Synthesis of novel sol-gel based hydraulic and bioactive endodontic cements

Synthesis of Novel Sol-Gel Based Hydraulic and Bioactive Endodontic Cements PhD Thesis presented by Xiaozhe Song Submitted for the degree of Doctor of Philosophy from the University of Seville Supervisor: Dra. María Aránzazu Díaz Cuenca Instituto de Ciencia de Materiales de Sevilla CSIC-US Tutor: Dr. Juan José Segura Egea Departamento de Estomatología Facultad de Odontología Universidad de Sevilla Seville, 2024 Doctoral thesis Synthesis of Novel Sol-Gel Based Hydraulic and Bioactive Endodontic Cements Supervisor Tutor Dra. María Aránzazu Díaz Cuenca Dr. Juan José Segura Egea Doctoral candidate Xiaozhe Song Seville, 2024 Acknowledgements Acknowledgements Time flies in the blink of an eye. In a twinkling, I have spent five years in Seville. This period has been significant for me, which is filled with moments of uncertainty, joy, and achievement. But most importantly, after these five years of exploring the path of scientific research, I have finally successfully completed my doctoral thesis. I want to express my gratitude to Dra. Aránzazu Díaz Cuenca, my supervisor, for hosting me in the Materials for Bioengineering and Tissue Regeneration group at the Institute of Materials Science (ICMS) and for her attentive guidance and support throughout the selection of the topic and the research process of this project. She closely monitors my research progress, offers valuable guidance, helps shape my ideas, and provides enthusiastic encouragement. Her meticulous approach, rigorous mindset, and practical wisdom have been extremely invaluable to me. She has not only been my academic mentor but also a guiding presence in my life journey. Despite the five-year duration of this project, the knowledge and support she has imparted will benefit me for a lifetime. Words cannot fully convey my appreciation for her. I would like to thank Professor Juan José Segura Egea, my tutor, for his invaluable generosity and assistance. His unwavering support and provision of essential experimental tools have been instrumental in my progress. His encouragement and support have been the driving force behind my advancement, and I am certain that I would not have completed my PhD without his guidance. I want to express my gratitude to Mari Carmen Jiménez Sánchez, a PhD student in the research group, for assisting me in getting acquainted with the lab's experimental setup when I initially joined, teaching me how to operate the equipment. She has been a guiding figure in my doctoral journey and continues to offer advice even after completing her own studies. Additionally, Diana Flores Barnett, a pre-doctoral student, Acknowledgements I have received encouragement, support, and empathy from you, and you were always there for me when I needed you. I am thankful to the technical persons at the ICMS, particularly Dr. José María Martínez Blanes, Dra. Cristina Gallardo López, Dr. Miguel Angel Avilés Escaño, and Dra. Mari Carmen Jiménez de Haro, for their expertise and knowledge, which help me to obtain accurate results. I would like to express my gratitude to Juan Pedro Espinós Manzorro, the director of the Institute of Materials Science, for his help during my stay. I am thankful to José Carlos Rivero Cabello for his selfless support in both my academic and personal endeavors. Additionally, for the administrative staff at cicCartuja, I am grateful for the assistance and consideration you provided over the last five years. The warmth and kindness I have experienced will always hold a special place in my memories. I would like to thank Dra. Victoria Areal Quecuty, Dra. Aurea Simon Soro, and María, Debera, and Carlos for their assistance during the setting time experiment in the Faculty of Dentistry in the University of Seville. Finally, to my parents, families, and husband, I am grateful for their understanding and support. Index Index Acknowledgements Abstract Resumen Chapter 1. General Introduction 1.1. Background 1.2. Commercial Calcium Silicate Cements Currently in Use 1.3. Current Research on Sol-gel Calcium Silicates for Application in Dentistry 1.3.1. Basics of the sol–gel synthesis technique 1.3.2. Progress in calcium silicate cements research 1.3.2.1. Compositions and synthesis routes 1.3.2.2. Final processing of materials routes 1.3.2.3. Therapeutic and clinical uses 1.4. Thesis Hypotheses, Objectives and Contents 1.4.1. Thesis hypotheses 1.4.2. Thesis objectives 1.4.3. Thesis contents 1.4.3.1. Chapter 1 1.4.3.2. Chapter 2 1.4.3.3. Chapter 3 1.4.3.4. Chapter 4 1.5. References Index Chapter 2. Sol-Gel Synthesis of Endodontic Cements: Setting Performance and Bioactivity In Vitro 2.1. Introduction 2.2. Materials and Methods 2.2.1. Bioactive endodontic cement (BEC) synthesis procedure 2.2.2. Ethanol post-synthesis treatment (EPT) 2.2.3. Material characterization 2.3. Results 2.3.1. Sol–gel synthesis variable influence in the BEC powder product standardization and optimization 2.3.2. Effect on the BEC properties of the ethanol-based post-synthesis treatment 2.3.2.1. Influence of EPT on the physicochemical properties of unhydrated sol–gel BEC material 2.3.2.2. Influence of EPT on hydration and the setting properties of the sol–gel synthesized BEC material 2.3.2.3. Influence of EPT on the bioactivity of the sol–gel synthesized BEC material 2.3.2.4. Influence of EPT on the biodegradation of the sol-gel synthesized BEC material 2.4. Discussion 2.5. Conclusions 2.6. References Chapter 3. Zinc-Inclusive BECs: Sol-Gel Synthesis and Characterization 3.1. Introduction 3.2. Materials and Methods Index 3.2.1. Synthesis of Zn-BEC using 28% molar Ca-substitution 3.2.2. Synthesis of Zn-BECs using 1, 3, and 5% molar Ca-substitution 3.2.2.1. Synthesis variables and route 3.2.2.2. Ethanol Post-Synthesis Treatment (EPT) 3.2.2.3. Characterization 3.3. Results 3.3.1. Zn-BEC using 28% molar Ca-substitution 3.3.1.1. XRD analysis of 28Zn-BEC precursor 3.3.1.2. XRD analysis of un-hydrated and hydrated heat treated 28Zn-BEC 3.3.2. Zn-BECs using 1, 3 and 5% molar Ca-substitution 3.3.2.1. Influence of DI H2O amount on the Zn-free BECs 3.3.2.2. Characterization of Zn-BECs using 1, 3 and 5% molar Ca-substitution 3.3.3. The influence of EPT on the composition and hydration characteristic of Zn-BECs 3.3.3.1. Crystal phase of Zn-BECs after EPT 3.3.3.2. FTIR analysis of Zn-BECs after EPT 3.3.3.3. Textural parameters of Zn-BECs after EPT 3.3.3.4. Setting time of Zn-BECs after EPT 3.3.3.5. XRD analysis of hydrated Zn-BECs after EPT 3.3.3.6. FTIR analysis of hydrated Zn-BECs after EPT 3.4. Discussion 3.5. Conclusions 3.6. References Chapter 4. Mesoporous BECs (MBECs): Synthesis and Hydration Performance 4.1. Introduction Index 4.2. Materials and Methods 4.2.1. Synthesis of the mesoporous BECs 4.2.2. Physico-chemical characterization of MBECs 4.3. Results 4.3.1. Wide-angle X-ray diffraction (WAXRD) analysis 4.3.2. Small-angle X-ray diffraction (SAXRD) analysis 4.3.3. FTIR analysis 4.3.4. Physisorption measurement 4.3.5. Setting time 4.3.6. Crystal phase of hydrated MBECs analysis 4.3.7. FT-IR analysis of hydrated MBECs 4.3.8. The pH variation 4.3.9. Weight loss 4.4. Discussion 4.5. Conclusions 4.6. References General Conclusions Appendix Appendix 1. Characterization Techniques and Methods Appendix 2. Abbreviations Appendix 3. Publications Chapter 1. General Introduction 3 toward linear or branched structures are also critical issues, which play a crucial role in determining the properties and performance of the final material. A major advantage of the sol–gel method is that it is possible to obtain materials with high purity and homogeneity at the molecular scale and to control the surfaces, interfaces and porosity of the materials obtained at the nanometric scale [31]. The sol–gel method produces homogeneous sols that can be converted into gels with a very high volume of nanopores. This nanoporosity is undoubtedly one of the most important characteristics of advanced bioceramics, as it translates into a higher specific surface area, greater reactivity and, therefore, faster kinetics in the bioactive response [32]. The sol–gel method can be used to synthesize bioceramics with different chemical compositions, which enables the production of a wide range of materials. Besides, it is a cost-effective technique, as it requires lower reaction temperatures and simpler equipment than other high temperature burning and thermal industrial processes. The low reaction temperature reduces the energy consumption and gas emissions, which contribute to its environmental sustainability. 1.2. Commercial Calcium Silicate Cements Currently in Use Calcium silicates, mainly Ca3SiO5 and Ca2SiO4, are the basic compounds in bioactive endodontic cements (BECs) [26,33]. BECs are bioceramics widely used in endodontics as restorative cements used in vital pulp therapy and endodontic sealers, due to their high biocompatibility, intrinsic osteoconductive activity and ability to induce regenerative responses as dentin bridges that promote better sealing of the pulp-capped site[34,35]. These calcium silicates compounds are capable of reacting with water at a physiological temperature, causing a hydraulic setting reaction. Originally the first product formulation was described as a powder composed of calcia, silica and alumina oxides and was then named mineral trioxide aggregate Chapter 1. General Introduction 4 (MTA), which is still a generic name used for BECs in dentistry [36]. In fact, MTA is based on Portland cement, which was revisited by Torabinejad et al. [37] for its use in endodontics. Despite its excellent properties, some problems in its clinical application, such as the long setting time, tooth discoloration, high cost and difficult handling, have driven the development of new formulations. The first clinically approved formulation was ProRoot MTA [38]. The initial setting time has been reported from 70 to 74 min [33]. In 2002, the gray ProRoot MTA (GMTA) was substituted by the new white ProRoot MTA (WMTA), free from tetracalcium aluminoferrite to reduce the problems concerning tooth discoloration [39]. MTA Angelus [40,41] followed with a similar composition based on Portland cement, but without the calcium sulphate dehydrate (gypsum). Further products marketed with shorter setting times are Biodentine [42-49], Endocem MTA [50-53], MTA Bio [54,55], EndoSeal MTA [56,57] and MTA Fillapex [58-63]. A setting time of as little as 0.3 min has been reported for TheraCal [64-68] because of the use of resin and light cure technology. The radiopacifying agent used is another important element that has been studied for the improvement of these products. ProRoot MTA contains about 2 at.