Chelate setting of alkali ion substituted calcium phosphates

Author's Accepted Manuscript

Chelate setting of alkali ion substituted calciumphosphates

Zeeshan Sheikh, Martha Geffers, Theresa Christel,Jake Barralet, Uwe Gbureck

PII: S0272-8842(15)00839-1DOI: http://dx.doi.org/10.1016/j.ceramint.2015.04.083Reference: CERI10490

To appear in: Ceramics International

Received date: 20 February 2015Revised date: 31 March 2015Accepted date: 16 April 2015

Cite this article as: Zeeshan Sheikh, Martha Geffers, Theresa Christel, Jake Barralet, UweGbureck, Chelate setting of alkali ion substituted calcium phosphates, CeramicsInternational, http://dx.doi.org/10.1016/j.ceramint.2015.04.083

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1

Chelate setting of alkali ion substituted calcium phosphates

Zeeshan Sheikh1,2*, Martha Geffers3*, Theresa Christel3, Jake Barralet1,4, Uwe

Gbureck3#

1 Faculty of Dentistry, McGill University, Montreal, Quebec, Canada

2 Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada

3 Department for Functional Materials in Medicine and Dentistry, University of Würzburg, Germany

4 Division of Orthopaedics, Department of Surgery, Faculty of Medicine, McGill University, Montreal,

Quebec, Canada

* Equal first authorship

# corresponding author: e-mail: [email protected] phone: 0049 931 20173550, Fax:

0049 931 20173500, postal address: Department for Functional Materials in Medicine and Dentistry,

Pleicherwall 2, 97070 Würzburg, Germany

Abstract

Ca2KNa(PO4)2 is an alkali substituted calcium orthophosphate that stimulates osteoblast

growth, shows excellent biocompatibility both in vitro and in vivo and is biodegradable

under in vivo conditions. We report the development and assessment of the physico-

chemical properties of Ca2KNa(PO4)2 cements which set by a chelating reaction with

phytic acid. This was beneficial to avoid formation of low soluble hydroxyapatite during

setting and in vitro ageing in PBS. The concentration of phytic acid used for cement

setting was found to have an inverse effect on mechanical properties. Cements with

lower concentration of phytic acid had higher compressive strengths, but lower

degradation speed in comparison to the higher concentration acid cements. Cement

degradation was predominantly identified to be a result of alkali phosphate dissolution in

conjunction with dissolution of phytic acid – calcium complexes formed during setting.

The high degradation speed with a mass loss of 35-63% over 28 days indicated their

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potential to be evaluated further for orthopaedic and dental bone repair and

regeneration applications as fully resorbable bone graft substitutes.

Keywords: Calcium phosphate cement, phytic acid, chelate setting

1. Introduction

Bone grafts are frequently used as substitutes for diseased, damaged or missing bone

tissue. This is imperative to prevent fibrous tissue ingrowth into the defect and to

maintain mechanical function. The most commonly used graft materials for orthopedic

and dental applications are autologous bone grafts which are considered to be the gold

standard [1,2]. Although they have a high biological acceptability [3,4], they have

disadvantages such as donor site morbidity, the need for a second surgery, increased

cost due to hospitalization and limited amounts that can be procured along with other

complications [3]. Research in the past few decades has focused towards exploring

suitable alternatives for the said purpose [5,6,7]. Amongst other available bone substitutes

based on natural or synthetic polymers, inorganic calcium phosphate bioceramics are

commonly used [8,9,10,11] since they are non-toxic and do not induce cell lysis in the

surrounding tissues after being implanted [12]. Calcium phosphates can be either applied

as sintered monoliths or granules or they can be used as self setting calcium phosphate

cement (CPC) formulations, which undergo a process of dissolution and precipitation

leading to a solidification and a strong material-bone interface [13,14]. Due to their

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relatively fast setting time and ease of manipulation, these materials are considered as

a good option as bone substitutes [8,9,15].

Ionic substitution of calcium phosphate compounds is widely used in bioceramics to

adjust either material properties (e.g. solubility or mechanical performance) or released

ions may be used to direct cellular response to the material such as osteoblast /

osteoclast activity [16] or the formation of blood vessels within the implant [17]. A higher

CaP biodegradability is achieved by sodium or potassium substitution [18] leading to

compounds such as CaKPO4, CaNaPO4 or Ca2KNa(PO4)2, which in addition stimulate

osteoblast growth and show excellent biocompatibility under in vitro and in vivo

conditions [19,20,21]. Sintered ceramics based on alkali substituted calcium phosphates

have also been demonstrated to have a superior degradation profile in vitro [22] and in

vivo [23]. However, the use of calcium alkali orthophosphates in self setting cements

leads to the formation of low soluble hydroxyapatite from the compounds during setting

[24]. This problem might be solved by using an alternative cement setting mechanism

based on the formation of calcium chelates as binder phase. Indeed, Berger et al. [25]

could demonstrate that the modification of the cement liquid with citric acid produced

Ca2KNa(PO4)2 cements which set without the formation of hydroxyapatite. However,

due to the limited calcium binding capacity of citric acid with only three carboxylic acid

groups, the obtained cements were mechanically weak and showed comparatively long

setting times [25].

