ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1700

Calcium Phosphate BasedBiomaterials for BoneAugmentation

JUN LUO

ISSN 1651-6214ISBN 978-91-513-0399-4urn:nbn:se:uu:diva-356982

Dissertation presented at Uppsala University to be publicly examined in Häggsalen,Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, Friday, 28 September 2018 at 13:15 forthe degree of Doctor of Philosophy. The examination will be conducted in English. Facultyexaminer: Professor Marc Bohner (RMS Foundation, Bettlach, Switzerland).

AbstractLuo, J. 2018. Calcium Phosphate Based Biomaterials for Bone Augmentation. DigitalComprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 1700. 55 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0399-4.

Basic (apatite-based) calcium phosphate cements (CPCs), and acidic (brushite and monetite-based) CPCs are used as bone replacement materials because of their bioactivity, mouldabilityand ability to harden in place. However, their application is limited by their inherent brittlenessand difficulties related to their handling. The current thesis aimed to provide solutions to theselimitations.

To assess the baseline, the mechanical properties of two promising experimental and twocommercially available apatite and brushite cements were investigated. The two experimentalCPCs exhibited significantly higher mechanical strengths than the two commercially availableones, warranting further advancement of the former towards clinical use.

The setting reaction of brushite cements was, for the first time, quantitatively studied inthe first seconds and minutes, using synchrotron X-ray diffraction. The reaction was found toinclude a fast nucleation induction period (<9 s), nucleation (<18 s), brushite content increaseand setting completion. The effect of the commonly used retardant citric acid – which usuallyalso gives stronger brushite cements - was also evaluated, providing important information forfurther cement development.

To overcome complicated usage and short shelf life of acidic CPCs, a ready-to-use acidicCPC was developed by mixing a monocalcium phosphate monohydrate (MCPM) paste and aβ-tricalcium phosphate (β-TCP) paste with suitable amounts of citric acid. The CPC showedadequate shelf life, good cohesion and mechanical performance.

To mitigate against the brittle behavior of CPCs, i) poly(vinyl alcohol) fibres were used toreinforce apatite cements, significantly improving the apatite matrix’s toughness and resistanceto cracking; ii) injectable, ready-to-use organic-inorganic composites with partly elastomericcompression behavior were designed based on silk fibroin hydrogels and acidic calciumphosphates, and their ability for antibiotic drug delivery was assessed.

In summary, insights into the functional properties of currently available CPCs as wellas the setting process of brushite cements were gained and several new calcium phosphate-based formulations were developed to overcome some of the drawbacks of traditional CPCs.Further studies, in particular of the biological response, are needed to verify the potential of thedeveloped materials for future use in the clinical setting.

Keywords: bone substitute materials, calcium phosphate cements, apatite, brushite, monetite,setting mechanism, premixed, fibre reinforcement, composite, silk fibroin, mechanicalproperties

Jun Luo, Department of Engineering Sciences, Applied Materials Sciences, Box 534, UppsalaUniversity, SE-75121 Uppsala, Sweden.

© Jun Luo 2018

ISSN 1651-6214ISBN 978-91-513-0399-4urn:nbn:se:uu:diva-356982 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-356982)

To my family

List of Papers

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

I Luo, J., Ajaxon, I., Ginebra, M. P., Engqvist, H., & Persson, C.

(2016). Compressive, diametral tensile and biaxial flexural strength of cutting-edge calcium phosphate cements. Journal of the Mechanical Behavior of Biomedical Materials, 60, 617-627.

II Luo, J., Martinez-Casado, F. J., Balmes, O., Yang, J., Persson, C., Engqvist, H., & Xia, W. (2017). In Situ Synchrotron X-ray Diffraction Analysis of the Setting Process of Brushite Cement: Reaction and Crystal Growth. ACS Applied Materials & Inter-faces, 9(41), 36392-36399.

III Luo, J., Engqvist, H., & Persson, C. (2018). A ready-to-use acidic calcium phosphate cement. (submitted)

IV Luo, J., Faivre, J., Engqvist, H., & Persson, C. (2018). The ad-dition of poly(vinyl alcohol) fibres to apatitic calcium phos-phate cement can improve its toughness. (submitted)

V Luo, J., Wu, D., Engqvist, H., & Persson, C. (2018). Silk fibroin hydrogels induced and reinforced by acidic calcium phosphate – A simple way of producing injectable, bioactive and drug-loadable composites for biomedical applications. (manuscript)

Reprints were made with permission from the respective publishers.

Author’s Contributions

My contributions to the papers included in this thesis are: Paper I Part of planning, experimental work, evaluation, major part of

writing

Paper II Part of planning, experimental work, evaluation, major part of writing

Paper III Major part of planning, experimental work, evaluation and

writing

Paper IV Part of planning, experimental work, evaluation, major part of writing

Paper V Major part of planning, experimental work, evaluation and

writing

Also published

Chen, S., Shi, L., Luo, J., & Engqvist, H. (2018). A novel fast-setting miner-al trioxide aggregate: Its formulation, chemical-physical properties and cyto-compatibility. ACS Applied Materials & Interfaces, 10 (24), 20334–20341.

Contents

Introduction ................................................................................................... 13

Aims and objectives ...................................................................................... 15

Background ................................................................................................... 16 Bone ......................................................................................................... 16 Bone substitute materials ......................................................................... 16 Chemistry of Calcium Phosphate Cements .............................................. 17

Apatite cements ................................................................................... 17 Brushite/monetite cements ................................................................... 18

Currently available apatite and brushite cements – Paper I .......................... 19

Setting mechanism of CPCs – Paper II ......................................................... 23 Apatite cements ........................................................................................ 23 Brushite cements ...................................................................................... 23 Controlling the setting reaction ................................................................ 25

Accelerating the setting process of α-TCP-based apatite cements ...... 25 Decelerating the setting process of brushite cements .......................... 25

Conclusion ................................................................................................ 28

Premixed CPCs – Paper III ........................................................................... 29 Conclusion ................................................................................................ 31

Fibre reinforcement – Paper IV .................................................................... 32 Conclusions .............................................................................................. 33

Organic/inorganic composites for enhanced mechanical behavior and bioactivity – Paper V .................................................................................... 34

Conclusion ................................................................................................ 35

Summary ....................................................................................................... 36

Future perspectives ....................................................................................... 37

Appendix ....................................................................................................... 38 Preparation methods ................................................................................. 38

Analytical techniques .................................................................................... 40 Mechanical assessment ............................................................................ 40

Compressive strength (CS) .................................................................. 40 Diametral tensile strength (DTS) ......................................................... 40 Biaxial flexural strength (BFS) ............................................................ 40

Setting time .............................................................................................. 41 Phase composition .................................................................................... 41 Porosity .................................................................................................... 42 Injectability............................................................................................... 43 Cohesion/water resistance ........................................................................ 43 Scanning electron microscopy (SEM) ...................................................... 43 Shelf life ................................................................................................... 43 Fourier transform infrared spectroscopy (FT-IR) ..................................... 44 Ultraviolet-Visible (UV-Vis) spectroscopy .............................................. 44

Svensk sammanfattning ................................................................................ 45

Acknowledgements ....................................................................................... 47

References ..................................................................................................... 49

Abbreviations

α-TCP Alpha-tricalcium phosphate β-TCP Beta-tricalcium phosphate BFS Biaxial flexural strength CDHA Calcium-deficient hydroxyapatite CPC Calcium phosphate cement CS Compressive strength DCPA Dicalcium phosphate anhydrous, or Monetite DCPD Dicalcium phosphate dihydrate, or Brushite DTS Diametral tensile strength FT-IR Fourier transform infrared spectroscopy HA Hydroxyapatite MCPM Monocalcium phosphate monohydrate Micro-CT Micro-computed tomography PVA Poly(vinyl alcohol) SEM Scanning electron microscopy SPP Disodium dihydrogen pyrophosphate SXRD Synchrotron X-ray diffraction XRD X-ray diffraction

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Introduction

Bone- and cartilage-related diseases affect millions of people worldwide, and are the most prevalent of human diseases [1].

