Chemical stability of grain boundaries in β-tricalcium phosphate ceramics β-tcp as bone substitute material

Transkript

1 UPTEC-K Examensarbete 30 hp Mars 2012 Chemical stability of grain boundaries in β-tricalcium phosphate ceramics β-tcp as bone substitute material Mirja Olsson

2 Abstract Chemical stability of grain boundaries in β-tricalcium phosphate ceramics Mirja Olsson Teknisk- naturvetenskaplig fakultet UTH-enheten Besöksadress: Ångströmlaboratoriet Lägerhyddsvägen 1 Hus 4, Plan 0 Postadress: Box Uppsala Telefon: Telefax: Hemsida: β Tricalcium phosphate (β-tcp, Ca 3 (PO 4 ) 2 ) is a commonly used bone substitute material due to its biocompatibility and resorption. This study focused on the production of almost fully dense β-tcp ceramics with varying degrees of impurities (Ca/PO 4 ratio, addition of 5% Mg). Three methods were used to produce the β-tcp ceramics, uniaxial pressing, slip-casting and isostatic pressing. In this study the isostatic pressing and sintering at 1150ºC for 20h and 15min, resulted in the densest β-tcp ceramics ( %). No significant differences of grain size and density could be detected between the samples produced with various compositions. These isostatically pressed samples sintered at 1150ºC were then dissolved in 0.08M acetic acid solution to simulate the in vivo resorption. It was found that the samples containing extra Mg dissolved slower. Attempts to determine the chemical composition of the grain boundaries were made without success. However, SEM observations of partly dissolved β-tcp ceramics revealed that the grain boundaries dissolved faster than the grains. The study was performed at the RMS foundation in Switzerland. Handledare: Marc Bohner Ämnesgranskare: Jöns Hilborn Examinator: Rolf Berger ISSN: , UPTEC-K12 002

3 Populärvetenskaplig sammanfattning Om man skadar sig eller har en sjukdom som gör att man har förlorat ben behöver detta ersättas för att kroppen ska läka på rätt sätt. En möjlighet är att använda sig av en keram som heter β-kalciumfosfat (β-tcp, Ca 3 (PO 4 ) 2 ). β-tcp är så pass lik kroppens eget ben att kroppen accepterar det som ersättningsmaterial, det heter att β-tcp är biokompatibelt. Efter ett tag så kommer kroppens egna celler att ha ersatt keramen med riktigt ben, genom att celler som heter osteoclaster äter upp keramen medan så kallade osteoblaster bygger upp nytt ben. Om resultatet blir lyckat eller inte beror bland annat på hur väl synkroniserad denna process är. Om osteclasterna äter upp keramen mycket snabbare än vad osteoblasterna kan bilda nytt ben blir det inget bra resultat. Därför har denna studie försökt att ta reda på om det är någon skillnad på hur snabbt β-tcp löses upp om man tillverkar det med varierande sammansättningar av kalcium (Ca) och fosfat (P) och om något ändras om man tillsätter magnesium (Mg). För att kunna undersöka detta var första steget att tillverka prover av β-tcp med hög densitet. Det är en relativ svår uppgift då proverna måste sintras * på en relativt låg temperatur för att inte omvandlas till α-tcp. Tre huvudsakliga metoder provades för att tillverka prover inför sintring, enaxladpressning, slamgjutning och isostatiskpressning av β-tcp pulver. Det visade sig att isostatisk pressning resulterade i de högsta densiteterna. Dessa prover sintrades i 1150 o C i 20 timmar och 15 minuter och uppnådde en relativ densitet på %. Fyra olika sorters prover tillverkades, två olika sammansättning gällande Ca/P molförhållandet nämligen Ca/P=1.49 och 1.5 samt med och utan extra Mg. För att testa om det var någon skillnad i upplösningshastighet mellan de olika sammansättningarna användes 0.08M ättikssyra. Det visade sig att prover med extra Mg upplöstes långsammare än de utan. För att försöka ta reda på varför undersöktes om det var någon skillnad i kornstorlek ** mellan proverna och om det var någon skillnad i kemisk sammansättning mellan kornen och korngränserna *** i respektive prov. Det visade sig att korngränserna löstes upp snabbare än själva kornen men om det var någon skillnad i den kemiska sammansättningen mellan kornen och korngränserna som var orsaken till detta kunde inte fastställas. Vad gäller kornstorleken så var det en stor variation inom respektive prov men mellan proverna var det ingen signifikant skillnad, därför kan inga slutsatser om kornstorlekens inverkan av upplösningshastigheten dras. För att ta reda på varför proverna med extra Mg löstes upp långsammare samt om den kemiska sammansättningen i korngränserna på de olika proverna är olika, behövs ytterligare experiment. *sintring: När fasta partiklar sammanfogas genom uppvärmning, dock under smältpunkten för materialet. **/***Korrn och korngränser: När man tittar i stor uppförstoring på ett material som β-tcp kan man se att det består av korn, och det som finns mellen kornen kallas för korngränser.