% Bi [39], which may not only produce tooth discoloration but also reduce its biocompatibility [69]. As an alternative to bismuth oxide, other compounds have been used, such as tantalum oxide Ta2O5, which is used in BioAggregate [70-74] and NeoMTA Plus [26,75-77]. ZrO2 is another agent widely used in products such as Endocem Zr [51,78], EndoSequence [79,80], iRoot SP [81-84], BioRoot RCS [85-88] and the previously mentioned Biodentine. MTA Repair HP, notable for its low setting time and fast bioactive response in vitro [25], contains CaWO4 as a radiopacifying agent and consists of tricalcium silicate nanoparticles with high aspect ratio, which provide to the precursor material an elevated surface area to maximize the hydration reaction [89,90]. Chapter 1. General Introduction 5 1.3. Current Research on Sol-Gel Calcium Silicates for Application in Dentistry The chemistry of the sol–gel technique offers great versatility and can be used for the preparation of a wide variety of bioceramic compositions and different macro-, micro- and nanostructure features for application in regenerative dentistry. 1.3.1. Basics of the sol–gel synthesis technique The sol–gel method is a wet-chemistry process, which involves several stages from the initial precursors solution (sol) to the gelation (gel) phase. The interest in and development of this process dates back more than 150 years, when it was discovered that hydrolysis under acidic conditions of the compound tetraethyl orthosilicate (TEOS) produced SiO2 in the form of a glass-like material [30,91]. A typical process starts with the hydrolysis and polycondensation reactions of the precursor alkoxide-type compounds. Silicon alkoxides represent the main network forming agents in sol–gel preparation methods and tetraethyl orthosilicate (TEOS) is still the most widely used silicate precursor, while water and/or ethanol are used as solvents. The formation of the silicate network follows a widely accepted two-stage process [30,92]: hydrolysis (Equation (1)) and condensation (Equations (2) and (3)). Hydrolysis and condensation may occur simultaneously as silanol groups on partially hydrolyzed molecules that undergo condensation: Si(OR)4 + 4 H2O → Si(OH)4 + 4 ROH (1) 2 Si(OH)4 → (OH)3Si-O-Si(OH)3 + H2O (2) Si(OR)4 + Si(OH)4 → (OH)3Si-O-Si(OR)3 + ROH (3) Chapter 1. General Introduction 6 By modifying the synthesis parameters, the properties of the final materials, such as the morphology and composition, can be controlled and designed. In general, the rate of hydrolysis is fast compared to that of condensation in strong acid conditions and, alternatively, a higher pH favors condensation. However, a higher and lower pH is able to promote condensation and hydrolysis and, in silica based systems, the reactions proceed as a result of acid catalysis at a pH < 2 and basic catalysis when the pH > 2, around the isoelectric point of silica at pH = 2 [93]. Silicon alkoxides tend to form a 3D gelled structure under acid conditions or individual particles under basic conditions [31,94]. The controlled growth of monodisperse silica spheres was first achieved by Stöber et al. [95], using a base-catalyzed sol–gel synthesis route involving silica alkoxide precursors and an ammonium hydroxide catalyst. Moreover, using appropriate template molecules a well-ordered hexagonal arrangement of mesopores is formed at low pH acid conditions [96]. Other silane oligomers capable of taking part in the hydrolysis and condensation reactions can be also used, apart from TEOS [97]. Besides, network modifier elements, such as calcium or magnesium, can be introduced in the form of inorganic salt or as alkoxide precursors [28,98,99]. Like silicon, phosphorous can be used a network former within the sol–gel process [28]. However, the σ-π double bond reduces the expected coordination number and produces a more relaxed network structure when compared to silica-based materials [31]. Besides, the low reactivity to acid-catalyzed hydrolysis of triethyl phosphate (TEP) should be noted, which is the most commonly used phosphorous precursor. Studies have shown a large loss of phosphorous for the TEP-prepared gels, most likely due to the much lower rate of hydrolysis of TEP than the silica precursor TEOS [100,101]. In short, by varying the different synthesis variables in the process, such as the silicon or phosphorus monomers, the salt precursors, the use of template molecules, co-solvents, pH Chapter 1. General Introduction 7 catalysts, the temperature and reaction times, as well as the different post synthesis treatments and processing routes, a large number of formulations in different useful morphologies and formats can be produced (Figure 1.1). Figure 1.1. Chemistry stages and processing strategies of sol-gel technologies. Although the synthesis of bioceramics using the sol–gel technique is considered safe, there are some health and safety hazards that must be considered. Thus, as mentioned, the synthesis involves the use of alkoxides, acids and solvents, which might require appropriate safety protocols, such as wearing protective clothing and gloves, working in a well-ventilated area and using fume hoods when necessary. Some sol–gel precursors and reaction by-products may release toxic fumes during the synthesis process. Besides, the sol–gel process may generate fine Chapter 1. General Introduction 8 particles or dust, which can pose a respiratory hazard if inhaled. It is, therefore, advisable to wear appropriate respiratory protection equipment, such as masks or respirators. 1.3.2. Progress in calcium silicate cements research 1.3.2.1. Compositions and synthesis routes Calcium silicates, particularly tri-calcium silicate, Ca3SiO5 (C3S), and di-calcium silicate, Ca2SiO4 (C2S), compounds are very relevant in dentistry as they are the fundamental components of hydraulic cements used in endodontic procedures [102]. Both C3S and C2S react with water to form calcium silicate hydrate (CSH), which through the polymerization network contributes to the self-setting properties and increased mechanical strength after aging [103]. However, it is well reported that C2S reacts with water at a slower rate than C3S [104] and also that C2S polymorphism in their beta (β) and gamma (γ) forms have important differences, with the beta form being the most reactive [105,106]. Zhao et al. [107] reported the sol–gel synthesis of pure C3S powders after a heat treatment at 1400 °C and above, using an initial Ca/Si molar ratio of 3, from Ca(NO3)24H2O (CaN) and TEOS as the precursor materials and HNO3 as a catalyst. The obtained C3S powder showed particles with some pores of about 1–5 µm and performed well as a self-setting workable paste, with good biocompatibility and surface bioactivity in vitro [108]. The synthesis and in vitro bioactivity of C2S compositions in their β and γ forms has also been studied using the same precursors, but modifying the Si/Ca molar ratio and the heat treatment temperatures [109,110]. More recently, the sol–gel synthesis parameters, such as the different mixing orders of reactants or the amount of nitric acid added, or the calcium silicates using a Ca/Si molar ratio of 3, has been evaluated and optimized [111]. The results from this study demonstrated that sol–gelderived powders can be achieved showing porous microstructures and with a setting time of Chapter 1. General Introduction 9 ~30 min, a value well below that specified for commercial silicate cements as detailed in Section 1.2. The synthesis of endodontic cements, including Al, Zn and F, has been investigated by Voicu et al., showing an increase in the C3S crystallite size and a shifting of its XRD peaks, which suggests the presence of Zn or/and F in the C3S lattice with a positive influence on the material’s grindability [112]. An interesting material with a 15 min setting time was obtained by these authors using TEOS, aluminum butoxide (C12H27O3Al), zinc acetate (Zn(CH3COO)22H2O) and CaN as reactive precursors and adequate thermal treatment of 1450 °C [113]. Successfully synthesized strontium-doped C3S up to Sr = 2 mol% with applicable setting times in clinical practice has been reported using TEOS, CaN and Sr(NO3)2 [114]. The synthesis of a magnesium–calcium silicate cement (Mg–CS) with Mg content of up to 10 mol% has been achieved using TEOS, CaN and Mg(NO3)26H2O as precursors, nitric acid as a catalyst and absolute ethanol as the solvent, followed by heat treatment at 800 °C for 2 h and ball milling for 6 h in ethyl alcohol using a centrifugal ball mill [115]. Using similar reactive compounds, different formulations including 1, 3 or 5 mol% of Mg to satisfy the (Mg + Ca)/Si molar ratio of 3, were also investigated after heat treatment at 1400 °C. It could be seen that the Mg ion incorporated into the C3S phase and residual Mg ion remained in the Mg(OH)2 phase, which plays the role of hydration accelerator, and the setting time was shortened [116]. C2S, C2S– xZn and C2S–xCu powders with different percentages (x = 5% or 10%) of Zn- or Cu-substituted Ca were also synthesized by a modified sol–gel method using a silica sol (SiO2, containing 25.5% SiO2), CaN and Zn(NO3)26H2O or Cu(NO3)23H2O with a nominal (CaO + ZnO + CuO)/SiO2 molar ratio of 1.8:1, an ethanol–water mixture and heat treatment at 800 °C for 3 h [117]. 1.3.2.2. Final processing of materials Workable pastes of C3S prepared by sol–gel using a liquid to powder ratio (L/P) of 0.8–1.2 mL Chapter 1. General Introduction 10 g−1 were self-setting and could be injected within 15–60 min [108]. Calcium silicate cements consisting of sol–gel-derived calcium silicate powder of SiO2–CaO ranged from 7:3 to 3:7 were processed using a 3.7 M ammonium phosphate solution (NH4)2HPO4–NH4H2PO4 with an L/P over the range 0.5–0.7 mL g−1, resulting in self-hardening to form apatite and a CSH gel within 9 min [118]. Sol–gel C3S based cements were processed using ball milling mixing in combination with a 20% radiopacifier phase, such as BaZrO3, CaZrO3 and SrZrO3, producing radiopaque materials, which were either comparable or else improved over the control MTA Angelus [119]. Nano powders of barium titanate (BT) and biocellulose (BC) were added (10 wt%) to calcium silicophosphate cements based on sol–gel synthesized calcium silicates, with a CaO/SiO2 molar ratio of 1 and an orthophosphoric acid solution partially neutralized with Al2O3 and ZnO. The addition of BT and BC nano powders determined the decrease in the setting time, whilst it did not significantly influence the mechanical properties of the resulting composites [120]. A recent paper published by our laboratory details how the use of the sol–gel route can be effective for obtaining endodontic cements with a majority of C3S content and C2S in its hydraulic beta form. Besides, the implementation of a novel post-synthesis treatment at room temperature using ethanol allows for a final product with a finer particle size and increased CaCO3 content, resulting in an improved material in terms of the setting time and bioactive response [121]. 