Bone autografts (i.e. autologous bone) are regarded as the gold standard for reconstruction/replacement of bone defects, followed by allografts (bone from donors or cadavers) [2]. These reconstruction options have the im-portant advantages of osteoinductivity (contain proteins which help facilitate bone healing) and osteoconductivity (provide scaffolds in which bone in-growth may occur) [3, 4]. However, there is a great need for synthetic bone substitute materials in the treatment of damaged and diseased bone due to the limitations associated with autograft and allograft substitutes (e.g. limited supply, high cost, pain at the site of harvest and the co-morbidities associat-ed with the harvesting process) [2, 5]. Ideally, the synthesized bone substi-tute materials should meet precise specifications, such as having good bio-compatibility, osteoconductivity, safety, good bonding with the human bone, suitable mechanical strength, porosity and a resorption rate in line with new bone formation, be easy to use and cost-effective [2]. Calcium phosphate cements (CPCs) are promising candidates for bone replacement materials due to their numerous advantages, in particular their chemical similarity to the mineral phase of bone (the main constituent of human bone is biological apatite), and their promotion of rapid bone for-mation at the surface [6, 7]. Generally, there are two main types of CPCs: basic CPCs (apatite-based) and acidic CPCs (brushite and monetite-based). Numerous commercially available CPC products have been developed over the last decades, most of which are apatite-based, such as Norian® Skeletal Repair System (SRS), due to their longer history and commonly better me-chanical properties than brushite and monetite-based cements.

CPCs may allow gradual replacement by newly formed tissue in vivo [8, 9]. However, brushite and monetite-based cements exhibit faster resorption rates than apatite cements due to their metastability in physiological condi-tions [10, 11]. In fact, apatite cements have been observed practically intact even after several years in vivo [8, 12]. Therefore, brushite and monetite cements have attracted attention as more resorbable replacement materials [13] for bone fracture treatment. Commercially available brushite cements, such as chronOS™ Inject, have been used to treat tibia, radius, fibula, and talus fractures [14].

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Whereas much research effort has been put into further improvement of basic as well as acidic CPCs, there is lack of a systematic comparison be-tween commercially available and recently developed in-house CPCs.

Furthermore, although apatite cements with different properties have been successfully synthesized, the development of brushite cements has been rela-tively slow. For brushite, the setting mechanism is not completely clear due to its fast and hard to follow setting process, thus limiting the development of novel brushite cements.

Although there are many commercial CPC products available, clinical ap-plication is limited by a number of disadvantages, including poor mechanical properties and difficulties associated with the handling processes. For exam-ple, the working time of the cements must be long enough to allow filling and/or shaping in clinics. This may be difficult to achieve for some brushite-based formulations. The intrinsic brittleness of CPCs also limits their use to non-load-bearing applications. It is therefore necessary to improve these properties in order to widen their applications. Moreover, infection in bone is one of the greatest problems encountered during postoperative recovery, and may result in loss of bone tissue and implant removal in a second operation [15]. Antibiotic CPC-based biomaterials, e.g. antibiotic-loaded CPCs, can be used to avoid or combat the infection [16].

In this thesis, different calcium phosphate based formulations were inves-tigated in order to provide a better understanding of the setting mechanism of brushite cement, and to optimize certain properties of CPCs and calcium phosphate based composites, namely mechanical, handling and drug delivery properties. More specific aims and objectives are presented in the next sec-tion.

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Aims and objectives

The aims of this thesis were to (i) study the mechanism of the setting reac-tion of acidic cements to inform further cement improvement, (ii) study and improve the mechanical properties of CPCs, and improve (iii) their handling and iv) drug delivery properties. More specifically, the thesis consists of five scientific papers aimed at addressing the following:

1) Comparatively characterize current commercially available and re-cently developed experimental CPCs (Paper I);

2) Understand the setting mechanisms of acidic cements, and use this in-

formation to construct cements with improved strength (Paper II);

3) Optimize CPCs to overcome some current limitations: a) for acidic CPCs, a ready-to-use system was developed to overcome the compli-cated handling process and poor cohesion (Paper III); b) for basic CPCs, fibre-reinforced formulations were developed to reduce brittle-ness and improve ductility (Paper IV);

4) Investigate CPCs as additives to reinforce other materials, such as silk fibroin hydrogels, to form composites, and assess both the mechanical properties and the potential usage of the composites as antibiotic car-riers (Paper V).

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Background

Bone Bone has a complex hierarchical microstructure [17]. It comprises a lamellar structure at the sub-microstructure (1–10 µm) level, with organic matter (mainly type-I collagen fibres) and mineral crystals [17, 18]. It can be divid-ed into cancellous (or trabecular) and cortical (compact) bone at the macro-structural level [17]. The mineral crystals of human bone consist of calcium phosphate salt in the form of hydroxyapatite minerals (Ca5(PO4)3OH), which contain ionic substitutions such as CO3

2-, or Na+, Sr2+, Mg2+, K+ [7, 19]. In human bone, the mineral phase (60-70 vol%) provides compressive strength, and the organic phase (10-20 vol%) provides tensile strength. Water (10-20 vol%) also affects the mechanical behavior of the mineral and the organic matrix [20, 21].

Bone is regarded as a structural scaffold for the human body which pro-tects vital organs and facilitates body movements along with joints, liga-ments and muscles [21].

The bones of our skeletal system may be exposed to multiple loading conditions, i.e. compression, tension, shear and a combination thereof during common, every-day activities [19]. However, traumatic incidents, some can-cers, and conditions such as osteoporosis can cause bone defects, such as voids or fractures, to occur. Whilst bone has the intrinsic ability to remodel and regenerate spontaneously, this is not possible for bone lesions above a certain, critical size (actual size dependent on site) [22]. Instead, these de-fects need to be treated with bone graft substitutes [23]. For example, augmentation with bone grafting has been used in foot and ankle surgery to try to avert nonunion and improve the osteoconductive processes [5].

Bone substitute materials Autograft and allograft substitutes are widely used to fill bone voids and repair bone fractures due to their potential osteoinductivity and osteoconduc-tivity [3, 4]. However, the limited material source, risk of disease transfer, infection, and chronic pain all restrict their application [24]. Synthetic bone graft substitutes, including metals (e.g. titanium [25]), polymers (e.g. poly(methyl methacrylate) [26]), ceramics (e.g. calcium phosphate, calcium

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sulphate, and bioglass) and composite materials (e.g. calcium phosphates in combination with polymers), have been developed for bone repair and re-generation over the past decades [27, 28].

Ceramic materials based on calcium salts have long been used as bone substitute materials and bone implant coatings owing to their excellent bio-compatibility, osteoconductivity and bioactivity [29]. Among ceramic mate-rials, calcium phosphate cements (CPCs) are some of the most researched materials due to their similarity in composition to human bone, with ad-vantages including those mentioned above, but also their ability to harden at body temperature in vivo [7, 30]. CPCs have been applied in clinics as bone void fillers after the first commercial calcium phosphate bone graft substi-tutes appeared on the market 40 years ago [7, 31].

Chemistry of Calcium Phosphate Cements CPCs are hydraulic cements formed by mixing one or several calcium phos-phate powders and an appropriate amount of water or aqueous solution to form a viscous paste. This paste can be easily manipulated and molded, and in some cases injected into bone defects, then self-set into a hard solid in situ [27, 30, 31].

Despite the numerous CPC formulations available, the end products can mainly be divided into two types [32]. The first are basic CPCs, or apatite cements (such as hydroxyapatite (HA), calcium-deficient hydroxyapatite (CDHA)). These are the most stable phases at a pH higher than 4.2. The second are acidic CPCs, divided into either brushite (dicalcium phosphate dihydrate, DCPD) or monetite cements (dicalcium phosphate anhydrous, DCPA). These are the most stable calcium phosphate phases at a pH lower than 4.2 [30, 32, 33].

The chemical process during the setting reaction includes two main steps: dissolution and precipitation [30], with less stable calcium phosphate reac-tants forming more stable CPCs [34]. The surface charge and the size of precipitates affect the material properties.

Apatite cements There are two main types of setting reactions to form apatite; hydrolysis and an acid-base reaction [31].

The hydrolysis reaction, whereby single calcium phosphate (α-tricalcium phosphate, α-TCP) dissolves in water to precipitate into apatite, is used in most commercial cement formulations and also used in this thesis to form apatite cements in Papers I and IV (equation (1)) [31, 32].

3 − ( ) + → ( )( ) (1)

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The acid-base reaction occurs when multiple calcium phosphates (basic reactants, normally tetracalcium phosphate, Ca4(PO4)2O, and acidic reac-tants, brushite/monetite) react with water (equation (2)) [31, 32]. ( ) + 2 → ( ) ( ) (2)

Many experimental and most commercially available basic CPCs are based on the hydrolysis of α-TCP, and experimental basic CPCs in this the-sis will focus mainly on α-TCP-based CPCs.