4 Table of Contents 1. Introduction Biomaterials Background and Aim Experimental: Materials and methods Synthesis of β TCP powders Characterization of β TCP powders Synthesis of dense β TCP samples Uniaxially pressed samples Slip casting Isostatically pressed samples Characterization of dense β TCP samples Density Sample composition Grain size determination Dissolution tests Grain boundary composition Results and Discussion Powder Characterization First synthesis of β TCP powders Second synthesis of β TCP powders Sample Characterization Density of samples produced with β TCP powders of the first synthesis Density of samples produced with β TCP powders of the second synthesis Composition of samples produced with β TCP powders of the second synthesis Grain size of samples produced with β TCP powders of the second synthesis Dissolution tests of samples produced with β TCP powders of the second synthesis Grain boundary composition Summary and Conclusions Future outlooks Acknowledgments References... 32

5 Dictionary β-tcp = β-tricalcium phosphate HA = hydroxyapatite ICP-OPS = inductively coupled plasma optical emission spectrometry SEM = scanning electron microscope SSA = Specific surface area XPS = X-ray photoelectron spectroscopy XRD = X-ray diffraction 1

6 1. Introduction 1.1 Biomaterials The concept of what a biomaterial is has evolved with time due to the progresses in the field of biomaterials science and medical technology. Traditionally, a biomaterial was considered to be a metal, polymer, ceramic material or a combination of these materials, used in a biological system [1]. But today, nanoparticles and quantum dots are also considered to be biomaterials. Nowadays, a biomaterial is said to be a substance that has been engineered to take a form which, alone or as a part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure, in human or veterinary medicine [2]. The properties of biomaterials should be designed to control the biological response. If the biological response is acceptable, the material is said to be biocompatible in that specific application [3] and it can be used in the body. Biomaterials are used for a wide range of applications, from contact lenses and dental prosthesis to artificial heart valves and vascular grafts. Another biomaterials application is the so-called bone grafting, where a surgeon replaces damaged or missing bone. Due to an ageing population and high risk sports there is an increase in bone-related diseases and bone fractures, which has enhanced the need for bone grafting and bone grafting substitutes [4]. 1.2 Background and Aim Today there are four main bone grafting procedures. The first one is to use bone autografts (the patient s own bone), which often requires more than one operation and often causes donor-site morbidity [5, 6]. Another way is to use bone or demineralised bone from human donors, so called bone allografts. However, to prevent disease transmission from the donor to the patient the bone needs to be processed, which often affects the properties [7]. A third way is to use xenografts, such as animal bone, or coral. The fourth alternative is to use a synthetic material as a bone graft substitute. Metals (e.g. Mg alloy), polymers (e.g. polylactides) and ceramics (e.g. calcium phosphate) can be used for that purpose. One of the most used ceramics for bone graft substitution is β-tricalcium phosphate (β-tcp, Ca 3 (PO 4 ) 2 ), due to its chemical similarity to bone, its biocompatibility and its resorption characteristics [8-10]. Since β-tcp is not soluble in serum [11], β-tcp resorption occurs by the action of osteoclasts and macrophages. However, recent findings at the RMS foundation suggests that spontaneous dissolution of β-tcp grain boundaries can occur in serum, which may lead to grain release and perhaps accelerated resorption. Therefore, it is highly relevant to study the grain boundary stability in order to better understand in vivo behaviour of β-tcp. Most β-tcp ceramics are produced from raw materials (e.g. calcium carbonate, calcium pyrophosphate, dicalcium phosphate, dicalcium phosphate dihydrate, calcium-deficient hydroxyapatite) that contain fairly large amounts of trace elements, such as Si, Sr, Mg, Mn, Fe, K, or Na. In most cases, the ions are included in the ceramic structure as doping agents [12]. 2

7 However, since trace elements have a tendency to segregate at grain boundaries, it is likely that some of these trace elements are also trapped at the grain boundaries. Such an effect might have a marked and relevant effect on the in vivo resorption. Beside the composition, another important parameter in the synthesis of β-tcp ceramics is the sintering profile, because it defines the ceramic density and the grain size [13]. Generally, smaller or finer structures should dissolve faster. However, a segregation of trace elements might depend on grain size, hence potentially changing grain boundary stability and ceramic resorption. Since it is not clear how a change of trace element content and grain size in a dense β-tcp ceramic would affect its dissolution, the present study aimed at studying the link between composition (trace element content), grain size, grain boundary stability and dissolution rate. In order to compare the dissolution rate of various samples, it is essential to produce samples with exactly the same geometry. This implies that samples should be free of pores or, in other words, that calcium phosphate sintering should be so good that all samples reach almost 100% relative density. Since Ito et al. [14] mentioned that dissolution rates should not be affected if the relative density differs by less than 2.6%, it is assumed that ideal samples should reach a relative density larger than 97%. Generally it is difficult to obtain fully dense samples of β-tcp, because the sintering temperature must not exceed the β α phase transformation temperature (1120ºC). Indeed, the change of density occurring during this phase transformation (from 3.07 to 2.86 g/cm 3 [13]) generates cracks and prevents densification One way to increase the β α transformation temperature and hence produce denser samples is to introduce impurities like magnesium [15, 16]. Considering the difficulties encountered in the past at the RMS foundation to produce β-tcp samples denser than 95%, a sub-aim of the present study was to establish a method to produce dense (> 97%) β-tcp samples. Beside the production of dense β-tcp samples, several other aspects of the study were poorly controlled such as the determination of the β-tcp grain size and dissolution rate, as well as the chemical analysis of grain boundaries. Therefore, in summary, the present project had the following aim and sub-aims: Aim: Study the link between composition (trace element content), grain size, grain boundary stability and dissolution rate of dense β-tcp ceramics Sub-aims: Produce dense (> 97%) β-tcp ceramics Establish a method to determine the grain size of β-tcp ceramics Establish a method to determine the dissolution rate of β-tcp ceramics Establish a method to determine the composition of grain boundaries The choise of which methods that were used in this study was manly based on what was thought to give the desierd information and results but it was also based on practical issues, like avilability of equiptment and costs. 3