1.3.2.3. Therapeutic and clinical uses Bioactivity, which implies the release of calcium ions, electroconductivity and the formation of an interfacial layer between the material and dentinal wall, is a common property of calcium silicates [33]. The hydration of sol–gel synthesized C3S yields a dissolvable CSH, whose ionic products have a stimulatory effect on relevant cell growth [108]. Fast setting and controllable degrading properties, as well as the stimulation of odontogenesis/angiogenesis, has been Chapter 1. General Introduction 11 reported for a magnesium–calcium silicate (Mg–CS) cement with a Mg content of up to 10 mol%. The Mg–CS cement has been shown to stimulate the proliferation of human periodontal ligament cells (hPDLCs) in vitro and actively promote the secretion of odontogenic (DSPP and DMP-1) and angiogenic (vWF and ang-1) proteins [115]. Also consistent with this, another study using Mg–CS with different amounts 1, 3, 5 mol% of Mg ion has been shown to promote osteogenic differentiation of human dental pulp stem cells (hDPSCs) [116]. Sol–gel synthesized C2S powders partially substituted with Zn or Cu were investigated systematically to examine their antibacterial activity in vitro and osteogenic activity in mandibular bone defects in vivo. The pure C2S cement showed a significant antibacterial response in comparison with the Zn-/Cu-substituted C2S cements in the initial several hours, but the latter could prolong the antibacterial efficacy. Also, maximum bone regeneration was consistently observed in defects filled with Zn-/Cu-substituted C2S cements [117]. Sol–gel synthesis of C3S including F and Zn ions in the crystal lattice has been correlated with the materials reactivity vs. water, as well as its mechanical and aesthetical properties [112]. Besides, the mixing of Sr2SiO4 particles synthesized by sol–gel to a cementing composite with C3S and C2S at a weight ratio of 30% by weight has been shown to give sufficient radio-opacity to an endodontic cement, as well as to enhance its bioactivity and tubule occlusion [122] 1.4. Thesis Hypotheses, Objectives and Contents 1.4.1. Thesis hypotheses Since its introduction in 1993 [123], the bioactive endodontic cements (BECs), has had a major impact on clinical application as well as on research into new and more advanced endodontic treatments. However, there are a number of issues such as long setting time, difficulty of Chapter 1. General Introduction 12 handling and tooth discoloration etc. that require improvement. Although the sol-gel method is an attractive chemical synthesis method for the preparation of bioceramics, this technique has not been very well developed for the synthesis of BECs. The working hypothesis is that BEC formulations with new physico-chemical properties and thus improved functionalities can be obtained by sol-gel synthesis procedures. Besides, the incorporation of different elements into the composition of the BEC can enhance their therapeutic benefits. Zinc has been shown to have very beneficial effects as a bactericidal element, inducer of bio-mineralization and other biological stimuli in the activation of enzymes and in cell proliferation and differentiation [124-126]. The hypothesis is that the zinc could be incorporated into BECs without interfering with its hydraulic properties but improving its bioactive and biological potential. Also, the combination of sol-gel method and surfactant agents acting as template or structuredirecting molecules make great progress in the development of bioceramics of improved textural properties such as surface area and nano-porosity glasses [32]. Hence, as the hydration activity of BEC is correlated to its surface area [127], the hypothesis is that mesoporous BECs could be prepared with improved textural parameters and hydration reaction. 1.4.2. Thesis objectives The overall objective of the Thesis is to synthesize BECs by the sol-gel method and to study the relationship between the physico-chemical parameters of the synthesized materials and their functionality in terms of response to hydration and bioactivity in vitro. To this end, the first objective has been to optimize the synthesis variables in order to: i) ensure a reproducible and homogeneous precursor material, as a step prior to the high-temperature for Chapter 1. General Introduction 19 month Follow-up. 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Introduction Bioactive endodontic cements (BECs) are dental materials widely used in endodontics for vital pulp therapy, coronal sealing during regenerative endodontic procedures isolating the intracanal treatment from the coronal restoration, apical barriers in immature teeth with necrotic pulp, and sealing of perforations or apical surgeries, among others [1,2]. BECs are powder formulations based on calcium silicates, mainly Ca3SiO5 and Ca2SiO4, which react with water and harden to form hydrated phases of good sealing abilities [1,3,4], antibacterial properties, and bioactive inducement of periapical healing and hard tissue formation [5]. Irrespective of their variable composition, the bioactivity, defined as the potential to stimulate the natural remineralization process at the material–tooth interface, is a common property of endodontic cements [1]. The international standard ISO 22317 [6] defines bioactive materials as those that—when implanted in a living organism—form a thin layer rich in calcium and phosphorus on the surface. Likewise, when crystallization of hydroxyapatite occurs on the material surface in simulated body fluid (SBF), the same bioactivity can be expected in vivo. Originally, this hydraulic tri/dicalcium Chapter 2. Sol-Gel Synthesis of Endodontic Cements 28 silicate-based product was described as “hydrophilic powder composed of calcia, silica and alumina oxides” and was named “mineral trioxide aggregate” (MTA), which is still a generic name commonly used for BECs in dentistry [4]. However, despite its excellent properties, there are some problems in its clinical application that require improvement. Among the main disadvantages of its use are long setting time, difficulty in handling in the clinical setting, and its aging or biodegradation behavior once applied, producing undesirable dental discolorations. In recent years, manufacturers have been trying to solve these problems with changes in the formulations [7]. Hence, there is much interest in determining which powder parameters, i.e., crystal phase composition [8], particle size [9], or the uses of additives [10], are the strongest contributors to cement setting performance and bioactivity. Particle size and specific surface area-related parameters have been shown to have an important effect on setting time and final consolidated microstructure. A larger surface area will increase powder reactivity towards hydration, promoting setting properties. In this respect, BEC formulations of high surface area have been synthesized using sol–gel procedures [11]. The sol–gel technique is a chemical procedure that consists in the successive hydrolysis and polycondensation of alkoxide precursors, typically tetraethoxysilane (TEOS), to develop colloidal particles (sol) and consequently convert them into a network (gel) of intrinsic nanoporosity [12], which confers to the material improved textural properties and functionalities [13,14]. A diversity of sol–gel materials in different configurations, i.e., powders, monoliths, foams, fibers, or coatings, are now under intensive investigation for biomedical applications [15-18]. Specifically in relation to endodontic cements, dicalcium and tricalcium silicates are the main components [3,19], and can be successfully improved in terms of textural properties by sol–gel synthesis [20,21]. However, the selection of the different synthesis variables, such as the type of reagents, their molar ratios, the sequence of incorporation and reaction times etc., is critical Chapter 2. Sol-Gel Synthesis of Endodontic Cements 35 Figure 2.3. XRD patterns of sol–gel BEC preparations using different reactive sources as specified in Table 1: (a) as-synthesized powders (without heating treatment); (b) after heat treatment at 1400 °C. The two peaks marked with asterisks could not be matched to any phase of the database records. Chapter 2. Sol-Gel Synthesis of Endodontic Cements 36 2.3.2. Effect on the BEC properties of the ethanol-based post-synthesis treatment Based on the results obtained in the previous section, the ACaNO-HNO formulation, showing the highest content of Ca3SiO5 as well as the absence of the non-hydraulic phase γ-Ca2SiO4, was selected to perform further ethanol post-synthesis treatment (EPT). 2.3.2.1. Influence of EPT on the physicochemical properties of unhydrated sol–gel BEC material Figure 2.4 displays particle size distributions for the as-synthesized BEC and also after EPT. For both samples, the graph shows a bimodal distribution of two maxima at 0.3–0.4 and 5–7 μm ranges. As expected, all the particles above 10 μm are completely removed after the EPT. Figure 2.4. Particle size distributions of the sol–gel synthesized powders: without post-synthesis treatment (ACaNO-HNO), and after EPT (ACaNO-HNO (EPT)). XRD analysis (Figure 2.5) indicates that EPT does not significantly affect formed calcium silicate phases, although it does result in the growth of new calcium carbonate crystals in the Chapter 2. Sol-Gel Synthesis of Endodontic Cements 37 form of the calcite polymorph, being those at the expense of the calcium oxide phase, as suggested by its diffraction peaks decreasing intensity. Figure 2.5. XRD patterns of unhydrated sol–gel BECs heat-treated at 1400 °C: without post-synthesis treatment (ACaNO-HNO), and after EPT (ACaNO-HNO (EPT)). This growth of calcium carbonate caused by EPT is confirmed by FT-IR analysis (Figure 2.6) showing the appearance of additional bands corresponding to calcite-specific vibrational modes at 874 and 713 cm−1 [22,23]. Besides, vibrational bands corresponding to Si-O asymmetric stretching (ν3) at 994, 936, 906, and 886 cm−1, and Si-O bending ν4 and ν2 at 520 and 456 cm−1, respectively, characteristics of Ca3SiO5 [3] are displayed in Figure 2.6. Interestingly, the presence of the band at 814 cm−1 associated with the Si-O symmetric stretching (ν1) indicates the ideal [SiO4]4− tetrahedron structure distortion of the Ca3SiO5 silicate phase [24]. This result suggests that Ca3SiO5 is in the form of a triclinic structure polymorph in good agreement with the XRD analysis (PDF 00-031-0301). The Si-O band at 846 cm−1 has been assigned to dicalcium silicate (belite) [25]. Chapter 2. Sol-Gel Synthesis of Endodontic Cements 38 Figure 2.6. FT-IR analysis of unhydrated sol–gel BECs heat-treated at 1400 °C: without