Brushite/monetite cements Brushite cements are commonly formed as a result of an acid-base reaction. The most common formulation consists of both an acidic calcium phosphate (monocalcium phosphate monohydrate; MCPM; Ca(H2PO4)2•H2O), a slight-ly basic calcium phosphate (β-tricalcium phosphate; β-TCP; β-Ca3(PO4)2) as the main powder phase, and water as follows: − ( ) + ( ) ∙ + 7 → 4 ∙ 2 (3)

Among the acidic CPCs, the precipitation of monetite is endothermic and

the precipitation of brushite is exothermic, and brushite typically precipitates faster than monetite [10, 35]. Therefore, under physiological conditions, it is normally easier to form brushite than monetite. However, monetite forms under some particular conditions, such as at very low pH or in a water-deficient environment [10]. Monetite can also form by dehydration of brushite at high temperatures [10].

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Currently available apatite and brushite cements – Paper I

Generally, at similar liquid to powder ratios, apatite cements have higher viscosities and longer setting times than brushite cements, primarily due to the slower reactant dissolution for apatite compared to that for brushite [36, 37]. A greater number of apatite-based CPCs, such as Norian® SRS, BoneSource® and Biobone®, have been commercially available than brushite based CPCs due to factors such as a longer history and traditionally higher mechanical strength [36, 38].

Brushite cements have, however, a unique advantage over apatite ce-ments. Apatite cement is relatively chemically stable and may still be present after years in vivo [8, 12], whereas brushite is generally 1-2 orders of magni-tude more soluble than apatite at a physiological pH (Table 1) [39]. Brushite and monetite can be resorbed within a few months [10, 40], which potential-ly permits fast replacement of the material with new bone, as has recently been observed in the skull [41]. Under physiological conditions, brushite is one of the precursor phases for apatite formation and the transformation of brushite to apatite may occur, depending on the formulation [7, 32, 42]. Sev-eral brushite and monetite based CPCs have been commercialized, including chronOS™ Inject, Eurobone®, and VitalOs® [38].

Table 1. Some common calcium orthophosphates [43, 44]. Ca/P molar ratio

Compound Formula Solubility 25°C, g·L-1

0.5 Monocalcium phosphate monohydrate (MCPM)

Ca(H2PO4)2·H2O 18

1.0 Dicalcium phosphate dihy-drate (brushite)

CaHPO4·2H2O 0.088

1.0 Dicalcium phosphate anhy-drous (monetite)

CaHPO4 0.048

1.5 Alpha-tricalcium phosphate (α-TCP)

α-Ca3(PO4)2 0.0025

1.5 Beta- tricalcium phosphate (β-TCP)

β-Ca3(PO4)2 0.0005

1.5-1.67 Calcium-deficient hydroxyap-atite (CDHA)

Ca10-x(HPO4)x(PO4)6-x(OH)2-x 0.0094

1.67 Hydroxyapatite (HA) Ca10(PO4)6(OH)2 0.0003

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The mechanical properties of CPCs have undergone extensive investigation, since an enhancement of the same could potentially expand their use to more clinical applications. The mechanical properties of CPCs are determined by their chemical composition, such as the ratio of calcium to phosphate content, and their microstructure, including porosity (pore fraction and size), mor-phology and crystal size [30].

For apatite cements (equation (1) and (2)), no or only very little water from the liquid is consumed during the setting reaction. The majority of wa-ter is used as a dispersant medium to form a workable paste. Porosity pre-dominately stems from unreacted liquid in the voids among the entangled crystals after setting. Therefore, porosity is closely related to the liquid to powder ratio of the starting reactants. A lower liquid to powder ratio leads to lower porosity and stronger cements but also a shorter working time and poorer handling properties. Higher porosity, interconnecting macro porosity and bigger pores are beneficial for fast resorption, tissue ingrowth and bone regeneration [45]. However, the mechanical properties decrease exponential-ly with an increase in porosity [44]. Since the water needed for brushite for-mation is higher than that for apatite formation (equation (3)), brushite ce-ments are generally less porous at similar liquid to powder ratios [46]. Brushite cements are generally considered to have lower mechanical strengths and less potential as structural biomaterials than apatite cements because of their intrinsically brittle plate-like crystals and loose packing [30, 47, 48]. Smaller brushite crystals could be more closely packed to form a dense crystal entanglement and induce fewer defects, which is beneficial for the mechanical properties [49]. This could be achieved through the addition of retardants to inhibit crystal growth. Different additives, as accelerators or retardants, influence the mechanical properties of both apatite and brushite cements by affecting the setting reaction and ultimately their microstructure.

Compressive strength testing is the most commonly used method to eval-uate the mechanical performance of CPCs [11]. While resistance to other loading modes may be more relevant to improve for these intrinsically brittle materials, compression tests are widely used due to their ease of implemen-tation. Also, there is little to compare with, as the tensile and flexural proper-ties of trabecular bone are difficult to measure and data on the same is hence scarce. It is generally assumed that a commercial CPC product should have at least a similar or higher compressive strength than trabecular bone for orthopaedic applications. However, the required mechanical properties may actually vary, as the mechanical properties of bone vary widely depending on position in vivo, age, gender and the stress imposed upon it [11].

As mentioned, other mechanical properties, such as tensile strength, are important to characterize, depending on the application. However, due to the difficulties associated with direct assessment of the tensile strength of CPCs [30], in most cases, alternative methods of measuring, such as flexural or diametral tensile tests are performed [30]. CPCs are brittle materials, and

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similar to most other brittle materials, the compressive strength of CPCs is commonly much higher than the tensile or bending strength [30].

To investigate the current status of commercial and experimental CPCs, Paper I evaluated the mechanical properties of a promising experimental brushite cement with high compressive strength [50] and an experimental fast setting apatite cement [51] in comparison with two commercially avail-able brushite- and apatite-based cements (chronOSTM Inject and Norian® SRS, respectively). In particular, their compressive, diametral tensile and biaxial flexural strengths were assessed in both wet and dry conditions (Fig-ure 1).

This study found that the two in-house cements exhibited higher mechan-ical strength than the two commercially available cements, and both the two in-house cements and Norian® SRS exhibited higher strength than human trabecular bone both under compressive and tensile conditions. However, chronOS™ Inject demonstrated a low mechanical strength, in the lower range of trabecular bone. Environmental conditions can also influence the mechanical behavior of CPCs (Figure 1). The mechanical strength of CPCs was commonly higher under dry conditions than under wet conditions, in agreement with earlier studies, and re-iterating the importance of evaluating these materials under relevant conditions. In fact, testing under wet condi-tions would be more representative of the in vivo scenario [11]. The results of this study supported further development of the experimental CPCs to-wards clinical application, as they demonstrated some advantages in compar-ison to existing cements.

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Figure 1. Mechanical properties of cements: (a) compressive strength (CS); (b) diametral tensile strength (DTS); (c) biaxial flexural strength (BFS). The result pre-sented is the average of between six to ten measurements per group. The error bars represent standard deviations of the mean [52]. (Reproduced with permission from Elsevier)

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Setting mechanism of CPCs – Paper II

A full understanding of the setting mechanism of CPCs would be beneficial for controlling hardening and microstructure formation in order to satisfy the clinical requirements. Many efforts have been made to study the setting pro-cess of CPCs.

Apatite cements For apatite cements, the setting reaction occurs in three stages: reactant dis-solution, new phase nucleation and crystal growth [31]. After mixing the liquid and powder phases, the calcium phosphate powders release calcium and phosphate ions during dissolution to produce a super-saturated solution. The nucleation of the new phases starts on the surface of reactant particles when the ionic concentration reaches a critical value. Once nucleation oc-curs, new phase crystal growth increases with the continuous dissolution of the reactants [31]. The precipitated needle-like or plate-like apatite crystals entangle to form a 3D network with increasing density during setting, providing the mechanical properties of the cement [30, 36].

Because of the low solubility of the reactants, during the first hours, the setting process is controlled by the dissolution kinetics of α-TCP. Once the reactants are surrounded by the new phase, the process is controlled by dif-fusion across this new phase [31].

Brushite cements The setting process of brushite is faster than apatite because of the high sol-ubility of the raw materials, especially acidic reactants like MCPM. The short setting time however causes difficulties in monitoring the reaction pro-cess.

