whitlockite, the second most abundant biomineral in bone: Nanocrystal synthesis in physiologically relevant conditions and biocompatibility evaluation, ACS Nano. 8 (2014) 634–641.

[58] A.L. Boskey, A.S. Posner, Magnesium stabilization of amorphous calcium phosphate: a kinetic study, Mater. Res. Bull. 9 (1974) 907–916.

[59] H. Ding, H. Pan, X. Xu, R. Tang, Toward a detailed understanding of magnesium ions on hydroxyapatite crystallization inhibition, Cryst. Growth Des. 14 (2014) 763–769.

[60] L.W. Fisher, J.D. Termine, Noncollagenous Proteins Influencing the Local Mechanisms of Calcification, Clin. Orthopeadics Relat. Res. 200 (1985) 362–385.

[61] J.P. Gorski, Biomineralization of bone: A fresh view of the roles of non-collagenous proteins, Front. Biosci. 16 (2011) 2598–2621.

[62] M.D. Mckee, B. Hoac, W.N. Addison, N.M.T. Barros, J.L. Millán, C. Chaussain, Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia, Periodontol. 2000. 63 (2013) 102–122.

[63] M.J. Olszta, X. Cheng, S.S. Jee, R. Kumar, Y.Y. Kim, M.J. Kaufman, E.P. Douglas, L.B. Gower, Bone structure and formation: A new perspective, Mater. Sci. Eng. R Reports. 58 (2007) 77–116.

[64] K. Lin, C. Wu, J. Chang, Advances in synthesis of calcium phosphate crystals with controlled size and shape, Acta Biomater. 10 (2014) 4071–4102.

[65] M. Sadat-Shojai, M.T. Khorasani, E. Dinpanah-Khoshdargi, A. Jamshidi, Synthesis methods for nanosized hydroxyapatite with diverse structures, Acta Biomater. 9 (2013) 7591–7621.

[66] J. Hu, M. Chen, X. Fang, L. Wu, Fabrication and application of inorganic hollow spheres, Chem. Soc. Rev. 40 (2011) 5472–5491.

[67] C.C. Yec, H.C. Zeng, Synthesis of complex nanomaterials via Ostwald ripening, J. Mater. Chem. A. 2 (2014) 4843–4851.

[68] X. Zhang, W. Zhang, Z. Yang, Z. Zhang, Nanostructured hollow spheres of hydroxyapatite: Preparation and potential application in drug delivery, Front. Chem. Sci. Eng. 6 (2012) 246–252.

64

[69] Y.S. Han, G. Hadiko, M. Fuji, M. Takahashi, A novel approach to synthesize hollow calcium carbonate particles, Chem. Lett. 34 (2005) 152–153.

[70] G. Qu, B. Tian, C. Su, Y. Tang, Y. Li, Bubble-assisted fabrication of hollow CoMoO4 spheres for energy storage, Chem. Commun. 54 (2018) 10355–10358.

[71] C. Zhou, Y. Zhao, T. Bian, L. Shang, H. Yu, L.Z. Wu, C.H. Tung, T. Zhang, Bubble template synthesis of Sn2Nb2O7 hollow spheres for enhanced visible-light-driven photocatalytic hydrogen production, Chem. Commun. 49 (2013) 9872–9874.

[72] H. You, L. Zhang, Y. Jiang, T. Shao, M. Li, J. Gong, Bubble-supported engineering of hierarchical CuCo2S4 hollow spheres for enhanced electrochemical performance, J. Mater. Chem. A. 6 (2018) 5265–5270.

[73] M. Mozafari, F. Moztarzadeh, A.M. Seifalian, L. Tayebi, Self-assembly of PbS hollow sphere quantum dots via gas-bubble technique for early cancer diagnosis, J. Lumin. 133 (2013) 188–193.

[74] J. Liu, D. Xue, Solvothermal synthesis of CuS semiconductor hollow spheres based on a bubble template route, J. Cryst. Growth. 311 (2009) 500–503.

[75] X. Zhang, Q. Zhao, Y. Tian, Y. Xie, Large scale fabrication of hollow palladium nanospheres by template-free approach, Chem. Lett. 33 (2004) 244–245.

[76] S. Tang, S. Vongehr, X. Wang, Y. Wang, X. Meng, Bubble-assisted growth of hollow palladium nanospheres with structure control allowing very thin shells for highly enhanced catalysis, RSC Adv. 4 (2014) 13729–13732.