post-synthesis treatment (ACaNO-HNO), and after EPT (ACaNO-HNO (EPT)). Figure 2.7. FEG-SEM observations at different magnifications of unhydrated ACaNO-HNO formulation: (a,c) secondary electron (SE) images of the material without post-synthesis treatment; and (b,d) SE images of the material after EPT. Chapter 2. Sol-Gel Synthesis of Endodontic Cements 39 Figure 2.7 displays FEG-SEM images at different magnifications of ACaNO-HNO formulation, comparatively for the as-synthesized and after EPT. The b, d micrographs, corresponding to the EPT material, show a less flat surface that appears decorated by aggregates with rhombic morphology characteristic of calcium carbonate crystals in the calcite polymorph [22]. An EDX analysis of these aggregates is displayed in Figure 2.8, indicating that these morphologies correspond to very rich calcium areas, which are at the same time deficient in silicon, thus confirming the calcite growth after EPT. Figure 2.8. FEG-SEM observations and EDX analysis of ACaNO-HNO (EPT): (a) low-magnification secondary electron (SE) image using 2 kV; (b) higher magnification corresponding to the area marked with the white square in image (a), white arrow in images (a,b) points to a characteristic aggregate formed by the EPT treatment; (c) low-magnification SE image of the area displayed in (a) using 20 kV; (d) EDX analysis of image (c) showing Si (red), Ca (green), and merged (yellow) element-distribution analysis. Physisorption measurements can help to quantify the structural and morphological changes Chapter 2. Sol-Gel Synthesis of Endodontic Cements 40 qualitatively observed above. In fact, the results indicate that the EPT produces a threefold increase in the values of the specific surface area and pore volume (nanopore) parameters (Table 2.2) Table 2.2. BEC samples’ textural parameters obtained by N2 physisorption. Sample SBET (m2 g-1) Vt (cm3 g-1) ACaNO-HNO 2.6 ± 0.3 0.006 ± 0.000 ACaNO-HNO (EPT) 6.9 ± 0.2 0.021 ± 0.001 2.3.2.2. Influence of EPT on hydration and the setting properties of the sol–gel synthesized BEC material The hydration process was analyzed using FT-IR (Figure 2.9). Both type samples show significant increase of carbonate bands at 1450, 875, 855, and 714 cm−1 built up from the reaction of atmospheric CO2 with calcium ad species. However, the distinctively ACaNO-HNO (EPT) sample shows stronger shifting of the Si-O asymmetric stretching (ν3) from ~950–1000 cm−1 to higher wave numbers ~1100 cm−1 as well as changes in the relative intensities of the out of plane (ν4) and in plane (ν2) bending vibrations [24], which are indicative of calcium silicate unit (SiO44−) dissolution and polymerization to form CSH [3]. Chapter 2. Sol-Gel Synthesis of Endodontic Cements 41 Figure 2.9. FT-IR analysis of hydrated BECs: without post-synthesis treatment (ACaNO-HNO), and after EPT (ACaNO-HNO (EPT)). The comparison of fracture surface FEG-SEM observations of studied hydrated materials indicates that the alcohol-processed sample shows lower porosity between the precursor microparticles, appearing as a more sealed surface (Figure 2.10). Figure 2.10. FEG-SEM observations on fracture surfaces of the hydrated ACaNO-HNO formulation: (a) without post-synthesis treatment; and (b) implementing EPT. The effect of the EPT was functionally evaluated by determining the initial and final setting Chapter 2. Sol-Gel Synthesis of Endodontic Cements 42 times as displayed in Table 2.3. The results indicate a very clear effect on the reduction of the setting time of the BEC material after EPT. Table 2.3. Initial and final setting times measured for the sol–gel BEC samples. Sample Initial setting (min) Final setting (min) ACaNO-HNO 26 ± 1 95 ± 7 ACaNO-HNO (EPT) 6 ± 0 53 ± 4 2.3.2.3. Influence of EPT on the bioactivity of the sol–gel synthesized BEC material Figure 2.11. FEG-SEM observations and EDX analysis of hydrated ACaNO-HNO formulation surfaces after 7 days SBF essay: (a,c) without post-synthesis treatment; and (b,d) after EPT. The surface microstructure observations and analysis of BEC samples after 7 days’ SBF soaking using FEG-SEM are displayed in Figure 2.11. Typical calcium phosphate (CaP) aggregates of spherical shape showing average diameters in the 1–2 μm range are observed for the untreated Chapter 2. Sol-Gel Synthesis of Endodontic Cements 43 sample. Comparatively, for the EPT sample, the observed growth in size of these spherical aggregates seems to culminate in their coalescence, and the lamellar formations of CaP appear more visible due to their larger size. To further characterize the CaP layer formed for both samples, FT-IR and XRD analyses were performed using material obtained by surface scraping. In comparison with the FT-IR analysis of the hydrated samples before the SBF essay (above, in Figure 2.6), both samples show an intensity increase of the carbonate bands at 1450, 875, 855, and 714 cm−1 as well as the calcium silica hydrate CSH band within the 1000–1100 cm−1 range. New specific phosphate phase bands at 1097, 960, 607, and 570 cm−1 are now detected in both samples (Figure 2.12). Figure 2.12. FT-IR analysis of hydrated BEC surface after 7 days SBF essay: without post-synthesis treatment (ACaNO-HNO), and after EPT (ACaNO-HNO (EPT)). As regards de CaP crystalline phase formation (Figure 2.13), the most intense XRD peaks for hydroxyapatite phase (PDF 00-009-0432) at 2θ = 31.7, 32,2 and 32.9 are clearly detected for Chapter 2. Sol-Gel Synthesis of Endodontic Cements 44 the EPT sample, whilst for the untreated sample, main signal matching with calcium hydrogen phosphate (PDF 00-017-0934) at 2θ = 24.8, 28.4, 32.1, and 33.3 are prevalent. Calcium carbonate in the form of calcite (PDF 01-086-2334) and aragonite (PDF 00-041-1475) are predominant. Figure 2.13. XRD analysis of hydrated BEC surface after 7 days SBF essay: without post-synthesis treatment (ACaNO-HNO), and after EPT (ACaNO-HNO (EPT)). 2.3.2.4. Influence of EPT on the biodegradation of the sol-gel synthesized BEC material Figure 2.14 shows the weight loss for the hydrated cements after exposure to the physiological solution. On day 1, the weight loss (%) was 3.39% ± 0.38% for ACaNO-HNO and 4.68% ± 0.13% for ACaNO-HNO (EPT) group. The weight loss increased linearly with time for both tested cement types during 28 d. Both samples exhibited similar degradation behavior. Chapter 2. Sol-Gel Synthesis of Endodontic Cements 51 terms of the in vitro evaluation for apatite-forming ability of implant materials [6], has been shown that effectively cloaks the foreign body from the tissue and allows wound healing [4]. Particularly, in vitro evaluation of hydroxyapatite-forming ability on the BEC surface in SBF is useful for evaluating its potential to stimulate the natural remineralization process at the material–tooth interface [45] and the regeneration of periradicular tissue [1]. The superficial formation of calcium phosphate thus promotes wound healing and formation of a reparative dentinal barrier whilst upregulating the differentiation of osteoblast, fibroblast, cementoblast, odontoblast, pulp cells, and many stem cells [46]. In odontoblast and cementoblast, high pH- sensing mechanisms are important for reactivating mineralization induced by an alkaline environment [47]. In this respect, our measurements of the SBF solution 7 days into the experiment indicate a significant increase in pH, which increases to values of 8.0 and 8.4 for the newly synthesized material and the EPT, respectively. Endodontic studies of the use of calcium hydroxide and its resulting alkaline environment created in the proximity of or adjacent to this material have been related to the influence of various mineralization enzymatic pathways as well as an antibacterial pH gradient [48]. Our results indicate that EPT stimulates more the bioactive response of the material, leading to increased growth and crystallization of the surface calcium phosphate layer. Both the volumetric expansion of the BECs and their ability to trigger the nucleation and growth of apatite deposits may reduce porosity and ensure a long-lasting seal. Furthermore, the strong presence of calcite crystals could have an additional advantage of the presented sol–gel BEC materials to attract fibronectin, which is responsible for cellular adhesion and differentiation [49,50]. The weight loss after 1 day is 3.39% ± 0.38% for ACaNO-HNO and 4.68% ± 0.13% for ACaNO-HNO (EPT). This may be due to the cement dissolving in SBF. BECs have high solubility [46]. Alqedairi et al. [51] reported the weight loss was 6.5% for GMTA and 4.6% for WMTA when immersed in distilled water for 24 h. Another study reported the solubilities of White MTA Angelus and MTA Bio were 3.47% and 3.51% respectively after 7 days [52]. When Chapter 2. Sol-Gel Synthesis of Endodontic Cements 52 dental cement is prepared, the mixing of powder and liquid leads to the incorporation of microscopic air bubbles, resulting in some degree of porosity [53] and solubilization of the components. On the other hand, the main components of BECs (C3S, C2S and CaO) have high solubility products in doubly deionized H2O, which are Ksp=9.6×10-23 for C3S and Ksp=4.3×10- 18 for C2S and Ksp=9.17×10-6 for CaO [54]. This also contributes to the high dissolution rate of BECs. However, the solubility occurs only on the exposed surface and the resulting dimension change is negligible. In addition, the higher solubility of BECs is beneficial for biomimetic remineralization and deposition of an interfacial layer. A higher solubility of ACaNO-HNO (EPT) compared to ACaNO-HNO is due to the lower pH values of the former, as the solubility of BECs is inhibited in alkaline environment [55]. In the initial stages, only a few calcium phosphates are deposited on the sample surfaces, so the weight loss is mainly caused by the dissolution of the samples. The samples treated with EPT showed a higher dissolution rate compared to untreated samples, resulting in greater weight loss. By the 7th day, SEM micrographs (Figure 2.11) observed that a significant amount of hydroxyapatite precipitated on both sample surfaces. As weight loss is influenced by both substrate dissolution and HA deposition, the weight loss of ACaNO-HNO is initially lower than ACaNO-HNO (EPT) on the 1st day, then higher on the 7th day, and ultimately follow a similar trend. In this study, both BECs released low levels of Si4+ ions (<20 ppm) of and high levels of Ca2+ (150-450 ppm). Our group previously measured Ca, P, and Si ion released from commercial MTA ProRoot White, NeoMTA Plus, and HP repair under the same experimental conditions [3]. These two new BECs, ACaNO-HNO and ACaNO-HNO (EPT) are more similar to HP repair which has higher specific surface area and great bioactivity. Si is a crucial trace element in bone formation and repair, playing a role in osteogenesis and stimulating stem cell proliferation and osteogenic differentiation [56]. Ca2+ ions are important regulators of cell functions and have Chapter 2. Sol-Gel Synthesis of Endodontic Cements 53 been shown to stimulate the proliferation human dental pulp cells (HDPCs) [57]. They also participate in pulp cell differentiation and hard tissue mineralization by modulating osteopontin (OP), bone morphogenetic protein-2 (BMP-2), and alkaline phosphatase (ALP) levels during pulp calcification, thereby inducing reparative dentin formation in endodontic treatment [58]. Therefore, the gradual release of Si4+ and Ca2+ during the degradation of the BECs contributes to odontogenic differentiation and biomineralization. When blended with water or aqueous substances, BECs undergo a hydration reaction, resulting in the formation of C-S-H and Ca(OH)2. The release of Ca(OH)2 is associated with the alkalizing effect. Furthermore, the ongoing interaction of Ca2+ with H+ or H3O+ ions from the remaining calcium silicate particles in the aqueous solution leads to a consistent rise in pH [59]. 