Many studies have focused on the setting process of brushite cements, for example, by monitoring heat release [35, 47], the pH value [53], and the chemical composition [54] during setting. The formation of brushite cement is commonly considered to include four stages: (1) precursor dissolution, (2) a supersaturated paste formation, (3) nucleation of a new phase in the paste,

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and (4) crystal growth of the new phase to form a solid mass with inter-locked crystals [10].

The whole setting process, including phase transformation and crystal growth, happens within minutes. It is therefore challenging to trace this pro-cess in real-time using traditional laboratory techniques. However, being able to evaluate the velocity of the reaction, the way phase transformation starts, and how crystals grow is crucial to understanding the mechanism and in optimizing the brushite formulations to better match their clinical applica-tions.

Paper II investigated the fast (on a time scale of seconds) reaction kinet-ics of brushite formed by precipitation of β-TCP and MCPM in real-time by employing synchrotron X-ray diffraction (SXRD). SXRD enables analysis of the crystal evolution from the very early stages in situ, with detailed struc-tural and morphological information by monitoring crystal evolution and phase transition [55, 56].

The reaction was found to include a fast nucleation induction period, nu-cleation, linear brushite content increase and slow brushite content increase, and setting completion. In Figure 2, a magnified profile of the intensity change of the strongest peak of brushite (corresponding to the (0 2 0) facets of the brushite crystal) from the setting reaction is shown. (Paper II)

Figure 2. Time-dependent X-ray diffraction (XRD) patterns of the brushite evolu-tion from the reaction between the powder mixture of MCPM: β-TCP molar ratio of 1: 1 and a liquid phase of distilled water.

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Controlling the setting reaction The reaction to form brushite is generally much faster than that of apatite [57]. A too-fast setting limits the working time, whereas a too-slow setting also causes problems, such as longer overall procedure duration [6] and poor cohesion which might result in a negative inflammatory response [58]. In clinical use, the working time of the cements must be long enough to finish the preparation process and allow filling and/or shaping [59]. For the con-ventional preparation process of CPCs, typically 10-20 min is a suitable range for the setting time to satisfy the clinical requirement [6]. Without modifications, apatite cements normally have a longer setting time than that. However, the setting time of the initial formulation to form brushite cement is much shorter (around 30 s) [60], which has been one of the main limita-tions of brushite cements.

Besides adjusting the liquid to powder ratio to provide an appropriate set-ting time for clinical applications, other factors can also affect the setting time, such as particle size, crystallinity, setting temperature and additives.

Accelerating the setting process of α-TCP-based apatite cements Factors that accelerate α-TCP dissolution or in other ways promote fast

setting of α-TCP-based apatite cements include fine particle size and low crystallinity of the α-TCP, accelerators, and high setting temperature [30, 32]. Fine particles have a much faster setting rate than coarse ones because of their higher specific area, which accelerates α-TCP dissolution [51]. Low crystallinity of α-TCP has demonstrated a higher reactivity and associated dissolution rate [61]. Many accelerators have been added in the liquid and/or powder phases to promote fast apatite setting, such as certain soluble phos-phate salts (e.g. Na2HPO4) as a source of phosphate ions [62] and apatite particles in the solid phase of apatite formulations as seeds to promote apa-tite formation [32, 51]. Some acids (e.g. acetic acid and MCPM) can also accelerate the setting reaction by lowering the pH, and thereby increasing the solubility of α-TCP. However, the effect strongly depends on the concentra-tion and type of acid [9, 30]. It is well known that temperature influences the kinetics of chemical reactions. Commonly, a high temperature accelerates the hydrolysis of α-TCP and thereby the reaction rate.

Decelerating the setting process of brushite cements Many strategies have been implemented to increase the setting time of brushite formulations by decelerating the dissolution of the reagents and/or the precipitation of brushite crystals, such as adding retardants (e.g. pyro-phosphates, citric acid) [10].

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In Paper II, the influence of a retardant, in this case citric acid, and the MCPM to β-TCP ratio in the setting reaction were also evaluated by SXRD (Table 2).

Table 2. Composition of the four groups of pastes [37] (Reproduced with permission from ACS Publications) Powder phase Liquid phase Group 1 MCPM: β-TCP = 45: 55 Distilled water Group 2 MCPM: β-TCP = 1: 1 Distilled water Group 3 MCPM: β-TCP = 45: 55 Citric acid solution (0.5M) Group 4 MCPM: β-TCP = 1: 1 Citric acid solution (0.5M)

Figure 3. Time-dependent weight fraction from the reaction of group 1 (a), group 2 (b), group 3 (c), and group 4 (d) [37] (Reproduced with permission from ACS Publi-cations).

Variation of the ratio between MCPM and β-TCP affected the setting rate of the cement and the ratio between brushite and monetite in the products (Figure 3). Furthermore, although citric acid did not retard the formation of brushite nuclei in the initial reaction, it significantly decreased the growth rate of the brushite content as well as the brushite crystal size (Figure 3, 4 and 5) [37].

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Figure 4. SEM images from the reaction of group 1 at 2 s (a, b), and 3 min (c, d).

Figure 5. SEM images from the reaction of group 3 at 2 s (a, b), and 3 min (c, d).

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Conclusion In Paper II, the fast setting mechanism of brushite formed from precipita-tion of β-TCP and MCPM was quantitatively analyzed in real time by in situ SXRD to evaluate the crystal evolution from the very early stages. The reac-tion was found to include a fast nucleation induction period, nucleation, line-ar brushite content increase and slow brushite content increase, and setting completion. Variation of the ratio between MCPM and β-TCP affected the setting rate of brushite as well as the ratio between brushite and monetite in the end products. With the presence of citric acid, the formation of brushite nuclei in the initial reaction was not retarded, but the growth rate of the brushite crystal size as well as the brushite content were significantly de-creased. This is the first time that the brushite setting process has been eval-uated in the initial seconds and minutes by SXRD. The findings are benefi-cial for improving our understanding of the setting mechanisms of these cements, and for providing a basis for further material development.

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Premixed CPCs – Paper III

Conventionally, bone substitute materials need to be shaped before being implanted in vivo through surgery. Minimally invasive surgery provides a simplified surgical procedure, smaller surgical incisions, a shorter treatment time and better patient compliance. CPCs have the ability to be filled and moulded into bone defects and harden in situ. These advantages of CPCs have shown great potential in minimally invasive surgery whereby calcium phosphate pastes are injected percutaneously in the body to fill the bone defects, such as in the treatment of fractures of radius and tibia which re-quires injection through a cannulated needle into the fracture site [11], or when used for bone augmentation through cannulated and/or fenestrated screws [63]. Good injectability is a premise for minimally invasive surgery. However, some issues, e.g. risk for phase separation during injection, limit the application of CPCs in minimally invasive surgery, as further detailed below.

For conventional CPCs, a calcium phosphate paste forms after mixing the calcium phosphate powder and the liquid phase. The use of any associated mixing system, commonly provided by the cement manufacturer, may be more or less difficult to use and prone to errors. Furthermore, once the calci-um phosphate powders and water come into contact, the hardening reaction starts and the viscosity of the pastes keeps increasing. Therefore, there is only a limited period of time during which the pastes can be injected. In addition, there is a risk for filter-pressing phenomena to occur (where liquid is preferentially extruded over powder), giving rise to e.g. final mechanical properties that are lower than expected [7].

In order to overcome the drawbacks of conventional CPCs, such as the complicated preparation process, limited injection window and lack of full injectability, premixed (= ready-to-use) CPCs consisting of one or two pastes have been developed [6].

In the one paste system, non-aqueous solvents are combined with the starting calcium phosphate powders to form a premixed paste, which can be injected directly into the bone defects. After injection, the body fluids pene-trate into the pastes to react with the calcium phosphate reactants and form CPCs. The hardening reaction therefore only occurs once the pastes come into contact with water (i.e. when they are injected into the bone defect). With suitable viscosity, the filter pressing phenomenon can also be avoided. On the negative side, the penetration of the body fluid is slow and volume-

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dependent [7, 64]. Furthermore, the penetration of body fluids into the ce-ment causes the interior of the paste to harden at a slower rate than the exte-rior, which weakens the mechanical properties of set cements. Moreover, for the acidic CPCs, the starting calcium phosphate powders (e.g. MCPM and β-TCP) can spontaneously partially set into monetite due to the inherent diffi-culties in eliminating moisture during manufacturing and storage, resulting in a short shelf-life.

In the two-paste system, reactive calcium phosphate reactants are separat-ed into two different premixed pastes formed by mixing with an aqueous solution and a non-aqueous solution, respectively. Mixing the non-aqueous and the aqueous pastes triggers the setting reaction to form CPCs [65]. How-ever, in acidic formulations, because of the high solubility and reactivity of the reactants, difficulties in achieving a good cohesion and sufficient me-chanical properties have so far hindered the development of two-paste acidic CPC systems.