[77] X. Li, Y. Xiong, Z. Li, Y. Xie, Large-scale fabrication of TiO2 hierarchical hollow spheres, Inorg. Chem. 45 (2006) 3493–3495.

[78] P.S. Epstein, M.S. Plesset, On the Stability of Gas Bubbles in Liquid‐Gas Solutions, J. Chem. Phys. 18 (1950) 1505–1509.

[79] G.H. Kelsall, S. Tang, S. Yurdakult, A.L. Smith, Electrophoretic beahviour of bubbles in aqueous electrolytes, J. Chem. SOC., Faraday Trans. 92 (1996) 3887–3893.

[80] M.R. Ghaani, P.G. Kusalik, N.J. English, Massive generation of metastable bulk nanobubbles in water by external electric fields, Sci. Adv. 6 (2020) 1–7.

[81] N.F. Bunkin, A. V. Shkirin, N. V. Suyazov, V.A. Babenko, A.A. Sychev, N. V. Penkov, K.N. Belosludtsev, S. V. Gudkov, Formation and Dynamics of Ion-Stabilized Gas Nanobubble Phase in the Bulk of Aqueous NaCl Solutions, J. Phys. Chem. B. 120 (2016) 1291–1303.

[82] H. Zhang, Z. Guo, X. Zhang, Surface enrichment of ions leads to the stability of bulk nanobubbles, Soft Matter. 16 (2020) 5470–5477.

[83] F. Eklund, J. Swenson, Stable Air Nanobubbles in Water: the Importance of Organic Contaminants, Langmuir. 34 (2018) 11003–11009.

[84] D.E. Yount, On the elastic properties of the interfaces that stabilize gas cavitation nuclei, J. Colloid Interface Sci. 193 (1997) 50–59.

[85] D.E. Yount, Skins of varying permeability: A stabilization mechanism for gas cavitation nuclei, J. Acoust. Soc. Am. 65 (1979) 1429–1439.

65

[86] F.E. Fox, K.F. Herzfeld, Gas Bubbles with Organic Skin as Cavitation Nuclei, J. Acoust. Soc. Am. 26 (1954) 984–989.

[87] Y. Yang, Z. Fang, X. Chen, W. Zhang, Y. Xie, Y. Chen, Z. Liu, W. Yuan, An overview of pickering emulsions: Solid-particle materials, classification, morphology, and applications, Front. Pharmacol. 8 (2017) 1–20.

[88] W. Xia, K. Grandfield, A. Schwenke, H. Engqvist, Synthesis and release of trace elements from hollow and porous hydroxyapatite spheres, Nanotechnology. 22 (2011) 1–10.

[89] J. Zhao, Y. Liu, W. Bin Sun, H. Zhang, Amorphous calcium phosphate and its application in dentistry, Chem. Cent. J. 5 (2011) 40.

[90] C. Combes, C. Rey, Amorphous calcium phosphates: Synthesis, properties and uses in biomaterials, Acta Biomater. 6 (2010) 3362–3378.

[91] N.C. Blumenthal, F. Betts, A.S. Posner, Stabilization of amorphous calcium phosphate by Mg and ATP, Calcif. Tissue Res. 23 (1977) 245–250.

[92] C. Qi, Y.J. Zhu, F. Chen, J. Wu, Porous microspheres of magnesium whitlockite and amorphous calcium magnesium phosphate: microwave-assisted rapid synthesis using creatine phosphate, and application in drug delivery, J. Mater. Chem. B. 3 (2015) 7775–7786.

[93] M. Iafisco, L. Degli Esposti, G.B. Ramírez-Rodríguez, F. Carella, J. Gómez-Morales, A.C. Ionescu, E. Brambilla, A. Tampieri, J.M. Delgado-López, Fluoride-doped amorphous calcium phosphate nanoparticles as a promising biomimetic material for dental remineralization, Sci. Rep. 8 (2018) 1–9.

[94] K. He, M. Sawczyk, C. Liu, Y. Yuan, B. Song, R. Deivanayagam, A. Nie, X. Hu, V.P. Dravid, J. Lu, C. Sukotjo, Y.P. Lu, P. Krá, T. Shokuhfar, R. Shahbazian-Yassar, Revealing nanoscale mineralization pathways of hydroxyapatite using in situ liquid cell transmission electron microscopy, Sci. Adv. 6 (2020) 1–12.