8 2. Experimental: Materials and methods 2.1. Synthesis of β-tcp powders β-tcp (Ca 3 (PO 4 ) 2 ) powders were produced by mixing calcium hydrogen phosphate powder (CaHPO 4, Calcium Phosphate GFS Chemicals, Art.N o : 1548, Lot.N o : P781262) and hydroxyapatite powder (Ca 5 (PO 4 ) 3 OH, Budenheim Tricalciumphosphate, Art.N o: : C5381, Lot.N o :A99068A) followed by several steps of solid-state sintering and milling to obtain a homogenous powder. During the solid-state sintering or thermal treatment (TT), the following reaction was supposed to occur: CaHPO 4 + Ca 5 (PO 4 ) 3 OH 2 Ca 3 (PO 4 ) 2 + H 2 O Two attempts to produce β-tcp powders were made. In Table 1 is an overview of the powders from the two different syntheses are presented. In the first synthesis powders with Ca/P molar ratios varying between 1.49 and 1.52 were produced (Table 2) In the second synthesis magnesium phosphate octahydrate (Fluka, Art N o :63732, Lot N o : ) was added to the mixture to obtain powers with a (Ca+Mg)/P ratio of 1.49 and 1.50 with and without 5mol% magnesium (Table 2). In the first synthesis the mixtures were first thermally treated (TT) at 900 o C for 4h. After the first TT at 900 o C the powder could not be sieved so it was put back in the furnace for a second TT, this time at 1300 o C for 4h. The TCP was then crushed to 1mm in a jaw crusher (Retsch BB51) and mixed with deionized water to obtain a thick paste. The paste was dried overnight in a furnace at 50 o C, followed by a TT at 1100 o C for 4h. Prior to the planetary milling step, the β- TCP was crushed to 0.5mm. To prevent the powder from agglomerating during milling, 10wt% fumaric acid (Fluka Analytical, Art.N o : , Lot.N o : BCBB8136) and ethanol (2ml/100g) were added. The powders were milled for 1h at a speed of 400 rpm. To burn off the fumaric acid the β-tcp was calcined at 400 o C for 4h and at 800 o C for 12h. For all calcination and TT steps a heating rate of 5 o C/min was used. 100g of the β-tcp with a molar ratio of 1.51 was milled an extra time in the planetary mill and then calcined at 400 o C for 24h. In the second synthesis, the powders were thermally treated at 1300 o C for 4h. Prior to planetary milling the TCP powders were crushed to 0.5mm with a crusher. To prevent agglomeration of the particles during milling fumaric acid and ethanol was added as described earlier. The powders were milled for 1h at 400rpm. To burn off the fumaric acid a calcination step at 400 o C for 20h was performed. Later on, the milling and the calcination steps at 400 o C were repeated a second time. 4

9 Table 1. An overview of the β-tcp powders produced in this study. Synthesis Nominal Ca/P Nominal (Ca+Mg)/P Extra Mg Denominations Times milled ratio _ _ _ _ *milled 1_ _ _ mol% 2_1.49+Mg _ mol% 2_1.50+Mg Calcinated ( o C) & 400 Table 2. Amount of initial reagents used to obtain β-tcp with different Ca/P and (Ca+Mg)/P molar ratios Powders Ca 5 (PO 4 ) 3 OH Mg(PO 4 ). 2 8H 2 O Nominal Ca/P ratio Nominal (Ca+Mg)/P ratio CaHPO 4 1_ g g - 1_ g g - 1_ g g - 1_ g g - 1_ g g - 2_ g g - 2_1.49+Mg g g 29.64g 2_ g g - 2_1.50+Mg g g 31.39g 2.2. Characterization of β-tcp powders X-ray powder diffraction patterns (XRD) were measured on a Panalytical CubiX powder diffractometer using graphite-monochromatized CuKα 1 radiation. A range from 5 to 60 2θ was scanned with a step size of and a counting time of 1 second per step. The phase content was quantified with Rietveld refinement using FullProf.2k version 5.00 [17]. In the Rietvel refinement the intensity of the peaks in the diffractogramas is compared with how the intensity should be for the different structures. Structural models were taken from Dickens et al. [18] for β-tcp, Sudarsanan and Young [19] for HA (hydroxyapatite), Boudin et al. [20] for β-calcium pyrophosphate (β-cpp) and M. Mathew et al. [21] for α-tcp. Some parameters are then refined in a computer program, in this case FullProf.2k version 5.00, and one can deduce the different phase quantitys. The specific surface area of the powders was analyzed by N 2 adsorption (BET-Method), using a Gemini 2360 device (Micromeritics USA). To get an overview of the powders morphology 5

10 scanning electron microscopy (SEM, Zeiss EVO MA 25) was applied. For that purpose, the powders were coated with gold or platinum, using a BAL-TEC SCD 050 Sputter Coater. The particle size distribution (PSD) of the powders was measured with a LS laser diffraction particle size analyzer from Beckman Coulter. To prevent the powders to agglomerate while measuring the PSD, 0.01M trisodium citrate dihydrate (Fluka, Art N o : 71466, Lot N o : ) was added and the sol was ultrasonicated for 12 min in a Bandelin Sonorex Synthesis of dense β-tcp samples Since it was not clear initially which method would give the highest β-tcp sample density, three synthesis methods were considered for green body production: uniaxial pressing, slip-casting and isostatic pressing. Uniaxial pressing is when the preassure comes from one direction and by isostatic pressing the preassure comes from all directions at ones. When producing sampels with slip-casting the powder is mixed with a solution and the slip (the mixture of powder and solution) is poured in to molds to dry. Details about these three methods are given hereafter Uniaxially pressed samples The powders from the first synthesis (powders 1_1.49 to 1_1.52; Table 1) were uniaxially pressed at 200MPa to obtain tablets with a diameter of 10mm and a height of mm. Each tablet contained 0.71g ± 0.02g. The samples were sintered according to two different sintering profiles ( Figure 1; Carbolite E3216 furnace). Figure 1a shows the one-step sintering (1S) profile where T1=1100 o C and the holding time is 20h15min. The second profile consisted of a two-step sintering (2S) with a first step at 1100 o C for 15min, and a second step at 800 o C for 20h. In both cases, half of the samples were inserted at the start of the sintering profile, i.e. at room temperature (RT) and the second half when the furnace temperature had reached T1=1100 o C. T 1 =1100 o C, for 20h,15min Cooling rate 10 o C/min down to 800 o C T 1 =1100 o C, for 15min Cooling rate 10 o C/min Temperature Heating rate 20 o C/min Temperature Heating rate 20 o C/min T 2 =800 o C, fo 20h Time Time a) b) Figure 1. (a) One-step sintering profile. (b) Two step sintering profile applied to uniaxiallypressed tablets made with powders 1_1.49 to 1_1.52 (Table 1). 6