When unset calcium phosphate pastes are delivered into bone defects, they tend to disintegrate upon early contact with body fluids or blood due to poor cohesion. This disintegration might result in severe problems, e.g. ce-ment embolism with potentially fatal outcomes [30, 66]. Therefore, CPCs should have sufficient cohesion to prevent disintegration in vivo. Commonly, a higher liquid to powder ratio not only leads to a decrease in mechanical strength and an increase in setting time, but also results in a lower viscosity of the unset pastes [67] and a reduction in cohesion [11, 32]. Increasing the viscosity of the paste can improve both cohesion and reduce the filter-pressing phenomenon at the same time.

In Paper III, we developed a ready-to-use acidic CPC based on the two-paste system from a series of ready-to-use calcium phosphate pastes (Figure 6). The ready-to-use acidic CPC formed via mixing of an MCPM paste and a β-TCP paste.

Figure 6. Schematic diagram of the ready-to-use acidic calcium phosphate cement.

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The MCPM paste was based on a water-immiscible liquid with two surfac-tants, and the β-TCP paste on a sodium hylauronate aqueous solution. A slightly higher level of β-TCP than MCPM (β-TCP: MCPM= 55: 45 in mo-lar ratio) was in the starting materials. The effect of citric acid as a retardant in the MCPM paste was also assessed. In the MCPM paste, the water-immiscible liquid, the synthetic short chain triglyceride Miglyol 812, with 8-12 C saturated fatty acids, was used as a hydrophobic carrier, and provided the necessary unreactive lubricant and carrier liquid. The two surfactants (castor oil ethoxylate 35 and hexadecyl-phosphate) promoted both compati-bility of the polar mineral particles with the hydrophobic carrier liquid [64], as well as stability of the MCPM paste. In the β-TCP paste, the addition of sodium hyaluronate enhanced not only the viscosity, but also the anti-washout ability of the calcium phosphate pastes [68].

Conclusion The cohesion properties of common premixed acidic CPCs are poor because of the high solubility of MCPM. In this system, with suitable amounts of citric acid, the ready-to-use acidic CPC showed sufficient cohesion, which is beneficial to its mechanical properties in vivo. Generally, acidic CPCs have a short shelf life because of the high reactivity between MCPM and β-TCP. In this system, MCPM and β-TCP were separated into two injectable pastes to prolong their shelf life. With the help of a dual chamber system, this ready-to-use acidic CPC formulation can be easily and quickly mixed and injected during operation. In this case, it could greatly facilitate clinical applications, i.e. by reducing surgery time, decreasing the risk of contamination, and en-suring repeatable results in a minimally invasive manner.

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Fibre reinforcement – Paper IV

Whilst CPCs have many advantages as bone substitute materials, their inher-ent brittleness limits their application to non-load-bearing applications. In fact, even though some cements are much stronger than trabecular bone un-der compression, they remain brittle materials with a low fracture toughness [69].

Many studies have therefore focused on improving the mechanical prop-erties of CPCs to extend their application to load-bearing or at least load-sharing applications. One of the main strategies is to use fibre reinforcement to form composite materials and thereby mitigate their brittle behavior [70]. In these composites the macroscopic mechanical behavior results from the mechanical properties of the cement matrix, the fibres and their mechanical interaction [71]. Different types of fibres, both degradable as well as non-degradable, polymeric (e.g. chitosan [72]) and ceramic (e.g. calcium silicate fibres [73]), have been investigated to improve the toughness and ductility of apatite cements [73, 74].

However, CPCs reinforced with biodegradable polymer fibres usually ex-hibit low elastic moduli and strength [71], and lose their strength too fast. This is because the dissolution of fibres in an aqueous environment results in an extremely porous CPC matrix, e.g. absorbable suture fibres (vicryl poly-glactin 910) reinforced chitosan- CPC composite gave 40.5 ± 5.8 MPa in flexural strength, which decreased to 9.8 ± 0.6 MPa after 35 days immersion in physiological solution and 4.2 ± 0.7 MPa at 119 days [75]. Combining polymer fibres of a lower degradability with CPCs may therefore be a valua-ble route to their reinforcement.

In Paper IV, the toughness of apatite cements was enhanced through poly(vinyl alcohol) (PVA) fibre reinforcement.

PVA is generally considered a biocompatible material. The mechanical properties and degradability of PVA are related to factors such as molecular weight, processing and crystallinity [76]. It also has a high affinity to water [77]. This hydrophilicity could be advantageous when used in water-based CPC matrices, as it provides a better integration with the matrix. In fact, PVA fibres have been found to increase the tensile strength and ductility without excessively decreasing the compression strength of a calcium alumi-nate cement [78]. However, PVA for fibre-reinforcement of CPCs had not yet been studied.

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Therefore, in Paper IV, the incorporation of a strong and stable PVA fi-bre (tensile modulus of 40 GPa and tensile strength of 1.83 GPa) in apatite cements was evaluated in terms of the resulting compressive strength, Young’s modulus, failure strain, diametral tensile strength, toughness (work of fracture), setting time, microstructure and inorganic composition. Differ-ent proportions of PVA fibres were incorporated and tested; 0%, 2.5%, 5% and 7.5% of the total weight of the final powder mixture (α-TCP powder and PVA fibres).

The addition of the PVA fibres did not significantly interfere with the formation of apatite, but reduced the compressive strength and setting time of the cements, most likely due to the PVA absorbing water [79]. The PVA fibres enhanced the fracture resistance of the apatite cement. Cement with 5 wt% of fibres (of powder mixture) could be considered a good compromise, with a compressive strength of 46.5 ± 4.6 MPa (compared to 62.3 ± 12.8 MPa without fibres and 0.1-14 MPa for human trabecular bone [80, 81]), a diametral tensile strength of 9.2 ± 0.4 MPa (compared to 7.4 ± 1.5 MPa without fibres) and a work of fracture of 9.1 ± 1.5 kJ/m2 (increased four times in comparison to cement without fibres).

Conclusions In Paper IV, a biocompatible, hydrophilic, but stable polymeric fibre, poly(vinyl alcohol) (PVA), with high mechanical strength was introduced to apatite cements. The presence of PVA fibres in the apatite cements signifi-cantly improved the toughness of the apatite and avoided the failure mode which is typically seen in non-reinforced CPCs. PVA fibre reinforcement can increase the material’s resistance to cracking, and seems to be a promis-ing alternative for bone replacement applications where a long-term solution is sought, e.g. in applications such as prophylactic femeroplasty, with highly loaded areas, where the long-term support function of the fixation is critical [82].

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Organic/inorganic composites for enhanced mechanical behavior and bioactivity – Paper V

An increasing trend is seen towards incorporating inorganic particles and/or networks into organic hydrogel matrices to reinforce the latter as well as to provide increased bioactivity [83-85]. Calcium phosphates are notable mate-rials as inorganic additions due to their high mechanical strength, good bio-compatibility and osteoconductivity, and ease of synthesis.

Silk fibroin, a natural fibrous protein that can be extracted from silkworm or spider, is a promising biomaterial due to its good biocompatibility and biodegradability, wide availability and nontoxic degradation products [86, 87]. Although silk fibroin-based hydrogels have been widely studied as a potential matrix for tissue engineering [86-88], their relatively poor mechan-ical performance is still a limitation for medical applications, especially as cartilage or bone fillers [89-91].