2.5. Conclusions The sol–gel synthesis technique is effective for obtaining BECs with majority C3S content in the triclinic form. The control of the set of synthesis variables tested allows obtaining a final material with an optimum formation of C3S and C2S in its beta form, thus avoiding the crystallization of C2S in its non-hydraulic gamma polymorph. 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[50] I.M.J. Faraco; R. Holland. Response of the pulp of dogs to capping with mineral trioxide aggregate or a calcium hydroxide cement. Dental Traumatology (2001), 17, 163-166. [51] A. Alqedairi; C.A. Muñoz-Viveros; E.A. Pantera, Jr.; M. Campillo-Funollet; H. Alfawaz; E.A. Abou Neel; T.S. Abuhaimed. Superfast Set, Strong and Less Degradable Mineral Trioxide Aggregate Cement. International journal of dentistry (2017), 2017, 3019136. [52] R.R. Vivan; R.O. Zapata; M.A. Zeferino; C.M. Bramante; N. Bernardineli; R.B. Garcia; M.A. Hungaro Duarte; M. Tanomaru Filho; I. Gomes de Moraes. Evaluation of the physical and chemical properties of two commercial and three experimental root-end filling materials. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology (2010), 110, 250-256. [53] M. Fridland; R. Rosado. Mineral Trioxide Aggregate (MTA) Solubility and Porosity with Different Water-to-Powder Ratios. Journal of Endodontics (2003), 29, 814-817. [54] L. Nicoleau; A. Nonat; D. Perrey. The di- and tricalcium silicate dissolutions. Cement and Concrete Research (2013), 47, 14-30. [55] S. Pushpa; C. Maheshwari; G. Maheshwari; N. Sridevi; P. Duggal; P. Ahuja. Effect of pH on solubility of white Mineral Trioxide Aggregate and Biodentine: An in vitro study. Journal of dental research, dental clinics, dental prospects (2018), 12, 201-207. [56] E.M.J.S. Carlisle. Silicon: a possible factor in bone calcification. (1970), 167, 279-280. [57] T. Takita; M. Hayashi; O. Takeichi; B. Ogiso; N. Suzuki; K. Otsuka; K. Ito. Effect of mineral trioxide aggregate on proliferation of cultured human dental pulp cells. International Chapter 2. Sol-Gel Synthesis of Endodontic Cements 59 endodontic journal (2006), 39, 415-422. [58] F. Rashid; H. Shiba; N. Mizuno; Y. Mouri; T. Fujita; H. Shinohara; T. Ogawa; H. Kawaguchi; H. Kurihara. The Effect of Extracellular Calcium Ion on Gene Expression of Bonerelated Proteins in Human Pulp Cells. Journal of Endodontics (2003), 29, 104-107. [59] F.R.J.M.t.a.i.d.f.p.t.a. Tay. Bioactivity of mineral trioxide aggregate and mechanism of action. (2014), 61-85. Chapter 3. Synthesis and Characterization of Zn-BECs 60 CHAPTER 3 Zinc-Inclusive BECs: Sol-Gel Synthesis and Characterization 3.1. Introduction Zinc is an important trace element in the human body. It is the constitute element of countless enzymes and play an essential role in various metabolic processes [1,2]. Zn acts on cartilage growth and bone growth. There is a broad consensus on the role of Zn on osteoblast cells. Zinc promoted osteoblast cell proliferation and differentiation [3]. Zn stimulates the proliferation and differentiation of osteoblasts and fibroblasts by managing the synthesis of DNA and RNA and promoting the secretion of alkaline phosphatase (ALP) [1]. Zn is abundant in bone and may act as a local regulator of bone. It has been reported on neonatal rats that Zn increases the number of tartrate resistant acid phosphatase (TRACP ) + ve calcitonin receptor (CTR) multinucleated cells (MNC) and that these effects are dependent on the presence of osteoblast like cells [4]. The effect of zinc on osteoblast differentiation is realized via stimulating the differentiation of alkaline phosphatase and the expression of osteogenic marker genes RunX2, Chapter 3. Synthesis and Characterization of Zn-BECs 67 Table 3.5. Reactive molar ratios for the preparation of Zn-BECs in the range of 0-5 mol% Ca substitution. Materials Ca:Si (Ca+Zn):Si Zn:Si H2O:TEOS H2O:HNO3 0Zn-BEC 3.00:1 3:1 0.00 31.25:1 268.77:1 1Zn-BEC 2.97:1 3:1 0.03:1 31.25:1 268.77:1 3Zn-BEC 2.91:1 3:1 0.09:1 31.25:1 268.77:1 5Zn-BEC 2.85:1 3:1 0.15:1 31.25:1 268.77:1 3.2.2.2. Ethanol Post-Synthesis Treatment (EPT) To further investigate the effect of decreasing particle size on the setting time of Znincorporated BEC, Zn-BECs were further milled and mixed with alcohol and sieved through a 10 µm mesh using the same procedure as described in Chapter 2, Section 2.2. 3.2.2.3. Characterization XRD, FTIR, and N2 physisorption techniques were used to characterize the physicochemical properties. Hydration, setting time and bioactivity performance were also evaluated as described in Appendix 1. Chapter 3. Synthesis and Characterization of Zn-BECs 68 3.3. Results 3.3.1. Zn-BEC using 28% molar Ca-substitution 3.3.1.1. XRD analysis of 28Zn-BEC precursor Figure 3.2. XRD pattern of 28Zn-BEC precursor. The 28Zn-BEC precursor contained mainly crystalline CaN (PDF: 00-026-1426) and, matching peaks of Zn(OH)NO3H2O (PDF: 01-084-1907) were also detected. 3.3.1.2. XRD analysis of un-hydrated and hydrated heat treated 28Zn-BEC The XRD patterns of un-hydrated powder and after 24 h of hydration of the 28Zn-BEC are shown in Figure 3.3. After calcination at 1400 °C, crystalline phases detected are Ca2Si (PDF: 00-028-0235), ZnO (PDF: 01-075-0576), γ-Ca2SiO4 (PDF: 01-087-1256), Ca3Si2O7 (PDF: 00- Chapter 3. Synthesis and Characterization of Zn-BECs 69 003-1105). It is important to note that hydraulic phases Ca3SiO5 and β-Ca2SiO4 were not identified. The main crystalline phases of un-hydrated 28Zn-BEC remained unchanged after hydration, except for generated CaCO3. This result suggests that hydraulic properties cannot be obtained using so high Zn inclusion. Figure 3.3. XRD patterns of un-hydrated and hydrated heat treated 28Zn-BEC. 3.3.2. Zn-BECs using 1, 3 and 5% molar Ca-substitution In order to evaluate the influence of the Zn content, a control material without Zn (0Zn-BEC) was also prepared and characterized. As a modification in the water amount, from 15 mL to 21 mL, respect to the standard BEC material selected in Chapter 2 was introduced; and, in order to the isolated determination of the incorporation of the different Zn contents, it was necessary to check the influence that this water addition parameter by itself may have on the final material properties. Thus, the first part of this selection will be devoted to a comparative characterization of the Zn-free control material and the selected ACaNO-HNO material of Chapter 2. 10 15 20 25 30 35 40 45 50 55 60                  ✓ ✓  ✓ ✓ ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ Hydrated a-CaCO3 Ca3Si2O7 ZnO Ca2Si                                        Intensity (n.u.) 2 Theta (degree)  -Ca2SiO4 Un-hydrated Chapter 3. Synthesis and Characterization of Zn-BECs 70 3.3.2.1. Influence of DI H2O amount on the Zn-free BECs Tables 3.6 and 3.7 revise reactive amounts and ratios used for 0Zn-BEC and ACaNO-HNO Znfree materials. As can be seen Ca:Si stoichiometry of C3S was adjusted more precisely to 3:1 for 0Zn-BEC. Table 3.6. Reactive amounts expressed in moles used for the synthesis of ACaNO-HNO and 0Zn-BEC. Material H2O HNO3 TEOS CaN ACaNO-HNO 0.8372 0.0031 0.0376 0.1125 0Zn-BEC 1.1721 0.0044 0.0375 0.1125 Table 3.7. Reactive molar ratios used for the synthesis of ACaNO-HNO and 0Zn-BEC. Material Ca:Si H2O:TEOS H2O:HNO3 ACaNO-HNO 2.98:1 22.24:1 268.77:1 0Zn-BEC 3.00:1 31.25:1 268.77:1 3.3.2.1.1. XRD analysis of Zn-free BEC precursors As can be seen from the Figure 3.4, the precursor of ACaNO-HNO was achieved amorphous, while 0Zn-BEC precursor showed the presence of crystalline calcium nitrate dihydrate (Ca(NO3)2 2H2O) (PDF 01-070-0053) and calcium nitrate (Ca(NO3)2) (PDF 00-007-0204). The presence of these crystallized calcium nitrates could be resulting from partly re-precipitation or nucleation and growth from solution during gel drying. Chapter 3. Synthesis and Characterization of Zn-BECs 71 Figure 3.4. XRD patterns of Zn-free BEC precursors synthesized using different DI H2O amount. 3.3.2.1.2. XRD analysis of heat treated Zn-free BECs Figure 3.5. XRD patterns of heat treated Zn-free BECs synthesized using different DI H2O amounts. Chapter 3. Synthesis and Characterization of Zn-BECs 72 Both materials showed the presence of T-Ca3SiO5, β-Ca2SiO4 and CaO. The CaO content is slightly higher for 0Zn-BEC than for ACaNO-HNO. 3.3.2.1.3. FT-IR analysis of heat treated Zn-free BECs Figure 3.6. shows that 0Zn-BEC and ACaNO-HNO have similar vibrational bonds, which are Si-O the asymmetric stretching (ν3) (996 cm-1, 938 cm-1, 906 cm-1, 883 cm-1), symmetric stretching (ν1) (846 cm-1, 812 cm-1), out-of-plane bending (ν4) (525 cm-1) and in-plane bending (ν2) (452 cm-1) [22,23]. ACaNO-HNO presented instead a well resolved wide vibrational band at 1500 cm-1 which is ascribed to carbonate species [24]. Figure 3.6. FTIR analysis of heat treated Zn-free BECs synthesized using different water amounts. 3.3.2.1.4. Textural parameters of heat treated Zn-free BECs The specific surface area and pore volume of 0Zn-BEC were remarkably reduced, more than Chapter 3. Synthesis and Characterization of Zn-BECs 73 half, compared to ACaNO-HNO. Table 3.8. Textural parameters of ACaNO-HNO and 0Zn-BEC. Material BET surface area (m2 g-1) Total pore volume (cm3 g-1) ACaNO-HNO 2.6 ± 0.3 0.006 ± 0.000 0Zn-BEC 1.2 ± 0.0 0.002 ± 0.000 3.3.2.1.5. FTIR analysis of hydrated Zn-free BECs Figure 3.7. FTIR analysis of hydrated Zn-free BECs synthesized using different water amounts. After 24 h of hydration, FTIR spectra of both 0Zn-BEC and ACaNO-HNO, consisted of the Si- O ν3 (996 cm-1, 938 cm-1, 906 cm-1), ν1 (846 cm-1, 812 cm-1), ν4 (525 cm-1) and ν2 (452 cm-1) vibrations from remnant calcium silicates and the CO32- vibrational bands. It should be noted that the intensity of the absorption peaks of Si-O in 0Zn-BEC is higher than that of ACaNO- HNO, meanwhile, more pronounced C-S-H peak between 1100-1200 cm-1 was found for Chapter 3. Synthesis and Characterization of Zn-BECs 74 ACaNO-HNO, indicating higher hydration reactivity of the latter [22,24]. 