11 Even though samples were generally pressed without additives, the 1_1.505 β-tcp powder was pressed both without and with 1.5wt% stearic acid (Fluka Chemika, Art.N o: : 73297, Lot.N o :430961). For that purpose, stearic acid was mixed with 0.71g (± 0.02g) of the β-tcp powder using a pestle and a mortar to distribute the acid evenly in the powder. Pressing was performed in the same way as described earlier. These samples were then sintered for 2h at 1100 o C, 1150 o C, 1200 o C and 1250 o C respectively. The samples were all inserted when the furnace reached the target sintering temperatures. To determine the effect of sintering temperature on ceramic shrinkage, tablets were also subjected to a one-step sintering performed at higher temperatures than 1100 o C. Samples where inserted into the furnace at T 1 =1150 o C, T 1 =1200 o C and T 1 =1250 o C. An overview of the samples produced with uniaxial pressing can be seen in Table 3. Table 3. Summary of the samples produced with uniaxial pressing. T 1 = 1100 C Inserted at + stearic 1S at Powder 1S 2S RT T 1 acid higher T 1_1.49 X X X X X 1_1.50 X X X X X 1_1.505 X X X X X X 1_1.51 X X X X X 2 milled X X X 1_1.51 1_1.52 X X X X X Slip-casting To produce green bodies by slip-casting a 1 w% polyacrylic acid (PAA, Aldrich Chemistry, Art.N o: : , Lot.N o :120M5074V) solution with ph~7 was prepared. The ph was adjusted with a 1 V% of a 25% ammonia solution (Merck GaA, Art.N o: : , Lot.N o :K ). For each sample 4.00 ± 0.00g of β-tcp powder was weighed. The powder was mixed with 0.95 ml of the 1% PAA solution by using a spatula and the vortex (VWR Digital Vortex MIXER). When the solution and the powder were properly mixed the slip was poured into cylindrical moulds (Ø = 13.5 mm; H = 22 mm) and left to dry at room temperature for 24h. The moulds, which consisted of a silicon mould lying on a calcium sulfate dehydrate (CSD) block, were closed hermetically with plugs so that the samples would not dry too quickly. For the same reason, the CSD block had been pre-soaked in water. When the samples had dried for 24h they were taken out from the moulds, put into an oven overnight at 60 o C to dry, and then sintered (see hereafter). 7

12 Due to time limitations only two samples of each Ca/P molar ratio ( ) were produced. These samples were then sintered 1100 o C for 1.5h. After measuring their densities the samples were sintered one more time at 1200 o C or 1300 o C for 4h Isostatically pressed samples Two series of samples were produced by isostatic pressing. In the first series, powder 1_1.52 was isostatically pressed into cylindrical blocks (Ø = 35.2mm ± 0.2mm, H = 22.8mm ± 0.7). These blocks were then cut into smaller pieces (approximately:10mm x 10mm x 4.5mm) and sintered. The samples were inserted into the furnace when the temperature had reached T 1 =1100 o C, T 1 =1125 o C, T 1 =1150 o C, T 1 =1175 o C, T 1 =1200 o C, T 1 =1225 o C and T 1 =1250 o C. The same procedure was followed for the second series of isostatically pressed samples. The differences were (i) the use of other powders namely all the powders produced in the second synthesis (from powder 2_1.49 to powder 2_ Mg; Table 1) and (ii) the use of only one sintering temperature (T 1 = 1150 o C). The sintering profile for the isostatically pressed samples is described in Figure 2. In order to maintain a uniform geometry during isostatic pressing (performed with a cold isostatic press, EPSI, Belgium), the samples were pre-pressed in a Struers ProntoPress-20 (Struers, Danemark). For each sample 50g of powder was pressed at 50kN for 3min, after a pre heating at 100 o C for 30sek. Isostatic pressing was performed in a 12.3 litre vessel and the samples were pressed at 2700bar for 20 seconds followed by a fast decompression, ca: 5 seconds. All samples were packed first in condoms and then in a polyethylene bag to prevent contact with the pressing fluid. Temperature T 1 for 20h,15min Cooling rate 10 o C/min down to 800 o C Time Figure 2. Sintering profile for isostatically pressed samples. The samples were inserted at T 1 =1150 o C Characterization of dense β-tcp samples The first step in characterizing the samples was to determine their density. The powder and sample preparation that had led to the highest density was then used to prepare new samples. The latter samples were then more extensively characterized, including dissolution rates, grain size determination and chemical composition of the grain borders. 8