Existing routes for improving the mechanical properties of silk-based hy-drogels are limited in biomedical applications due to e.g. toxicity of chemi-cal crosslinking reagents involved [92, 93] and/or harsh treatment methods which are unsuitable for in vivo use (e.g. high temperatures [94], addition of hexafluoroisopropanol [95]) [96, 97].A low pH value can trigger the fast gelation of silk fibroin [88, 98, 99]. In Paper V, we developed a novel route to reinforce silk fibroin hydrogels through the manufacture of acidic calcium phosphate/silk fibroin composites. The influence of the calcium phosphate content in the composites was evaluated by varying the weight ratio of acidic calcium phosphate to silk fibroin (2/2, 3/2, 4/2, 5/2, and 6/2). The potential of these composites as drug vehicles was also evaluated because of their good biocompatibility, biodegradability and porosity. The composites were formed from a dual-paste organic-inorganic composite system based on silk fibroin and acidic calcium phosphates. In this system, the acidic calcium phosphates (brushite and monetite) were formed through blending MCPM paste and β-TCP/silk fibroin paste. The acidic environment during brushite formation induced the silk fibroin gelation. The formed silk fibroin hydrogel was also reinforced by the acidic calcium phosphate crystals. The compo-sites displayed a partly elastomeric compression behavior. With an increase in calcium phosphate content, the mechanical strength of the composite in-creased significantly, at a relatively low calcium phosphate content (ratio between acidic calcium phosphate to silk fibroin from 2/2 to 4/2). However,

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at a higher calcium phosphate content, i.e. from 5/2 to 6/2, the increase was not obvious, presumably because a higher brittle calcium phosphate content increased the brittleness of the composites and hindered silk β-sheet for-mation due to high concentration of Ca2+ [100]. Vancomycin was used in the drug release tests. In the drug release tests, vancomycin-loaded samples showed similar drug release behavior for all calcium phosphate contents. Most of the vancomycin (80.0- 94.0 wt%) in all groups was released within 14 days. The release rate of vancomycin was higher than some previous reports based on CPCs [101, 102]. The fast release speed and high release rate may be beneficial in specific applications, and might decrease the risk of mutational resistance due to having antibiotic concentrations at subinhibitory levels for too long a period [31, 103, 104].

Conclusion Composites with a partly elastomeric compression behavior were formed in the dual-paste system. The mechanical properties of the composite were easily varied by altering the amount of the calcium phosphate reactants. While the measured stresses were low (between 0.2 and 1.2 MPa at 10% strain, between 1.1 and 2.9 MPa at 60% strain), they were in the range of failure stresses of trabecular bone (0.1-14 MPa [80, 81]), and the material formulations permitted large, mainly elastic deformations to take place, when tested up to 60% strain. The dual-paste system provides a potential method for developing silk fibroin-based composites with suitable mechani-cal properties for biomedical applications, e.g. bone fillers and drug vehicles.

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Summary

Calcium phosphate-based cements and composites have attracted great inter-est and have been extensively used in both bone repair and replacement. The work presented in this thesis focused on investigating different calcium phosphate-based cements and composites, the mechanism of the brushite setting process, and the development of strategies to improve the selected properties of CPCs.

The results of this thesis provided a systematic comparison between ex-perimental and commercially available CPCs, which is valuable for re-searchers in further developing the experimental CPCs. Furthermore, the thesis reveals more detailed information of brushite formation and provides a theoretical basis for the design of improved brushite formulations.

To improve selected properties of CPCs, we developed three new CPC-based formulations: 1) a ready-to-use acidic CPC, formed by mixing an MCPM paste and a β-TCP paste, to overcome the complex handling process in the operating theatre and improve the cohesion and shelf life of the acidic CPCs; 2) PVA fibre-reinforced apatite cements to mitigate the intrinsic brit-tleness of CPCs; 3) acidic calcium phosphate/silk fibroin composites, formed by mixing an MCPM paste and a β-TCP/silk fibroin paste, to reinforce silk fibroin hydrogels and confer bioactivity.

In this thesis, different calcium phosphate based formulations were evalu-ated. It provides a better understanding for the setting reaction of brushite cement and several potential methods to optimize certain properties of calci-um phosphate based biomaterials, i.e. mechanical, handling and drug deliv-ery properties. The findings are beneficial for designing new calcium phos-phate based biomaterials to further match their application in bone augmen-tation.

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Future perspectives

There are still many properties to investigate and improve, and many possi-ble applications to evaluate for the investigated biomaterials. For example, other relevant mechanical properties, such as bending strength and fatigue properties will need to be studied for potential future use in load-bearing or load-sharing applications.

The biocompatibility and osteoconductivity of CPCs has been widely ver-ified. However, upon addition of new additives in CPCs, biological evalua-tions are needed to evaluate and further approach clinical use, including bio-compatibility tests in vitro and in vivo.

For ready-to-use CPCs, the properties of the products are acceptable after a low-temperature (4 °C) 3-month shelf-life test. In further work, it may be important to improve the long-term shelf life of the ready-to-use CPCs at higher temperatures (e.g. normal room temperature) to widen their clinical application.

For the fibre-reinforced CPCs, further knowledge on improvement of the interfacial adhesion between the fibres and the apatite would be of interest, as this is crucial to the final mechanical properties as well as the in vivo deg-radation behaviour.

The mechanical strength of the silk fibroin/calcium phosphate composites is acceptable as both bone fillers and drug vehicles. However, it was still in the low range of trabecular bone. Therefore, an improved method and/or new type of calcium phosphate might be useful in obtaining silk fibroin/calcium phosphate composites with a higher mechanical strength, and should be con-sidered in further work.

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Appendix

Preparation methods The CPCs and calcium phosphate based composites presented in this thesis were prepared in different ways.

In Paper I, two experimental in-house apatite and brushite cements and two commercially available brushite and apatite based cements were pre-pared to evaluate the mechanical properties of CPCs and their current status. The experimental brushite cement was prepared by mixing a powder phase including MCPM (sieved to < 75 µm), β-TCP with disodium dihydrogen pyrophosphate (SPP) acting as a retardant and a liquid phase, citric acid so-lution [50]. The formulation was optimized for high mechanical strength, suitable setting time, injectability and porosity [50]. The experimental apatite cement was prepared from a powder phase including a fine α-TCP powder provided by a collaborator, and HA seeds as a nucleation agent [51]. A sodi-um hydrogen phosphate (Na2HPO4) solution was used as the liquid phase to accelerate the setting reaction. The produced cements showed a final me-chanical strength after 24 hours. The commercially available brushite and apatite based cements, chronOS™ Inject and Norian® SRS, were prepared as-received according to the instruction from the manufacturer.

In Paper II, to evaluate the influence of the reactant ratio between MCPM and β-TCP and citric acid in the setting reaction of acidic CPCs, MCPM (< 75 µm) and β-TCP with two different ratios in the powder phase were mixed with water or citric acid solution to form brushite cement (Table 2).

In Paper III, a ready-to-use acidic dual-paste system consisting of an MCPM paste and a β-TCP paste was developed. The MCPM paste was pre-pared by mixing MCPM powder and a water-immiscible liquid with two surfactants in a planetary ball mill. The two surfactants (castor oil ethoxylate 35 and hexadecyl-phosphate) can improve the stability of the pastes and the compatibility between the polar calcium phosphate powder and nonpolar liquid and cohesion properties. In addition, the two surfactants can also im-prove the water balance in the paste once in contact with water. Different amounts of citric acid were added in MCPM pastes to control the setting speed. The β-TCP paste was prepared by mixing β-TCP powder and a sodi-um hyaluronate aqueous solution. Sodium hyaluronate was added to increase viscosity and improve stability and cohesion.

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In Paper IV, PVA fibre-reinforced apatite cements were developed. Dif-ferent amounts of PVA fibres (0.9 mm in length and 13 µm in diameter) were introduced in the powder phase of α-TCP powder to form a homoge-nous mixture by using a shaker-mixer. This mixture was combined with a Na2HPO4 solution to prepare an apatite-based composite. The produced composites were allowed to set for 7 days before testing.

In Paper V, acidic CPCs (brushite and monetite) were used to trigger the gelation of silk fibroin hydrogel to form inorganic/organic dual setting com-posites. The composites were formed via mixing an MCPM paste and a β-TCP/silk fibroin paste. MPCM pastes were formed by manually mixing MCPM and distilled water. β-TCP/silk fibroin pastes were prepared by man-ually mixing β-TCP powder and silk fibroin solution. The silk fibroin solu-tion was prepared according to an established method [105].

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Analytical techniques

Mechanical assessment Compressive strength (CS) CS is the most widely investigated mechanical property of CPCs (Papers I, III, IV and V). The CS (σ) of a material is the maximum stress that the ma-terial can sustain before failure under a compressive load. It is evaluated by applying an increasing load to a cylindrical sample with a specific cross-sectional area (A) until it breaks. The sample size most commonly used is 6 mm in diameter and 12 in height, in accordance with the ISO 5833 standard for evaluation of acrylic bone cements. While this standard was developed for acrylic bone cements used to fixate joint prostheses, it is also commonly applied to other bone cement types, although at a lower load rate for ceramic cements, due to their brittleness. The maximum force (F) is measured by load cells fitted to a materials testing machine (in our case a Shimadzu AGS-X). The CS is calculated according Equation 4 as follows.