3.3.2.1.6. Setting time The mean and standard deviation of the initial and final setting times of ACaNO-HNO and 0Zn- BEC are shown in Table 3.9. As can be seen, the setting time of 0Zn-BEC was found significantly higher in comparison to ACaNO-HNO. Table 3.9. Initial and final setting times of ACaNO-HNO and 0Zn-BEC. Material Initial setting (min) Final setting (min) ACaNO-HNO 26 ± 1 95 ± 7 0Zn-BEC 45 ± 1 106 ± 3 3.3.2.1.7. Bioactivity evaluation The FEG-SEM micrographs of the surface of hydrated cements after 7 days of immersion in the SBF are presented in Figure 3.8. Although both surfaces were completely covered by a new formed CaP phase, it was important difference in the size and morphology of this growing phase. Typical calcium phosphate aggregates of spherical shape with average diameters in the 1-2 µm range were observed for ACaNO-HNO sample. Comparatively, the spherical aggregates of 0Zn-BEC are smaller in size, and the lamellar formations of CaP could not be well resolved at the higher magnification due to their small particle size. The FTIR analysis of the deposited phase on the surfaces of 0Zn-BEC and ACaNO-HNO are shown in Figure 3.9. The double peaks at 570 cm-1 and 607 cm-1 ascribed to PO43- vibrations, which were detected in both materials, are used for identifying crystalline CaP, proving that the Chapter 3. Synthesis and Characterization of Zn-BECs 75 coating deposited on the surface of the BEC samples was hydroxyapatite. Figure 3.8. FEG-SEM observations and EDX analysis of hydrated Zn-free BECs surface after immersing in SBF for 7 days. (a) and (b) ACaNO-HNO; (c) and (d) 0Zn-BEC. In summary, ACaNO-HNO prepared using 15 mL DI H2O shows a lower CaO content, and a larger specific surface area, which correlates with a lower setting time, and a higher bioactivity response. Chapter 3. Synthesis and Characterization of Zn-BECs 76 Figure 3.9. FTIR spectrum of hydrated Zn-free BECs surface coating after immersing in SBF for 7 days. Chapter 3. Synthesis and Characterization of Zn-BECs 83 times among the samples. 3.3.3.2.6. Crystal phase of Zn-BECs after hydration Figure 3.15. XRD patterns of Zn-BECs with different levels of Zn content following a 24-hour period of hydration. The XRD patterns of the Zn-BEC formulations following 24 h hydration period are depicted in Figure 3.15. In all instances, the peak observed at 2θ=18.02° was attributed to Ca(OH)2 (PDF 00-044-1481). The peaks at 2θ=29.48°, 39.48°, and 43.18° were linked to calcite CaCO3 (c- CaCO3). Notably, for 0Zn-BEC, 1Zn-BEC and 3Zn-BEC, the intensities of peaks associated with T-Ca3SiO5, M-Ca3SiO5, β-Ca2SiO4, and CaO diminished post-hydration. Furthermore, there was no substantial alteration in the γ-Ca2SiO4 in 3Zn-BEC. However, in the case of 5Zn- BEC, apart from a faint diffraction peak at 2θ=18.02° related to Ca(OH)2, there was no significant intensity change observed for M-Ca3SiO5, γ-Ca2SiO4 and ZnO, indicating that the Chapter 3. Synthesis and Characterization of Zn-BECs 84 hydration process of 5Zn-BEC was impeded. 3.3.3.2.7. FTIR analysis of Zn-BECs after hydration Figure 3.16. FT-TR analysis of Zn-BECs with different Zn content levels following 24 hours of hydration. After 24 hours of hydration, the observed adsorption peak at 1644 cm-1 in 0Zn-BEC, 1Zn-BEC, and 3Zn-BEC corresponded to the ν2 vibration mode of O-H [24,30]. Additionally, the peaks at 1796 cm-1, 1354 cm-1 to 1580 cm-1, 875 cm-1, 714 cm-1 were corresponding to CO32- [24,31], as depicted in Figure 3.18. Furthermore, in 0Zn-BEC and 1Zn-BEC, the intensity of the Si-O ν3 absorption peak between 883-996 cm-1 of T-Ca3SiO5 decreased and shifted to a higher range between 1000-1100 cm-1. The Si-O ν3 vibration peak at 937 cm-1 and 890 cm-1 in 3Zn-BEC also shifted towards a higher wave number of 965 cm-1, indicating the dissolution of [SiO4]4- group and polymerization to form C-S-H [30]. However, in 5Zn-BEC, only small absorption peaks of Chapter 3. Synthesis and Characterization of Zn-BECs 85 CO32- at 1354 cm-1 to 1560 cm-1 were observed, while the absorption intensity of the Si-O ν3, ν4, and ν2 remained unchanged after 24 hours of hydration, indicating no hydration behavior occurred in 5Zn-BEC. 3.3.3.2.8. In vitro bioactivity evaluation After immersing the specimens in simulated body fluid (SBF) for a period of 7 days, spherical apatite aggregates were observed to form on the surfaces of 0Zn-BEC, 1Zn-BEC, and 3Zn-BEC. Upon closer examination using SEM at high magnification, small rod-shaped apatite particles were detected on the surface of 1Zn-BEC, while the apatite on the surface of 0Zn-BEC and 3Zn-BEC exhibited a densely packed internal structure (see figure 3.17). Conversely, the surface of 5Zn-BEC displayed a hydrated porous surface without any visible deposit coating. EDX analysis revealed that the deposits on the surface of 0Zn-BEC, 1Zn-BEC and 3Zn-BEC primarily consisted of calcium (Ca) and phosphorus (P), indicating the presence of hydroxyapatite. In contrast, the coating on the surface of 5Zn-BEC was predominantly composed of Ca, with no significant peaks of P observed. Chapter 3. Synthesis and Characterization of Zn-BECs 86 Figure 3.17. FEG-SEM images & EDX analysis of the surfaces of hydrated Zn-BECs with varying levels of Zn at different magnifications following immersion in SBF solution for 7 days: (a,b) 0Zn-BEC; (c,d) 1Zn-BEC; (e,f) 3Zn-BEC; (g,h) 5Zn-BEC; The outer layer of the specimen was removed using a surgical spatula, and the subsequent FTIR Chapter 3. Synthesis and Characterization of Zn-BECs 87 analysis was conducted (see Figure 3.18). The observed absorption peaks at 607 cm-1 and 570 cm-1 were indicative of crystalline CaP, implying that the deposited coating is hydroxyapatite. Distinct phosphate bands were identified in 0Zn-BEC, 1Zn-BEC, and 3Zn-BEC, while no discernible PO43- absorption peak was evident in 5Zn-BEC. Notably, the phosphate intensity in 1Zn-BEC was notably higher than that of the other two samples, suggesting that 1Zn-BEC exhibited higher bioactivity. The remaining peaks in the infrared spectra of Zn-BEC cements after 7 days of incubation in SBF reflect the Si-O ν3 vibrations of all samples, including 5Zn- BEC, which shifted to higher wave numbers of approximately 1000-1100 cm-1. This suggests that all cements underwent a favorable hydration reaction during the 7-day immersion in SBF. Figure 3.18. FTIR analysis of hydrated Zn-BEC surfaces after 7 days SBF assay. Figure 3.19 illustrates the pH variations of four Zn-BEC cement specimens following a 7-day immersion in SBF solution. It was evident that the pH of the SBF consistently increased over Chapter 3. Synthesis and Characterization of Zn-BECs 88 time for all samples. At equivalent time intervals, the pH of 0Zn-BEC was the lowest, followed by 1Zn-BEC and 3Zn-BEC, with 5Zn-BEC exhibiting the highest pH. Figure 3.19. The pH value alterations of hydrated Zn-BECs after immersing in SBF solution for a duration of 7 days. Figure 3.20 illustrates the weight loss of four types of Zn-BECs following immersion in SBF solution for a duration of 7 days. Specifically, the weight loss of 0Zn-BEC, 1Zn-BEC, and 3Zn- BEC specimens were determined to be 7.73%, 8.67%, and 7.43%, respectively. In contrast, the 5Zn-BEC sample exhibited a marginal increase of 1.03% during the same immersion period. The weight of 5Zn-BEC increased, possibly because it continued to react with water in SBF, causing its weight to rise. This was supported by the FTIR analysis of its surface after 7 days in SBF, which indicated that the hydration reaction of 5Zn-BEC was complete. This suggested that while a high Zn content initially slowed down the hydration reaction of BEC, it eventually continues over time. Chapter 3. Synthesis and Characterization of Zn-BECs 89 0%, 1% and 3% Zn inclusive-BECs had similar weight loss, indicating that Zn incorporation had no effect on the degradation rate of BEC when the Zn content was below 3%. Similar to commercial BEC, the dissolution rate of BEC prepared by sol-gel method was high. The higher dissolution rate may result in the presence of internal voids after implantation into the tooth thus causing a poor seal. However, at the same time, BEC has the ability to deposit hydroxyapatite, which fills the internal voids and avoids infiltration. Figure 3.20. Weight loss of hydrated Zn-BECs following immersion in SBF solution for a period of 7 days. The Si ion concentrations of four Zn-BECs following a 7-day immersion in SBF solution are presented in Figure 3.21. As the Zn content increases, there is a corresponding increase in Si ion release, indicating a higher dissolution rate, consistent with observed pH variations. Notably, no Zn ion release was detected in the SBF solution using ICP-AES. Chapter 3. Synthesis and Characterization of Zn-BECs 90 Figure 3.21. Si ion concentration in hydrated Zn-BECs after being immersed in SBF solution for a duration of 7 days. Chapter 3. Synthesis and Characterization of Zn-BECs 91 3.3.3. The influence of EPT on the composition and hydration characteristic of Zn-BECs 3.3.3.1. Crystal phase of Zn-BECs after EPT Figure 3.22. XRD patterns of Zn-BEC powders following EPT with different levels of Zn content. Following the process of EPT, the XRD patterns of Zn-BEC powders are depicted in Figure 3.22. In comparison to untreated Zn-BECs, the diffraction intensities of the peaks of CaO at 37.35° and 53.86° exhibited a decrease in all samples, while the intensity of the diffraction peak at 29.40°, identified as calcite-CaCO3 (c-CaCO3), emerged. Corresponding to the CaO content in untreated Zn-BEC, 0Zn-BEC displayed the highest c-CaCO3 content, which gradually decreased with increasing Zn addition. Negligible c-CaCO3 was observed in Zn-BEC with 5% Zn addition. T-CaiO5, M-CaSiO5, β-CaSiO4, γ-Ca2SiO4, and ZnO were still present in Zn- BEC containing 0-5% Zn after EPT. Chapter 3. Synthesis and Characterization of Zn-BECs 92 3.3.3.2. FTIR analysis of Zn-BECs after EPT The FTIR spectra of the different Zn-BEC powders following EPT are shown in Figure 3.23. The FTIR spectra of the Zn-BECs subsequent to EPT exhibited similar Si-O vibrations in comparison to the untreated Zn-BEC powders (Figure 3.16). Additionally, carbonate bands at 1490-1420 cm-1, 874 cm-1, and 714 cm-1 were observed for all four materials [22,24]. An attenuation in both the intensities and quantities of CO32- absorption peaks was noted with increasing Zn content, indicating a decrease in CaCO3 content. The c-CaCO3 content in 5Zn- BEC (EPT) was remarkably reduced compared to 0Zn-BEC (EPT). Figure 3.23. FTIR spectra of unhydrated Zn-BECs