13 2.4.1 Density To measure the density of the sintered samples a method based on Archimede principle was used. The samples were impregnated in ethanol (99.8%, Dr. Grogg Chemie AG, Switzerland, Art.N o: : G003, Lot.N o : ). For that purpose, the samples were dipped in ethanol and submitted to vacuum (Pump Struers Epovac). After impregnation, the samples were placed in a sample holder and the change of weight was measured. This change of weight (m s ) corresponded to the Archimedes force, i.e. the volume of the sample and sample holder multiplied by the liquid density. Three measurements were performed for each sample. To calculate the sample volume (V s ) following equation was applied: ms mh = Vs ρ EtOH Where m h is the Archimedes weight of the sample holder and ρ EtOH is the density of ethanol, 0.79g/cm 3. When the V s is calculated the dry weigt (g) of the sampel is divided with the volume (V s ) of the sampel to calculate the density of the sample Sample composition The crystalline phase composition of β-tcp samples was determined by XRD whereas inductively coupled plasma optical emission spectrometry (ICP-OES) was applied to determine their chemical composition. The samples were prepared on single-crystal Si plates for XRD data collection, because the amount of material was limited. However, the fit of the intensities was accurate enough to allow phase quantification by Rietveld refinement. The ICP-OES was performed at Interlabor Belp AG (Belp, Switzerland) Grain size determination To determine their grain size, the dense β-tcp samples were first embedded in Technovit 5071 resin and then polished using a Struers TegraPol 21 machine (Details in Table 4). To dissolve the resin the polished samples were immersed in acetone (Acetone EP, Brenntag Schweizerhall AG, Switzerland, Art N o : , LotN o : ). After de-embedding, the samples were thermally etched at 1050 o C for 30min, coated with gold (BAL-TEC SCD 050 Sputter Coater.), and then analysed by SEM. To determine the average grain size of the samples, a modified version of the intercept procedure proposed by ASTM (ASTM standard E112-10) was applied. For each sample three images (instead of 6) at a magnification of 1000 (instead of 8000) times were used. This standard is originally meant for metallic materials but was nevertheless used in this study. To approximate the grain size a template with four lines was put over the image and the intersections along the line with grain boundaries were counted. How to count and from there calculate the grain size was established using the ASTM standard. These images were also used to estimate the porosity of the samples. For that purpose, a grid was placed on top of the images and the number of intersections that were over a pore were counted and compared with the total number of intersections. 9

14 Table 4. The six different polishing steps used to prepare the samples for SEM and grain size determination. Step Paper Suspension Time Pressure 1 MD Piano 220 Water 3min 240N 2 MD Allegro DP-P 9μm 6min 30s 220N 3 Plan DP-P 9μm 6min 30s 200N 4 Plan DP-P 3μm 8min 30s 200N 5 Dur DP-P 1μm 6min 30s 180N 6 Chem Ops-U 4min 180N Dissolution tests Dissolution tests were performed on isostatistically pressed samples obtained with calcium phosphate powders from the second synthesis, i.e. Ca/P: 1.49 and 1.5 with and without 5mol% magnesium (denominations: 2_1.49 to 2_ Mg see Table 1). The samples were dissolved in an 0.08M acetic acid solution (VWR International, Art.N o: : , Lot.N o : K ) set at a ph of 4.52±0.02. This ph value was selected to simulate the ph change that osteoclasts cause during bone resorption [22]. To stabilize the ionic strength and hence make the ph measurements more reliable, a 0.01M sodium chloride solution (NaCl, CarlRoth, ArtN o : , Lot.N o : ) was added. The ph value was adjusted with sodium hydroxide solution (5M, NaOH, Sigma-Aldrich, Art N o : 71690, Lot N o : SZBA188OV). For each dissolution test 100ml of this acetic acid solution was used. The samples were inserted into a nylon cell strainer (BD Falcon, 70μm, Lot.N o : , Ref: ) hung in the dissolution solution (Figure 3). The stirring rate of the magnetic bar was set at 400rpm. The temperature was 25 ± 2 o C. Three different dissolution times were selected: 23h, 46h and 92h. When the dissolution tests were stopped, the samples were cleaned in water in an ultrasonic bath (Sondex Super Digital) four times and then once in ethanol. The loose particles released from the samples surface during cleaning were centrifuged to separate them from the water and then cleaned once with ethanol. After both the loose particles and the samples had been cleaned, they where dried over night at 30 o C in vacuum. Afterwards the weight of the cleaned samples could be determined. ICP-OPS was used to determine (i) the composition of the sample before dissolution (see also section 2.4.2), (ii) the composition of the sample after dissolution and cleaning, (iii) the composition of the loose particles, and (iv) the composition of the dissolution solution. Attempts to analyze the loose particles and the partly dissolved samples were also made with X-ray photoelectron microscopy (XPS). XPS-measurements were performed with a Kratos Axis Nova (Kratos Analytical, Manchester, UK). The samples were analysed on two spots each. The following settings were used: X-ray source: Monochromatic AlKα irradiation ( ev), 225 W, angle of incidence 54.6 (with respect to the surface normal). Finally, the partially dissolved samples before and after cleaning were observed by SEM. 10

15 Sample Figure 3. Dissolution setup Grain boundary composition A direct and an indirect method were used to identify the composition of the grain boundaries. The direct approach consisted of comparing the surface composition of a broken and a polished β-tcp piece, hoping that the broken piece would present an inter granular mechanical failure. Surface analysis was to be performed by XPS according to the method described in section The second approach used the ICP-OPS results described in section Specifically, the composition of the loose particles was compared to the composition of the cleaned samples and undissolved samples. This approach can only work if the grain boundaries dissolve faster than the grains. 3. Results and Discussion This section is divided into two main parts, one devoted to the characterization of the powders (section 3.1) and the second one devoted to the characterization of the sintered β-tcp samples (section 3.2) Powder Characterization First synthesis of β-tcp powders In the XRD diffractograms of powders 1_1.49 to 1_1.52, it can be seen that hydroxyapatite (HA) was not present in the powder with a nominal Ca/P ratio lower than 1.5 (Figure 4). In the powders with a Ca/P ratio larger than 1.5 the HA peak became more intense and the intensity of the β-tcp peaks decreased. Rietveld refinement was applied to the XRD results to determine the quantitative phase composition. From the phase composition it was possible to calculate the (Ca+Mg)/P ratio of the powders by assuming that all phases were stoichiometric and that no amorphous phases were present. The results showed a slightly larger Ca/P molar ratio than 11