= / (4)

Diametral tensile strength (DTS) The test for DTS is similar to that of CS to obtain the maximum force (F) before failure, only the sample is placed on its edge, and a disk is used rather than a cylinder. This test can induce tensile forces perpendicular to the load-ing direction [106]. The sample shape for DTS, used in Paper I and IV, was a disk with 8 mm in diameter (d) and 3 mm in height (h). The DTS (σ) is calculated according to Equation 5 as follows.

= (5)

Biaxial flexural strength (BFS) BFS was measured using a piston-on-3-ball test (ASTM Standard F394-78) [107] in the above universal testing machine to obtain the maximum force (F). Disc samples of diameter 8 mm and height 3 mm were centered and supported on three steel spheres with a diameter of 3.18 mm, positioned

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120° apart on a circle with a diameter of 10 mm. The entire test fixture was placed in the universal testing machine. The BFS (σ) was calculated using Equation 6, 7 and 8 as follows.

= −0.2387 ( − )/ 2 (6)

where d is the thickness at the sample center (mm);

= (1 + ) ln( ) + [ ]( ) (7)

= (1 + ) [1 + ln( ) ] + (1 − )( ) (8)

v is Poisson's ratio; A is the radius of support circle; B is the radius of the loaded area; and C is the sample radius. A Poisson’s ratio of 0.27 was used in Paper I [108].

Setting time The setting time is commonly defined as the time needed for the calcium phosphate pastes to harden enough to withstand a certain applied pressure [30]. The Gillmore needle test is the most commonly used method to assess the setting time of CPCs. The measured times include the initial setting time (the time when a thick Gillmore needle, giving rise to a local pressure of 0.31 MPa, no longer creates a visible mark on the sample) and the final set-ting time (the time when a thin Gillmore needle giving rise to a local pres-sure of 5.04 MPa, no longer creates a visible mark on the sample) [106, 109]. The setting time was tested in Papers III and IV.

Phase composition The phase composition information is important in order to evaluate the extent of the reaction. XRD in combination with Rietveld refinement can help assess the relative phase quantities in CPCs, and was used in Papers I, III, IV. XRD is a technique used to determine the phase composition of crystal-line materials. The XRD measurement is based on Bragg’s law:

= 2 sin (9) Where λ is the wavelength of the incident X-ray, d is the lattice plane dis-tance and θ is the angle between the incident beam and the measured lattice plane.

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Rietveld refinement, a technique that can be used for quantitative phase analysis [110], was used to quantify the phase composition in Papers I, III, IV. In Rietveld refinement, a theoretical XRD pattern is calculated based on crystal parameters retrieved from crystallographic databases. This pattern is compared to the measured pattern. The calculation process is then iterated to improve the calculated pattern until it matches the measured pattern. The refined parameters can be obtained from the calculation.

Porosity

There are several methods to evaluate the porosity of CPCs, such as helium pycnometry [111], water evaporation [111] and micro-computed tomogra-phy (micro-CT, for larger pores only) [112].

Helium pycnometry is a porosity method used to measure the skeletal density (density excluding pores). During the measurement, helium is pumped into an empty chamber with well-calibrated volume and the same chamber filled with sample material. The pressure difference between the empty chamber and the chamber with the samples is evaluated. The dried cement samples are crushed into small pieces to properly fill the chamber before analysis. Afterwards, the porosity of the sample is calculated using the skeletal density and the apparent density (density with pores) obtained from, e.g. Archimedes’ principle in Paper I and III.

The water evaporation method is based on a hypothesis that all pores in wet samples are filled with water, and the volume of the pores is equal to the volume of the water in the wet samples [111]. The volume of the water can be evaluated from the division of the water content (obtained from the mass of the samples before and after drying) with the density of water. The porosi-ty is calculated from the division of the volume of the evaporated water with the apparent volume of the samples in Papers I and III. The apparent vol-ume of the samples can be obtained from the division of the mass of the samples after drying with the apparent density.

Micro-CT is a non-destructive method to image the microstructure of the samples. It utilizes X-rays to image the samples in 2D to get a series of 2D images by rotating the samples and then reconstruct the 2D images to create a 3D model. Even closed pores can be detected through micro-CT. However, the resolution of the micro-CT is limited to around 5 µm. In CPC samples, most pores are in the range of nano- to a few micrometers [111, 113], which cannot be detected by micro-CT. This technique was applied in Paper V.

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Injectability Injectability tests were used in Paper III to evaluate the feasibility of MCPM and β-TCP pastes to be injected from a syringe. The force during injection can reflect the viscosity and stability of the pastes. Injectability was evaluated by monitoring simultaneously the extrusion force and plunger displacement of the pastes extruded from a 3 mL plastic syringe at different crosshead speeds in a universal testing machine [64, 114].

Cohesion/water resistance Cohesion is the ability of CPCs to be injected into and harden in a static aqueous solution without disintegrating into small pieces [115].

A modified method was used in Paper III to evaluate cohesion properties of CPCs based on previous work [64]. The MCPM and β-TCP paste in Pa-per III were mixed and then injected in distilled water. The samples were photographed immediately after injection and after 24 h to obtain qualitative results. The set specimens were removed from the distilled water after 24 h, rinsed with fresh distilled water and dried at 37 °C. The entire liquid was collected and centrifuged to secure the precipitate, which was dried at 37 °C. The mass of the set specimens and precipitates were weighed after drying to acquire quantitative results.

Scanning electron microscopy (SEM) In SEM, a focused beam of accelerated electrons is used to scan the surface of samples and generate signals (e.g. backscattered electrons and secondary electrons) that can be detected and analyzed to give structural and chemical information. The samples were coated with a thin layer of Au/Pd to reduce charging on the sample surface before analysis. In Papers I-V, secondary electrons were used to provide morphological information of CPCs.

Shelf life Shelf life is the length of time that the CPCs or their precursors can be stored and still result in adequate properties, as compared to the freshly prepared products. For conventional CPC formulations, calcium phosphate powders are stable individually and available off-the-shelf [7].

However, brushite based formulations commonly have shorter shelf-lives than apatite based formulations due to the inherent difficulties in removing and limiting access to moisture during manufacturing and storage, and the

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high reactivity between MCPM and β-TCP, which favors the formation of monetite [65, 116]. In Paper III, MCPM and β-TCP were stabilized in dif-ferent liquid phases for 3 months at 4 °C, separately. The stability of these pastes was assessed through the injectability evaluation. Chemical reactivity of the pastes was also evaluated by comparing the setting time and compres-sive strength of the set cements and fresh pastes.

Fourier transform infrared spectroscopy (FT-IR) The wavelengths of infrared radiation (IR) absorption bands can be used to evaluate specific types of chemical bonds. Therefore, IR spectroscopy is used to identify these groups and analyze the compound structure. In Paper V, an IFS 66v/S spectrometer (Bruker, USA) was used to obtain IR spectra. The spectrum was collected from 4000 to 400 cm-1 and the resolu-tion was 4 cm-1. A blank scan was recorded for background removal.

Ultraviolet-Visible (UV-Vis) spectroscopy The energy of UV or visible light can excite non-bonding electrons or π-electrons of molecules to higher anti-bonding molecular orbitals. Therefore, when the electrons of molecules are easily excited, light with a suitable wavelength can be absorbed by these molecules. In Paper V, the released vancomycin hydrochloride in the eluate was ana-lyzed by UV-vis spectroscopy at 280 nm using a UV-1800 UV/Vis spectro-photometer (Shimadzu, Japan).

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Svensk sammanfattning

Kalciumfosfatcement (CPCs) används som benersättningsmaterial på grund av deras injicerbarhet, nedbrytningsförmåga, förmåga att härda på plats, osteokonduktivitet och kemiska likhet med mineralfasen hos ben. Det finns två huvudtyper av CPCs: basiska CPC (apatitbaserade) och sura CPC (brus-hit- och monetitbaserade). De övergripande syftena med denna avhandling var att 1) undersöka olika kalciumfosfatbaserade cementformuleringar för att utvärdera deras nuvarande status; 2) bättre förstå härdningsmekanismen för brushitcement, vilken är viktig för materialets vidare utveckling och använd-ning i klinik; och 3) optimera vissa egenskaper hos CPCs och kalciumfosfat-baserade kompositer för att uppfylla ytterligare kliniska krav, med fokus på mekaniska, hanterings- och läkemedelsutsöndringsegenskaper.