[95] A.L. Boskey, A.S. Posner, Conversion of amorphous calcium phosphate to microcrystalline hydroxyapatite. A pH-dependent, solution-mediated, solid-solid conversion, J. Phys. Chem. 77 (1973) 2313–2317.

[96] N.J. Mordan, P.M. Barber, D.G. Gillam, The dentine disc. A review of its applicability as a model for the in vitro testing of dentine hypersensitivity, J. Oral Rehabil. 24 (2008) 148–156.

[97] D.G. Gillam, N.J. Mordan, H.N. Newman, The Dentin Disc surface: a plausible model for dentin physiology and dentin sensitivity evaluation, Adv. Dent. Res. 11 (1997) 487–501.

[98] O.W. Reeder, R.E. Walton, M.J. Livingston, D.H. Pashley, Dentin Permeability: Determinants of Hydraulic Conductance, J. Dent. Res. 57 (1978) 187–193.

[99] R.J.M. Lynch, R. Navada, R. Walia, Low-levels of fluoride in plaque and saliva and their effects on the demineralisation and remineralisation of enamel; role of fluoride toothpastes, Int. Dent. J. 54 (2004) 304–309.

[100] N. Reznikov, R. Shahar, S. Weiner, Three-dimensional structure of human lamellar bone: The presence of two different materials and new insights into the hierarchical organization, Bone. 59 (2014) 93–104.

66

[101] R.K. Nalla, A.E. Porter, C. Daraio, A.M. Minor, V. Radmilovic, E.A. Stach, A.P. Tomsia, R.O. Ritchie, Ultrastructural examination of dentin using focused ion-beam cross-sectioning and transmission electron microscopy, Micron. 36 (2005) 672–680.

[102] A. Koblischka-Veneva, M.R. Koblischka, J. Schmauch, M. Hannig, Human dental enamel: A natural nanotechnology masterpiece investigated by TEM and t-EBSD, Nano Res. 11 (2018) 3911–3921.

[103] E. Beniash, C.A. Stifler, C.Y. Sun, G.S. Jung, Z. Qin, M.J. Buehler, P.U.P.A. Gilbert, The hidden structure of human enamel, Nat. Commun. 10 (2019) 1–13.

[104] L. Schroeder, R.M. Frank, High-resolution transmission electron microscopy of adult human peritubular dentine, 526 (1985) 449–451.

[105] F. Caruso, S. Mantellato, M. Palacios, R.J. Flatt, ICP-OES method for the characterization of cement pore solutions and their modification by polycarboxylate-based superplasticizers, Cem. Concr. Res. 91 (2017) 52–60.

[106] E.D. Eanes, J.D. Termine, M.U. Nylen, An electron microscopic study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite, Calcif. Tissue Res. 12 (1973) 143–158.

[107] J. Yu, J. Zhang, A simple template-free approach to TiO2 hollow spheres with enhanced photocatalytic activity, Dalt. Trans. 39 (2010) 5860–5867.

[108] X. Wang, J. Feng, Y. Bai, Q. Zhang, Y. Yin, Synthesis, Properties, and Applications of Hollow Micro-/Nanostructures, Chem. Rev. 116 (2016) 10983–11060.

[109] J. Daintith, Henry’s law., in: A Dict. Chem., Oxford University Press, 2008. [110] M. Alheshibri, J. Qian, M. Jehannin, V.S.J. Craig, A History of

Nanobubbles, Langmuir. 32 (2016) 11086–11100. [111] A.R. Studart, A. Nelson, B. Iwanovsky, M. Kotyrba, A.A. Kündig, F.H.

Dalla Torre, U.T. Gonzenbach, L.J. Gauckler, J.F. Löffler, Metallic foams from nanoparticle-stabilized wet foams and emulsions, J. Mater. Chem. 22 (2012) 820–823.

[112] A. Stocco, E. Rio, B.P. Binks, D. Langevin, Aqueous foams stabilized solely by particles, Soft Matter. 7 (2011) 1260–1267.

[113] G. Mohamedi, M. Azmin, I. Pastoriza-Santos, V. Huang, J. Pérez-Juste, L.M. Liz-Marzán, M. Edirisinghe, E. Stride, Effects of gold nanoparticles on the stability of microbubbles, Langmuir. 28 (2012) 13808–13815.