16 anticipated (Figure 5). The deviations from the nominal (Ca+Mg)/P ratios could either be a systematic error in the phase quantities obtained from Rietveld refinement or an inaccuracy actually present in the samples (e.g. due to imperfect chemical compositions of the raw materials, biased precursor powder mixtures due to moisture content, etc.). * β-tcp * HA Ca:P = 1.52 Intensity (a.u.) Ca:P = 1.51 Ca:P = Ca:P = 1.50 Ca:P = Diffraction Angle ( 2θ) Figure 4. XRD diffractograms from β-tcp powders with different Ca/P molar ratios. From bottom to top, the nominal Ca/P molar ratio varied from 1.49, 1.50, 1.505, 1.51, and 1.52 (denominations: 1_1.49 to 1_1.52 see Table 1). 12

17 Ca/P measured Ca/P nominal Figure 5. Comparison between nominal and measured Ca/P molar ratios of powders 1_1.49 to 1_1.52 (Table 1). The XRD ratios were measured by applying a Rietveld refinement analysis on the XRD results. Three measurements were performed for each powder. The standard deviations are shown on the chart, but are so small that they are hidden behind the square symbols.the straight line denotes a situation where the nominal values are true. 13

18 The specific surface area (SSA) measurements were all in a narrow range, between 2.29 and 2.66 (Fig 6). However, the powder with a Ca/P molar ratio under 1.5 had a SSA value significantly lower than the values obtained for the other powders (p < 0.02) The β-tcp powder that was milled a second time in the planetary mill had a much larger specific surface area, namely 6.60 ± 0.08 m 2 /g. The difference in SSA value between these powders is probably related to the use of a relatively high calcination temperature (800ºC) for the samples milled only once. Indeed, β-tcp sintering generally starts below 800ºC SSA (m 2 /g) Ca/P measured Figure 6. Specific surface area measurements of the β-tcp powders obtained with different Ca/P molar ratios and milled once in the planetary mill. Powders 1_1.49 to 1_1.52 (Table 1). SEM measurements revealed that most powders had very similar morphologies (Figure 7a-f). All powders consisted of a rather broad particle size distribution. Apart from the powder milled twice, all powders presented a rather smooth appearance, probably due to the calcination step at 800ºC (sintering). The particle size distribution measurements based on volume fraction (Table 5) shows quite illogical results when comparing the different powders to each other, especially D90. For example, the results of powder 1_1.50 are very similar to those of powder 1_51 milled twice, even though there is a big difference in the SSA. Furthermore, the mean particle sizes of powder 1_1.50 are very different from those of powder 1_1.51 (milled once), even though there is only a small difference in SSA. This could be due to the extremely small amount of powder that was used when measuring PSD, which would mean that the measurement is not representative for the powder. It can also mean that there are still agglomerates in the powders. The diffraction particle size analyzer at the RMS Foundation is still rather new and further adjustments to the measuring protocol might be required to obtain more reliable results. 14

19 a) 1_ 1.49 b) 1_1.50 c) 1_1.505 d) 1_1.51 e) 1_1.52 f) 2*milled 1_ 1.51 Figure 7. SEM images of the β-tcp powders with different Ca/P molar ratios. (a-e) Milled once in the planetary mill and then thermally treated at 400 and 800ºC. (f) Milled a second time in the planetary mill, and then thermally treated at 400ºC. Powders 1_1.49 to 1_1.52 (Table 1) 15

20 Table 5. The PSD results from the first synthesis. Powder D10 (µm) D50 (µm) D90 (µm) 1_ ± ± ± _ ± ± ± _ ± ± ± _ ± ± ± _ ± ± ± *milled 1_ ± ± ± Second synthesis of β-tcp powders The XRD diffractograms of the powders from the second synthesis show that HA can only be found in the powder with a Ca/P molar ratio of 1.5 without the extra magnesium (Figure 8). Using Rietveld refinement small amounts of α-tcp ( w%) could be detected in the two powders without the extra 5mol% Magnesium. Rietveld refinement was also applied to analyze the resulting (Ca+Mg)/P molar ratios (Table 6). As seen from the powders of the first synthesis, the powders of the second synthesis had a slightly larger Ca/P molar ratio than anticipated. However, this effect was restricted to powders produced without extra Magnesium. Attempts to determine the Mg concentration from XRD data was also made. In order to do this the (Ca+Mg)/P ratio was fixed to The results verify that there is more Mg in the powders where extra Mg was added but also that there is some Mg in the other two powders (Table 6). The analysis of the SSA values shows that the powders produced in the second synthesis had much higher SSA values than those of the powder produced in the first synthesis (Table 6). The values ranged between 11.6 and 13.2 m 2 /g. When looking at the SEM images, the morphology of the powders from the second synthesis was similar to that of the powder milled twice in the first synthesis (Figure 9). Again the PSD results were quite illogical with a big variation of D90 between the powders (Table 7). Also, when comparing the PSD of the powders from the first and second synthesis, one would expect lower results for the powders of the second synthesis but this was not observed. 16

21 β-tcp * HA Ca:P = 1.5+Mg Intensity (a.u.) * Ca:P = 1.5 Ca:P = 1.49+Mg Ca:P = Diffraction Angle ( 2θ) Figure 8. XRD diffractograms from β-tcp powders with and without extra magnesium. Powder denominations: from 1_1.49 to 1_ Mg (see Table 1). Table 6.Comparison between the nominal and measured Ca/P molar ratios of the powders of the second synthesis. The measurements were performed by the XRD and Rietveld refinement was applied to determine the crystalline composition of the powders. The last column shows the SSA measurements. Sample Ca/P nominal value (Ca+Mg)/P nominal value (Ca+Mg)/P measured Mg content in β-tcp SSA (m 2 /g) 2_ ± Ca 2.97 Mg 0.03 (PO 4 ) ± _1.49+Mg ± Ca 2.97 Mg 0.03 (PO 4 ) ± _ ± Ca 2.97 Mg 0.03 (PO 4 ) ± _1.50+Mg ± Ca 2.97 Mg 0.03 (PO 4 ) ±