De mekaniska egenskaperna hos två nyligen utvecklade experimentella apatit- och brushitcement och två kommersiellt tillgängliga apatit (Norian® Skeletal Repair System) och brushitcement (ChronOS™ Inject) undersöktes systematiskt. De två experimentella CPC: erna visade sig uppvisa signifikant högre mekaniska styrkor än de två kommersiellt tillgängliga CPC: erna, och även högre än styrkan hos humant trabekulärt ben. Denna studie är värdefull för vidareutveckling av de experimentella CPC: erna mot kliniska tillämp-ningar.

Härdningsmekanismen för brushitcement studerades kvantitativt under de första sekunderna och minuterna av reaktionen för första gången, med hjälp av synkrotron-röntgendiffraktion. Reaktionen visade sig innefatta en kort period av kärnbildningsinduktion, kärnbildning, och ökning av brushitande-len fram till avslutning av härdningen. Genom att variera reaktantförhållan-det förändrades både härdningshastigheten hos brushiten samt förhållandet mellan bildad brushit och monetit, vilket påverkar de slutliga mekaniska egenskaperna hos cementet. Citronsyra, som vanligen används som retar-dant, minskade signifikant tillväxthastigheten för brushitkristallerna och brushitinnehållet under härdningen men hade ingen effekt på kärnbildningen.

Traditionellt är det svårt och dyrt att blanda CPC i operationssalen. Därför är det bättre att förbereda icke-reaktiva kalciumfosfatblandningar, för att direkt kunna injicera de färdiga pastorna i bendefekter. Denna metod kan uppnås med sura CPCs, eftersom två kalciumfosfatreaktanter, monokalcium-fosfatmonohydrat (MCPM) och β -tricalciumfosfat (β -TCP), vanligtvis in-går i härdningsreaktionen för sura CPCs. En sur CPC, redo för direktan-vändning, utvecklades därför genom att blanda en MCPM-pasta och en β -

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TCP-pasta. Kohesionsegenskaperna för sura CPC: er som är färdiga att an-vända är generellt dåliga på grund av den höga lösligheten hos MCPM. Detta kan orsaka sönderdelning in vivo, vilket resulterar i dåliga mekaniska egen-skaper och kan även ha negativa biologiska effekter. Med lämpliga mängder citronsyra som retarderande medel utvecklades emellertid en injicerbar, klar att använda, sur CPC med tillräcklig hållbarhet, god kohesion och mekanisk prestanda. Detta cement har potential för förenklad klinisk tillämpning med repeterbara resultat och kan appliceras på ett minimalt invasivt sätt med hjälp av ett tvåkammarsystem.

För att motverka CPC: s spröda beteende användes biokompatibla poly (vinylalkohol) (PVA) fibrer med höga mekaniska styrkor för att förstärka apatitcement. Tillsatsen av de hydrofila PVA-fibrerna förbättrade signifikant apatitmatrisens seghet och motståndskraft mot sprickbildning, vilket resulte-rade i ett lovande kandidatmaterial för långsiktiga benersättningstillämp-ningar, såsom profylaktisk femeroplastik.

Oorganiska CPC kan också tillsättas till organiska polymera hydrogeler för att erhålla förbättrade funktioner, såsom bioaktivitet och kontrollerad läkemedelsleverans. En injicerbar organisk/oorganisk komposit baserad på silkfibroin-hydrogeler och sura kalciumfosfater utvecklades. Kompositen bildades genom att blanda en MCPM-pasta och en β -TCP / silkfibroinpasta. Efter blandning bildades sura kalciumfosfater (brushit och monetit). Den sura miljön under brushit- och monetitbildningen resulterade i silkfibroin-gelering. De mekaniska egenskaperna hos kompositmaterialet varierades genom tillsats av olika mängder sura kalciumfosfatpartiklar i 3D-silkfibroin-nätverket och olika β – ark - halt i silkfibroinet. Förmågan hos dessa kompo-siter att användas för lokal leverans av antibiotika utvärderades också. Lä-kemedelsladdade prover visade liknande leveransbeteende för alla kalcium-fosfatinnehåll och frisatte mer än 80 viktsprocent av läkemedlet inom två veckor.

Sammanfattningsvis har skillnader mellan experimentellt utvecklade och kommersiellt tillgängliga CPC: er undersökts, vilket gav detaljerad informat-ion om de experimentella CPC: ernas mekaniska egenskaper och deras rele-vans för eventuell vidareutveckling. Vidare studerades härdningsmekan-ismen för sura CPC, vilket är viktigt för att förstå hur man kan använda och förbättra dessa material för att bättre matcha de kliniska tillämpningarna. Flera nya CPC-baserade formuleringar har också utvecklats för att övervinna några av problemen med traditionella CPC, t.ex. komplicerad användning och kort hållbarhet för sura CPC, och deras inneboende sprödhet. Ytterligare biologiska utvärderingar behövs för att verifiera potentialen hos de utveck-lade materialen för eventuell framtida användning i klinisk miljö.

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Acknowledgements

Many people have supported, helped and encouraged me during my time in Uppsala University. I would like to express my warmest gratitude to all of you.

First and foremost, I would like to express my sincere gratitude to my main supervisor Cecilia, for your invaluable support and patient guidance throughout my PhD. Thank you for giving me the freedom to do the projects that I’m interested in and teaching me how academic writing should be done. I would also like to thank my co-supervisor Håkan, for the opportunity to start research and for giving me an overall viewpoint on my projects. I learned a lot from you on how to conduct research towards to clinical appli-cations. Special thanks to Wei for your kind help in my experiments and papers, and your positive responses every time I had questions.

To Céline, my former officemate, for your wonderful company and a great time in the office, I really enjoy talking to you. To Sara, for your en-couragement and warmest hugs. To Ingrid, for helping me so much during my first year and my first project in Uppsala, which gave me a good start. To Dan, for always helping with my experiments and listening patiently. To Le, for sharing your travel experiences, and study and work information. To Michael, for your help whenever I met problems in the lab and your great sense of humor. To Luimar, for sharing your fun life experience. To Caro-line, for your time and for helping me with micro-CT and the mechanical testing machine. To Alejandro, for your time and help with ICP and for keeping our lab so tidy. To David, for always helping me with Swedish files and for the Swedish Summary in this thesis. To Susan, for helping me im-prove my English writing. To Anna, for wonderful discussions about both research and life. To Yi, Charlotte, Christina and Gry for helping me with the cell study. To Philip, for valuable discussions and suggestions about my projects. To Wei L, Camilla, Amina, Lee, Oscar, Susanne, Torbjörn, Shiuli, Marjam, Sara, Johanna, Erik, Bing, Jonatan and other members of the MiM group, thank you for providing a pleasant work environment, where I have spent four fantastic years. To Stefan, for helping me with the 3D printing. To all other members in our division, I am encouraged by every greeting and warm smile.

Many thanks to my other co-authors Prof. Maria-Pau Ginebra, Dr. Fran-cisco Javier Martinez-Casado, Dr. Olivier Balmes, Julien Faivre. Thanks for

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your great contribution in my thesis. Special thanks to Dr. Gerard Insley for valuable discussions and providing materials needed for the experiments.

All the teachers and assistants who have helped me in the past four years. Your knowledge and support are essential to this thesis.

Many thanks to other friends in Ångström Laboratory and elsewhere in Uppsala: Song Chen & Shu Li, Xi Lu, Yuanyuan Han, Zhen Qiu & Zhicheng Wang, Liyang Shi & Jingyi Hong, Yanjun Zan & Yan Ji, Peng Zhang & Yan Guo, Huan Wang, Rui Sun & Lei Tian, Lu Wu & Pengfei Li, Kai Hua, Changqing Ruan, Zhaohui Wang, Chao Xu & Hongmei Yang, Shengyang Zhou & Hongli Yang, Xueying Kong, Ruijun Pan & Qiuhong Wang, Mingzhi Jiao & Yurong Hu, Wen Huang, Meiyuan Guo & Yuhan Ma, Shihuai Wang, Yi Ren & Xiaowen Li, Chunze Yuan & Lin Li, Hailiang Fang, Xingxing Xu, Fengzhen Sun, Shaohui Chen for all the happiness we have experienced to-gether.

Special thanks to my friends from PSE, Sichuan University: Duo Wu & Dongming Liu, Shuqin Cao & Chong Wang, Huan Xu. I cannot forget your help after I made plans to move abroad and the time you shared with me in Sweden.

At last but not least, the most important support to my PhD study is from my family: 谢谢我的家人，尤其感谢我的爸爸妈妈和杨佼佼。

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1700

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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”.)

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