heat-treated at 1400 °C following EPT with different Zn contents. Chapter 3. Synthesis and Characterization of Zn-BECs 99 to the crystalline lattice of C3S is 4.7% by weight (4.7 wt%). The inclusion of Zn results in a reduction in the surface area of BECs. Zn is utilized as a mineralizer in Portland cement to lower its melting point [41]. Consequently, the molten C3S phase can be achieved at a lower calcination temperature, leading to a denser structure upon cooling to ambient temperature. Lin et al. [15]. and Voicu et al. [12] examined the microstructure of Zn-inclusive C3S using SEM, illustrating the compact morphology and a decrease in pore sizes of C3S. The setting time of BECs is influenced by various factors including their composition [42], particle size [43], water-to-powder ratio [44-46], and additives [47]. The prolonged setting times (initial: 45 min) observed in this chapter, in comparison to that of BEC in chapter 2 (initial: 26min), can be attributed to the higher water to powder ratio. The increased quantity of DI H2O during the synthesis process results in Zn-free BEC with a reduced surface area, necessitating a lower amount of Milli-Q H2O for achieving a paste with similar viscosity and consistency. For comparative purposes, the same water-to-powder ratio was utilized, leading to an excessive water and consequently an extended setting time [44,45]. Additionally, the incorporation of Zn reduced the contents of β-C2S and CaO, and altered the crystalline form of C3S, which solely has a prolonged setting time. Zhao et al. [36] prepared pure C3S using the sol-gel method and measured its setting time, with initial setting and final setting of 120 min and 180 min, respectively. Zhao et al. [37] also prepared pure C3S using precipitation method with initial and final setting times of 70 ± 5 min and 130 ± 6 min respectively. Grech et al. [48] have reported a final setting time of tricalcium silicate bone cement with 20% ZrO2 substitution as 290 min. Conversely, it has been reported that the high-temperature crystalline form of C3S exhibits accelerated hydration activity [39]. The combined influence of these factors results in a slightly longer setting time for Zn-BECs. However, the final setting of Zn-BECs (<2 h) remained Chapter 3. Synthesis and Characterization of Zn-BECs 100 significantly shorter than that of ProRoot MTA (4 h) [22]. XRD and FTIR analyses of Zn-BEC after 24 hours of hydration indicated that 0Zn-BEC, 1Zn- BEC and 3Zn-BEC exhibited similar levels of hydration, while the hydration process of 5Zn- BEC is hindered. Zn is known to significantly impede the hydration reaction of cement [34,38]. Incorporation of low levels of Zn did not affect the reactivity of C3S, but as the Zn content increased, it prevented its hydration reaction [38]. Zn forms insoluble Ca(Zn(OH)3)2 2H2O with Ca2+ and OH- ions in the solution, reducing the concentration of Ca2+ and inhibiting formation of portlandite and saturation of pore solution, thus further hindering the hydration of cement grains [32]. The precipitation of portlandite and C-S-H gel is inhibited until all Zn has been converted into insoluble Ca(Zn(OH)3)2 2H2O [32]. Similarly, Eltohamy et al. [49] demonstrated that the addition of Zn slowed down the cement setting reaction. Lin et al.[15] investigated the hydration properties of Zn-C3S and found that there were no significant heat releases after the initial rapid exothermic reactions when Zn addition reached 4.8%, indicating the inhibitory effect of Zn on Zn-C3S hydration. The findings of this study revealed that Zn had no impact on the hydration process of BEC when the Zn content was 1% and 3%, but it inhibited the hydration reaction of BEC when the Zn content was 5%. The assessment of bioactivity in vitro demonstrated that a hydroxyapatite coating was formed on the surfaces of 0Zn-BEC, 1Zn-BEC and 3Zn-BEC cements after 7 days of immersion in SBF, while the formation of apatite on 5Zn-BEC cements surface was hindered. It is widely acknowledged that the inclusion of Zn in BGs hinders their bioactivity [50-52]. Zn can function as both a network modifier and a network-forming agent in the Si-O network, which reduces degradation of the glassy network and consequently diminishes its bioactivity [52]. The rapid release of Si ions from the glass is a critical factor in determining its high bioactivity [53]. However, it is not the case for Zn-BECs, as it was observed that the release of Si ions from 5Zn- Chapter 3. Synthesis and Characterization of Zn-BECs 101 BEC was the highest among the four samples. The inhibitory effect of Zn on the bioactivity of BEC can be attributed to the fact that Zn binds to phosphate ion (PO43-) in the SBF solution to form an insoluble precipitate, depleting the PO43-. In aqueous solution, the solubility product of Zn3(PO4)2 is 9.1 × 10-3, which is four orders of magnitude lower than that of Ca3(PO4)2 (2.0 × 10-29), indicating that the Zn2+ ion binds more readily to the PO43- ion than Ca2+. Kanzake et al. [54,55] investigated the effect of Zn2+ ions on the growth of HA and found that a solution containing very small amounts of Zn2+ ions inhibited HA growth. In this study, the concentration of Zn in the SBF was measured and found to be undetectable, which supports this perspective. However, when the Zn content was less than 5%, Zn-BEC exhibited good bioactivity, particularly 1Zn-BEC, which displayed larger HA crystal particles. The immersion of Zn-BECs in SBF led to the release of calcium hydroxide, resulting in a rise in solution pH. The increased solubility of the cement is attributed to greater porosity [44,45], which in turn is influenced by a higher water/powder ratio. In specimens with higher Zn content, the smaller surface area leads to greater porosity when mixed with the same volume of water, consequently resulting in higher solubility. This increased solubility led to the release of more OH- ions upon contact with SBF, thereby causing a more rapid increase in pH. The treatment of Zn-BECs with ethanol after synthesis leads to a reduction in particle size and an increase in specific surface area. This treatment also results in a significant decrease in setting time, an increase in hydration activity and a reduction in the inhibitory effect of high Zn content on Zn-BEC hydration. The decrease in setting time is attributed to the reduction in particle size [43] and the introduction of CaCO3 [56,57]. Previous researches have demonstrated that the incorporation of CaCO3 in calcium silicate cements can serve as a site for crystallization of calcium silicate hydration. The reduction in setting time of Zn-BECs treated with ethanol is primarily due to the decrease in particle size and the presence of CaCO3. Chapter 3. Synthesis and Characterization of Zn-BECs 102 The reduction in setting time is most pronounced for 0Zn-BEC, followed by 1Zn-BEC (EPT) with limited CaCO3, and then 3Zn-BEC (EPT) and 5Zn-BEC (EPT) with very little CaCO3. 3.5. Conclusions At a high level of Zn incorporation (28%), the composition of Zn-BEC primarily comprises ZnO and γ-C2S, with absence of hydrophilic C3S and β-C2S. Low levels of Zn incorporation do not impact the formation of hydraulic C3S, but the inclusion of Zn facilitates the formation of C3S and modifies its crystalline structure, reducing the quantity of β-C2S and CaO. The integration of Zn does not affect the setting time of Zn-BEC. Excessive Zn integration (5%) impedes the hydration reaction of C3S, while 1% and 3% Zn integration does not have an adverse effect on the hydration of Zn-BECs. Low levels of Zn addition (≤3%) do not have a negative impact the apatite deposition on the surface of Zn-BEC cements, while high levels of Zn addition (5%) prevent the formation of apatite on the cement surface. Zn integration elevates the pH of the SBF solution after soaking and increases the concentration of ionic dissolution. 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Wasielewsky; G.S. dos Santos; A. Bernardi; E.A. Bortoluzzi; L.d.F.R. Garcia. Effect of the addition of nanoparticles of CaCO3 and different water-to-powder ratios on the physicochemical properties of white Portland cement. Microscopy Research and Technique (2021), 84, 592-601. [57] M.G. Parvan; G. Voicu; A.I. Badanoiu; A.I. Nicoara; E. Vasile. CO(2) Sequestration in the Production of Portland Cement Mortars with Calcium Carbonate Additions. Nanomaterials (2021), 11, 875. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 114 Table 4.1. Reactive amounts expressed in moles used for MBECs synthesis. Materials DI H2O HNO3 TEOS CaN 2.0CaSi 0.8372 0.0031 0.0375 0.0750 1.8CaSi 0.8372 0.0031 0.0375 0.0675 1.6CaSi 0.8372 0.0031 0.0375 0.0600 1.5CaSi 0.8372 0.0031 0.0375 0.0563 1.3CaSi 0.8372 0.0031 0.0375 0.0488 Table 4.2. Reactive molar ratios used for MBECs synthesis. Materials Ca:Si H2O:TEOS H2O:HNO3 2.0CaSi 2.0:1 22.32:1 268.77:1 1.8CaSi 1.8:1 22.32:1 268.77:1 1.6CaSi 1.6:1 22.32:1 268.77:1 1.5CaSi 1.5:1 22.32:1 268.77:1 1.3CaSi 1.3:1 22.32:1 268.77:1 4.2.2. Physico-chemical characterization of MBECs XRD and FTIR were employed to examine the chemical composition and functional groups of the five sample powders and the hydrated cements. N2 physisorption was utilized to measure the specific surface area and textural parameters. The setting time was assessed using Gillmore needle. Further details regarding the experimental procedures can be found in Appendix 1. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 115 4.3. Results 4.3.1. Wide-angle X-ray diffraction (WAXRD) analysis Figure 4.1. WAXRD patterns of heat treated MBEC powders with the different Ca:Si molar ratios. The crystal structures were identified using X'Pert HighScore Plus and the International Center for Diffraction Data PDF-2 2002. The WAXRD patterns are shown in Figure 4.1. As can be seen there is little variation in the crystal formation with varying the different Ca:Si ratios. The major diffraction peaks within 2θ=32-34° correspond to β-C2S (PDF:00-033-0302). 2θ=37.3° and 53.8° are matched with calcium oxide (CaO) phase (PDF: 00-037-1497). The intensity ratios of CaO to β-C2S are lower with decreasing the Ca:Si ratio. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 116 4.3.2. Small-angle X-ray diffraction (SAXRD) analysis Figure 4.2. SAXRD patterns of the MBEC powders with the different Ca:Si molar ratios. SAXRD is a powerful experimental technique used to study the structure of highly ordered mesoporous materials [7,41]. The SAXRD patterns for 1.5CaSi and 1.3CaSi formulations showed a well-resolved diffraction peak in the small-angle range (2θ=1.18°) which can be indexed to the (100) diffraction of a two-dimensional hexagonal (p6mm) lattice (Figure 4.2) [2,42]. It was observed that increasing in Ca content produces both a decrease in intensity and an increase of the peak width, suggesting a loss of nanopore order. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 117 4.3.3. FTIR analysis Figure 4.3. FTIR analysis of the heat treated MBEC powders with the different Ca:Si molar ratios. Figure 4.3 presents the FT-IR spectra of the as prepared MBEC powders. In all cases the spectrum appeared dominated by the characteristic bands of the CO32- groups and the Si-O bonds. Intense absorption bands were shown at 997, 925, 895 and 850 cm-1 that corresponded to the asymmetric stretching (ν3) vibrations of the Si-O bonds and the band at 520 cm-1 was attributed to the out-of-plane bending (ν4) of the Si-O bond in β-C2S phase [43]. The absorption peaks at 1480-1420 and 875 cm-1 were assigned to CO32- group [44-46], originating from hydration reaction of CaO that leads to the formation of Ca(OH)2, which, exposed to the air, formed calcium carbonate (CaCO3) [47]. A slight decrease in intensity of the main peaks at 1480-1420 and 875 cm-1 corresponding to the CO32- group was observed with decreasing Ca content, which was in good agreement with the decreasing CaO amount observed by XRD patterns. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 118 4.3.4. Physisorption measurement Figure 4.4. N2 adsorption-desorption isotherms of the MBEC powders with different Ca:Si molar ratios. Figure 4.5. BJH pore size distributions obtained by the isotherm adsorption branch of the MBEC powders with different Ca:Si molar ratio. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 119 Figures 4.4 and 4.5 show the N2 adsorption-desorption isotherms and pore size distributions for the synthesized MBEC powders. The results indicated the Type IV isotherms, characteristic profile of mesoporous materials and the hysteresis loops resulting from capillary condensation observed for the nanopore structure were of the H1 type, characteristic features of cylindrical nanopores [1,48]. Barret-Joyner-Halenda (BJH) pore size distribution of the specimens proved the preservation of mesoporous structure with an average pore diameter close to 4 nm. Table 4.3. Specific surface area, total pore volume and pore size distribution of the MBEC powders measured by the N2 adsorption-desorption isotherms. Materials BET surface area1 (m2 g-1) Total pore volume2 (cm3 g-1) Pore size3 (nm) 2.0CaSi 28.78 0.0458 3.5 1.8CaSi 29.44 0.0565 3.9 1.6CaSi 22.60 0.0416 3.9 1.5CaSi 24.65 0.0557 4.1 1.3CaSi 30.71 0.0478 3.9 1 Specific surface area are calculated by the BET method. 2 Total pore volume is estimated by the N2 adsorbed amount at 0.99 relative pressure. 3 Pore size distribution is obtained by the BJH method using the isotherm adsorption branch. Table 4.3 summarizes the BET surface area (SBET), total pore volume (VT) and average pore size obtained by N2 physisorption of five MBEC powders. As can be seen, the specific surface area was in the range between 22.6 to 30.7 m2 g-1. It was evident from the table that there was no correlation between the resolution of the low angle peak and the textual parameters. The MBEC powders containing different CaO content had similar specific surface area, total pore volume and average pore size, which were remarkably higher compared to standard BEC and Zn-BECs in Chapter 2 and Chapter 3. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 120 4.3.5. Setting time Figure 4.6. Setting times of the MBECs with the different Ca:Si molar ratios. Table 4.4. Initial and final setting times of the MBECs with the different Ca:Si molar ratios. Materials Initial setting (min) Final setting (min) 2.0CaSi 91 ± 4 226 ± 6 1.8CaSi 82 ± 4 219 ± 5 1.6CaSi 76 ± 1 213 ± 3 1.5CaSi 64 ± 1 199 ± 1 1.3CaSi 82 ± 2 217 ± 6 Figure 4.6 and Table 4.4 show the initial and final setting times of the MBEC materials. After mixing with Milli-Q water, the initial setting time of MBEC materials was shortened from 91 Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 121 ± 4 min to 64 ± 1 min as the Ca:Si ratio was decreased from 2:1 to 1.5:1. However, the initial setting time was prolonged to 82 ± 2 min when the Ca:Si ratio was further decreased to 1.3:1. The final setting time followed the same tendency. Among the MBECs synthesized by different Ca:Si ratios, 1.5CaSi possessed the shortest setting time of 64 ± 1min. 4.3.6. Crystal phase of hydrated MBECs analysis Figure 4.7. XRD patterns of the MBECs after 1 day of hydration using a liquid to powder ratio of 0.9 mL g-1. Figure 4.7 presents the XRD patterns of the MBECs after hydration. As shown, the hydration products were CaCO3 in the allomorphs calcite (c-CaCO3) (PDF: 00-086-2334), Ca(OH)2 (PDF: 00-044-1481) and β-Ca2SiO4. c-CaCO3 was also identified in the set form of MBECs. Similar phenomenon was reported for BioAggregate [49]. Peaks corresponding to β-Ca2SiO4 were strongest in 2CaSi, and decreased in the materials 1.8CaSi, 1.6CaSi, 1.5CaSi. For 1.3CaSi, all Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 122 β-Ca2SiO4 reacted with water and no residual β-Ca2SiO4 peaks were observed. β-Ca2SiO4 react with water to form calcium silicate hydrate (C-S-H) and Ca(OH)2 [40]. The exposure of Ca(OH)2 to air forms CaCO3. Since the C-S-H is amorphous or semicrystalline, it cannot be detected by XRD. 4.3.7. FT-IR analysis of hydrated MBECs Figure 4.8. FT-IR spectra of the MBECs after hydration for 24 h using a liquid to powder ratio of 0.9 mL g-1. Figure 4.8 displays the FTIR analysis of the MBECs after hydration. The absorption peaks of CO32- group (centered at 1450 cm-1 and 875 cm-1) [44-46] appeared in all specimens, being the highest intensity band for 1.3CaSi. The asymmetric stretching vibrations (ν3) (997 cm-1, 925 cm-1, 895 cm-1, 850 cm-1) and out-of-plane bending vibrations (ν4) (520 cm-1) [43] of Si-O from the as-sintered β-Ca2SiO4 powders weakened from 2.0CaSi to 1.3CaSi suggesting the Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 123 dissolution of β-Ca2SiO4 crystals. No apparent C-S-H bands were observed in 2CaSi to 1.5CaSi, probably owing to the slow hydration rate of β-Ca2SiO4 [45]. For 1.3CaSi, spectra of Si-O bands in range of 850-1000 cm-1 shifted toward high wavenumbers 1000-1100 cm-1 suggesting rearrangement of [SiO4]4- and polymerization of silica to form C-S-H [46]. No peaks associated with unreacted β-Ca2SiO4 were found. It could be clearly seen that the remnant β-Ca2SiO4 content in MBEC cements after hydration decreased with decreasing Ca content. There was a relatively large amount of residual β-Ca2SiO4 still present in 2.0CaSi, and there was no significant difference in the detection of unreacted β-Ca2SiO4 for 1.8CaSi to 1.5CaSi. β-Ca2SiO4 reacted completely with water to form C-S-H in 1.3CaSi. The MBEC powder with the lowest Ca content (1.3CaSi) shows the highest hydration reactivity. 4.3.8. The pH variation Figure 4.9. pH value changes of SBF medium during immersion of set MBEC disks for a period of 7 days. Chapter 4. Synthesis and Hydration Performance of Mesoporous BECs 130 are prepared by the sol-gel method combined with organic templates. XRD and FTIR analyses show that the composition of MBECs with varying Ca:Si molar ratios primarily consist of β- C2S and CaO. A trend is observed wherein the CaO content decreases as the Ca:Si molar ratio decreases. The sol-gel method is demonstrated to be effective in preparing stable and highly reactive β-C2S powders at low temperatures without the addition of stabilizers. The SAXRD patterns indicate that the diffraction peaks become progressively sharper as the CaO content decreases. The introduction of ordered mesopore structure using P123 as a structure-directing agent is confirmed when Ca:Si=1.5. N2 adsorption-desorption isotherms show a two to threefold increase in specific surface area. 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Cytocompatibility and antibacterial properties of capping materials. The Scientific World Journal (2014), 2014, 181945. General Conclusions 137 General Conclusions The findings from this Doctoral Thesis work can be summarized in the following general conclusions: ➢ Bioactive endodontic cements (BECs) with high content of hydraulic C3S are successfully obtained by optimizing the sol-gel synthesis routes. The main components of these BEC powders are hydraulic Ca3SiO5 (C3S) and β-Ca2SiO4 (β-C2S). The implementation of a novel additional ethanol post-synthesis treatment (EPT) at room temperature allows for a final BEC product with a finer particle size and increased CaCO3 content, resulting in an improved material in terms of setting time and bioactive response. ➢ The sol-gel synthesis approach of BECs incorporating zinc (Zn-BECs) indicates that at a high level of Zn inclusion (28% molar), the composition of Zn-BEC primarily comprises ZnO and γ-C2S, with absence of hydraulic C3S and β-C2S. When the Zn inclusion is lower (1% to 5% molar), it promotes the formation of C3S and reduces the amount of β-C2S and CaO, resulting in C3S becoming the main phase. The addition of Zn alters the crystalline structure of C3S, causing it change from triclinic to monoclinic, this later being the actual polymorph found in commercial BECs. The inclusion of Zn does not affect the setting time of Zn-BECs. Excessive Zn inclusion has a negative impact on the hydration reaction and General Conclusions 138 apatite deposition ability, while low levels of Zn addition have a similar or even better hydration rate and bioactivity compared to the Zn-free BEC. Besides, further EPT treatment reduces the particle size of Zn-BECs, introduce CaCO3, increases their surface area, and consequently reduces their setting time and enhances their hydration performance. ➢ Mesoporous BECs (MBECs) with major components of β-C2S and CaO are successfully prepared by the sol-gel method combined with a structure-directing agent. These materials of varying Ca:Si molar ratios, are primarily composed of β-C2S and CaO. A trend is observed where the CaO content decreases as the Ca:Si molar ratio decreases. All MBECs showed monodisperse populations of nanopores, leading to increased surface area and reduced setting time. Among the BECs, Ca:Si=1.3:1 formulation exhibits the highest hydration reactivity. Based on their chemical composition and hydration behavior, it is suggested that both Ca:Si=1.3:1 and Ca:Si=1.5:1 show potential for future use in endodontic applications. Appendix 1. Characterization Techniques and Methods 139 APPENDIX 1 Characterization Techniques and Methods 1.1. Thermal Treatment Procedure After obtaining the dried gel, grind it in the agate mortar to ensure uniformity of the precursors. The resulting fine powders are then placed in a 100 mL alumina crucible (HDN ceramics) with a lid and heated in a programmable furnace (MOD. JM 3/16, OBERSAL). Figure 1 illustrates the sintering profiles of standard BEC and Zn-BEC, while Figure 2 depicts the sintering profile of MBEC.