[114] Z. Du, M.P. Bilbao-Montoya, B.P. Binks, E. Dickinson, R. Ettelaie, B.S. Murray, Outstanding stability of particle-stabilized bubbles, Langmuir. 19 (2003) 3106–3108.

[115] T.S. Horozov, Foams and foam films stabilised by solid particles, Curr. Opin. Colloid Interface Sci. 13 (2008) 134–140.

[116] A.L. Oliveira, R.L. Reis, P. Li, Strontium-substituted apatite coating grown on Ti6Al4V substrate through biomimetic synthesis, J. Biomed. Mater. Res. - Part B Appl. Biomater. 83 (2007) 258–265.

[117] V. Uskoković, S. Marković, L. Veselinović, S. Škapin, N. Ignjatović, D.P. Uskoković, Insights into the kinetics of thermally induced crystallization of amorphous calcium phosphate, Phys. Chem. Chem. Phys. 20 (2018) 29221–29235.

67

[118] J.M. Sedlak, R.A. Beebe, Temperature programmed dehydration of amorphous calcium phosphate, J. Colloid Interface Sci. 47 (1974) 483–489.

[119] Y. Kojima, K. Sakama, T. Toyama, T. Yasue, Y. Arai, Dehydration of Water Molecule in Amorphous Calcium Phosphate, Phosphorus Res. Bull. 4 (1994) 47–52.

[120] H. Pan, X.Y. Liu, R. Tang, H.Y. Xu, Mystery of the transformation from amorphous calcium phosphate to hydroxyapatite, Chem. Commun. 46 (2010) 7415–7417.

[121] M.J. Larsen, E.I.F. Pearce, Saturation of human saliva with respect to calcium salts, Arch. Oral Biol. 48 (2003) 317–322.

[122] F.R. Tay, S.M. Kwong, A. Itthagarun, N.M. King, H.K. Yip, K.M. Moulding, D.H. Pashley, Bonding of a self-etching primer to non-carious cervical sclerotic dentin: interfacial ultrastructure and microtensile bond strength evaluation, J. Adhes. Dent. 2 (2000) 9–28.

[123] T. Sui, J. Dluhoš, T. Li, K. Zeng, A. Cernescu, G. Landini, A. Korsunsky, Structure-Function Correlative Microscopy of Peritubular and Intertubular Dentine, Materials (Basel). 11 (2018) 1493.

[124] H. Ryou, E. Romberg, D.H. Pashley, F.R. Tay, D. Arola, Importance of Age on the Dynamic Mechanical Bahviour of Intertubular and Peritubular Dentin, J. Behav. Biomed. Mater. 42 (2015) 229–242.

[125] M. Goldberg, Understanding dental caries: From pathogenesis to prevention and therapy, 2016.

[126] S.P. Humphrey, R.T. Williamson, A review of saliva Normal composition, flow, and function. Humphrey, Williamson. 2001. Journal of Prosthetic Dentistry.pdf, J. Prosthet. Dent. 85 (2001) 162–169.

[127] E.D. Eanes, J.L. Meyer, The Influence of Fluoride on Apatite Formation from Unstable Supersaturated Solutions at pH 7.4, J. Dent. Res. 57 (1978) 617–624.

[128] K. Markowitz, D.H. Pashley, Discovering new treatments for sensitive teeth: The long path from biology to therapy, J. Oral Rehabil. 35 (2008) 300–315.

[129] D.J. White, M.A. Lawless, A. Fatade, A. Baig, R. von Koppenfels, H. Duschner, H. Götz, Stannous fluoride/sodium hexametaphosphate dentifrice increases dentin resistance to tubule exposure in vitro, J. Clin. Dent. 18 (2007) 55–59.

[130] F. García-Godoy, R.D. Trushkowsky, A diagnostic device to record dentin hypersensitivity, Am. J. Dent. 26 (2013) 27–29.

[131] C.R. Gernhardt, How valid and applicable are current diagnostic criteria and assessment methods for dentin hypersensitivity? An overview, Clin. Oral Investig. 17 (2013) 31–40.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 2024

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science andTechnology, Uppsala University, is usually a summary of anumber of papers. A few copies of the complete dissertationare kept at major Swedish research libraries, while thesummary alone is distributed internationally throughthe series Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology.(Prior to January, 2005, the series was published under thetitle “Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-437696

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2021