22 a) 2_1.49 b) 2_1.49+Mg c) 2_1.50+Mg d) 2_1.50+Mg Figure 9. SEM images of the β-tcp powders with and without extra magnesium. Table 7. PSD results from the second synthesis Powder D10 (µm) D50 (µm) D90 (µm) 2_ ± ± ± _1.49+Mg 0.53 ± ± ± _ ± ± ± _1.50+Mg 0.42 ± ± ±

23 3.2. Sample Characterization In the previous section (3.1.), the results of the powder characterizations were presented. In the present section, the results of the sintered β-tcp samples are presented. When the density results are presented the relative density refers to fully dense β-tcp, even though at higher temperatures the samples also contained some α-tcp that has a lower density Density of samples produced with β-tcp powders of the first synthesis Uni-axially pressed samples The green body density of the uniaxially pressed samples was 2.09 ± 0.09 g/cm 3, which corresponds to 68 ± 3 % of the theoretical density of β-tcp. When comparing the density of samples sintered according to one (1S) and two step (2S) sintering at 1100 o C it appears that the one-step sintering resulted in significantly (p<1x10-13 ) denser samples for all the Ca/P molar ratios (Figure 10). Also, significantly (p<3x10-8 ) higher densities were obtained when the samples were inserted at the sintering temperature into the furnace. In other words, the densest samples were produced from β-tcp powder with a Ca/P of 1.5 inserted at 1100ºC and kept 20h at the latter temperature (one-step sintering). Adding 1.5 wt% stearic acid to the powder before pressing did not seem to improve the densification of the samples (Figure 11). At 1100 o C no difference could be seen but at 1150 o C and at 1150 o C the samples with 1.5 wt% stearic acid have a significantly lower density (p<0.012). Increasing the temperature of the one-step sintering to T > 1100ºC led to a strong increase of the relative densities: most powders reached a value above 2.9 g/cm 3, almost independently of the sintering temperature and Ca/P molar ratio (Figure 12). For the samples sintered at 1150 o C, 1200 o C and 1250 o C the β-tcp powder with the nominal Ca/P=1.49 had the highest density. Milling the 1_1.51 powder once or twice did not significantly affect the density of β-tcp samples produced with this powder and sintered at 1150 o C (Figure 13) 19

24 Density (g/cm 3 ) % 95% 90% 85% 80% 75% 70% Relative density Ca/P Nominal Ca/P 65% Figure 10. Density of β-tcp samples after one (1S) and two step (2S) sintering. The samples were obtained by uniaxial pressing using the β-tcp powders from the first synthesis (powders 1_1.49 to 1_1.52 see Table 1). Symbols: ( ) 1S inserted at room temperature; ( ): 1S samples inserted at T 1 =1100 o C; ( ) 2S inserted at room temperature; ( ) 2S inserted at T 1 =1100 o. Density (g/cm 3 ) % 95% 90% 85% 80% 75% 70% Relative density Temperature ( o C) 20 65% Figure 11. Effect of sintering temperature and stearic acid content on the density of β-tcp samples. The samples were obtained by uniaxial pressing using the 1_1.505 (Table 1) β-tcp powder from the first synthesis. Sintering time: 2h. Stearic content: ( ) 0%; ( )1.5 wt%.

25 Density (g/cm3) % 95% 90% 85% 80% 75% 70% Relative density Ca/P measured Nominal 65% Figure 12. Effect of sintering temperature and Ca/P molar ratio on the density of samples sintered for 20h15min at: ( )T 1 = 1100 o C; ( ) T 1 = 1150 o C; ( ) T 1 = 1200 o C; and ( ) T 1 = 1250 o C. The samples were obtained by uniaxial pressing using a β-tcp powder from the first synthesis (powders 1_1.49 to 1_1.52 see Table 1). Density (g/cm 3 ) % 95% 90% 85% 80% 75% 70% Relative density Temperature ( o C) 21 65% Figure 13. Effect of sintering temperature and number of milling steps on the density of samples sintered for 20h15min at T = 1100 to T = 1250ºC. The samples were inserted into the furnace at the sintering temperature. The samples were obtained by uniaxial pressing using a β-tcp powder from the first synthesis. Symbols: ( ) Milled once in the planetary mill: ( ) Milled twice in the planetary mill (powder 1_1.51 see Table 1).

26 Slip-Casting The density of sintered slip-casted samples can be seen in Figure 14. The density for the samples sintered at 1100 o C were quite low compared to the uniaxially pressed ones. Density (g/cm3) % 95% 90% 85% 80% 75% 70% Relative density Ca/P Nominal Ca/P 65% Figure 14. Density of sintered slip-casted samples. Symbols: ( ) Sintered at 1100 o C for 1.5h; ( ) Sintered at 1200 o C for 4h; ( ): Sintered at 1300 o C for 4h. (powders 1_1.49 to 1_1.52 see Table 1) Isostatically pressed samples The samples prepared through isostatic pressing reached a higher density after sintering than those prepared with the other two methods, i.e. uniaxial pressing (Figure 15) and slip casting (Fig 14). 22

27 Density (g/cm 3 ) % 95% 90% 85% 80% 75% 70% Relative density Temperature (C o ) 65% Figure 15. Effect of sintering temperature and pressing method on the β-tcp sample density. All samples were prepared with powder 1_1.52 (Table 1), inserted at T 1 into the furnace, and sintered for 20h15min. ( ): Prepared with uniaxial pressing; ( ): Prepared with isostatic pressing Density of samples produced with β-tcp powders of the second synthesis Based on the measured densities of the samples prepared from the first synthesis, the samples from the second synthesis were only prepared through isostatic pressing. All samples were sintered at 1150 o C for 20 hours and 15 minutes. In comparison with the theoretical value of β- TCP (3.07g/cm 3 ) these samples reach a relative density between 97.7 and 99.2 % (Figure 9). Table 8. Density results, measured with the method of Archimedes, for isostatically pressed samples obtained with powders from the second synthesis Samples Density (g/cm 3 ) Relative density (%) 2_ ± ± 0.1 2_1.49+Mg 3.00 ± ± 0.1 2_ ± ± 0.1 2_1.50+Mg 3.05 ± ± Composition of samples produced with β-tcp powders of the second synthesis In the diffractograms of isopressed and sintered (1150 o C) samples obtained with powder of the second synthesis, it can be seen that HA is only present in the sample produced with powder 2_1.49. Regarding α-tcp formation it could be traced only in very low amounts (0.2 w%) for 2_1.50. The ICP screening shows that there are many trace elements in the sintered samples (Appendix 1), like potassium (K), strontium (Sr), manganese (Mn), sodium (Na), 23

28 aluminum (Al), iron (Fe) and magnesium (Mg). The amounts of magnesium in the samples are fairly large also in the samples where no extra magnesium was added. β-tcp * HA Ca:P = 1.5+Mg Intensity (a.u.) * Ca:P = 1.49 Ca:P = 1.5 Ca:P = 1.49+Mg Diffraction Angle ( 2θ) Figure 16. Diffractograms of sintered (1150 o C) and isopressed samples obtained with powders of the second synthesis Grain size of samples produced with β-tcp powders of the second synthesis The grain size distribution within the same samples was quite broad (Figure 17). For example, in the sample shown in Fig 17a, the smallest grains had a diameter of 0.6µm whereas the biggest grains had a diameter close to 8µm. No significant difference in average grain size could be established between the different samples (Table 9). The porosity measurements indicate that the densities of the samples varied between 96.6 and 98.7%. These value are slightly lower than the values obtained with the Archimedes method (97.7 and 99.2 %). This is probably due to the thin film of liquid that is on the samples when measuring with the Archimedes method, which could result in slightly higher density results. 24

29 a) 2_1.49 b) 2_1.49+Mg c) 2_1.50 d) 2_1.50+Mg Figure 17. SEM images of samples that have been polished and then thermally etched at 1150 o C for 30min. These samples were produced with the powders from the second synthesis that were isostatically pressed. Table 9. Average grain size and porosity measurements from SEM images of isopressed samples from the second powder synthesis. Samples Average grain size (µm) Porosity (%) Relative density based on porosity (%) 2_ ± ± ± 0.8 2_1.49+Mg 2.68 ± ± ± 0.8 2_ ± ± ± 0.8 2_1.50+Mg 2.98 ± ± ± Dissolution tests of samples produced with β-tcp powders of the second synthesis In order to compare the dissolution rate of the samples the results are presented as a function of the initial surface area (mm 3 ) divided by the weight loss in grams. The measurements of the surface area are highly simplified, ignoring pores and irregularities. Generally, the dissolved amount increased almost linearly with time (Fig 18). The samples without extra magnesium 25

30 dissolved faster than the other ones (p<1x10-13 ), particularly in the first 46 hours (Figure 18). After 92 h there was no longer a big difference between samples 2_1.50 and 2_1.50+Mg. The SEM images of the unclean surfaces after dissolution, give a picture of how the loose particles on top of the samples differed between the different compositions (Figure 19). For 2_1.49 it almost looked like there would be one preferred direction of dissolution, which could not be seen for the other samples. The SEM images of the cleaned surfaces looked more similar when comparing the different samples. The gaps seen between grains suggested that grain boundaries dissolved faster than did the grains Rate ((g/mm 3 )*10 3 ) Time (h) Figure 18. Dissolved amount as a function of the initial surface area. The samples were dissolved for 21h, 46h and 92h. ( ): 2_1.49; ( ): 2_1.50; ( ): 2_1.50+Mg ( ); 2_1.49+Mg 26

31 a) 2_1.49 b) 2_1.49+Mg c) 2_1.50 d) 2_1.50+Mg Figure 19. SEM images of uncleaned samples after 72h of dissolution. 27

32 a) 2_1.49 b) 2_1.49+Mg c) 2_1.50 d) 2_1.50+Mg Figure 20. SEM images of cleaned samples after 72h of dissolution. To be able to compare the ICP screening results after 23h of dissolution of the partly dissolved sample, the loose particles, the dissolution solution and the screening of the undissolved samples, all concentrations were normalized. Specifically, all Mg, Fe, Mn and Sr concentrations were divided by the P concentration (Table 10). These values were then compared in a statistical analysis to see if there was a preferential dissolution of any of the ions. This approach revealed that there was a significant difference in the Mg content of loose particles and dissolution solution (p<0.046). There was more Mg in solution than in the loose particles, indicating a preferential dissolution of Mg compared to phosphate ions. For the other ions no significant difference could be found between the dissolution solution and the loose particles (p>0.05). When comparing the undissolved sample with the partly dissolved ones for the same kind of sample, no significant difference could be detected. The XPS measurements showed no significant differences in Mg, Fe, Mn or Sr content when comparing undissolved samples with the partly dissolved ones or the loose particles after dissolution. The XPS results were normalized by putting the Ca at% content of the samples to 1. For this application the XPS seem so be a less sensitive method than ICP screening and the amount of Fe, Sr and Mn is on the border of the detection limit for XPS in this application. 28

33 Table 10. Normalized ICP screening results after 23h of dissolution. Mg/P Fe/P Mn/P Sr/P 2_1.49 Undissolved sample Partly dissolved sample Loose particles Dissolution solution _1.49+Mg Undissolved sample Partly dissolved sample Loose particles Dissolution solution _1.50 Undissolved sample Partly dissolved sample Loose particles Dissolution solution _1.50+Mg Undissolved sample Partly dissolved sample Loose particles Dissolution solution