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Here we report the development of a calcium phosphate cement which is based on the

reaction of an alkali substituted, silica doped calcium phosphate (Ca2KNa(PO4)2) and

inositol phosphate (C6H6(OPO3H2)6; IP6), also known as phytic acid [26,27,28]. Phytic acid

is found in rice, corn, wheat, and soybean [29], and has strong chelating capability

towards calcium ions due to its six phosphate groups. We hypothesized, that this setting

mechanism would prevent the formation of low soluble hydroxyapatite and hence

maintain a high degradation ability of the materials after cement setting. In particular, we

have evaluated the physico-chemical properties of such chelate setting alkali ion

substituted calcium phosphate cements depending on both phytic acid concentration

and prolonged ageing in PBS solution.

2. Experimental

2.1 Method and materials

2.1.1 Synthesis

Ca2KNa(PO4)2 was prepared by melting CaHPO4, Na2CO3 and K2CO3 in a molar ratio

of 4:1:1 in a platinum crucible at >1550 °C. Silica substituted Ca2KNa(PO4)2 was

produced accordingly by adding 3 wt.% or 6 wt.% SiO2 to the raw powder mixture prior

to sintering, while the sodium and potassium content was decreased in the same

stoichiometric manner as silica was added. The glass melts were quenched in water

and manually crushed and sieved with 355 µm pore size-mesh followed by ball milling in

a planetary ball mill for 10 min at 200 rpm.

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In order to investigate the effect of concentration of the chelating agent, cements were

produced by mixing the Ca2KNa(PO4)2 powders with phytic acid having 20, 30, 40 and

50% concentration respectively with a powder of liquid ratio of 2 g/ml. The cement

pastes were mixed on a glass slab for 30 s and the cement slurry was cast into silicone

molds forming hardened cement cuboids (~6 х 6 x 12 mm) and cement discs (~15 mm

diameter, 2mm thickness). The samples were allowed to set for 24 h at 37°C ± 1˚C in a

water bath for 24 hours. At the end of the incubation period, the samples were removed

from the mould, dried and weighted until constant weight was reached.

2.1.2 Characterisation

The phase purity was confirmed for the alkali substituted calcium phosphate cement

samples set by phytic acid using X-ray diffraction (XRD). XRD data was collected

(Siemens D5005 X-ray diffractometer) with Ni filtered CuKα radiation (lamda = 1.54A)

with 2 dimensional area detector at 40 kV and 40 mA. A step size of 0.02° was used to

measure from 10 to 40° over a count time of 2 s per degree. Fourier transform infrared

(FTIR) spectra of the products were recorded within a wave number range of 4000-

650 cm-1 via attenuated total reflexion mode (Nicolet is10, Thermo Scientific, Waltham,

Massachusetts, USA). The compressive strength (wet) of the prepared cement blocks

was measured before and after in vitro ageing in PBS solutions. Before testing,

geometrical measurements of the sample were made in triplicate and the samples were

weighed. Samples were mounted on the testing machine (Zwick/ Roell Z010) so that the

long axes of the cement cylinders were perpendicular to the lower anvil. A compressive

force was then applied to the upper surface of the cement samples at a constant

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crosshead displacement rate of 1 mm/min until failure occurred. The applied load was

measured using a 10kN load cell (Zwick/Roell Z010). Mean strength was determined

from the average of 6 measurements. After testing in compression, cement fragments

were retrieved, weighed and dried in a vacuum desiccator at a temperature of 37°C.

The cement fragments were then ground to powder using a pestle and mortar. The true

density of the powder was determined using a helium pycnometer (Accupyc 1330,

Micromeritics). The volume of each sample was measured 10 times following 10 purges

of the measurement chamber with helium. The relative porosity (bulk porosity) of the

cements was calculated from apparent and true density measurements. The specific

surface area (SSA) of cements was determined by using the Brunauer–Emmett–Teller

(BET) method with helium adsorption–desorption (Tristar3000, Micromeritics).

Bioceramic microstructure was observed using scanning electron microscopy imaging

(Hitachi S-4700 FE-SEM; Tokyo, Japan), at an accelerating voltage of 2 kV.

2.1.3 In vitro ageing

After initial characterisation was completed, the samples were stored at 37 ± 1°C and

~100% relative humidity for 24 h. The cement discs prepared were immersed in PBS

solutions (n=5). The samples were aged at a liquid to cement volume ratio (LCVR) of 60

as used by Grover et al. [30] for 28 days at 37 ± 1°C. Dynamic ageing protocols were

achieved by refreshing the liquid every 24 h throughout the experiment to remove any

dissolution products. To quantify the amount of mass loss over time, the samples were

weighed daily. The sample weight loss was measured according to:

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∆W = (W1 – W2)/W1

(weight before in vitro incubation was measured as W1, weight after incubation washed

by distilled water and dried until constant was measured as W2). After a time period of 7

and 28 days, all the samples were removed from the solutions and tested in

compression and characterised for changes in phase composition, SSA, density and

porosity. Ion concentrations (Ca, P and Si) in the used aging PBS solution were

determined using inductively-coupled-plasma mass-spectrometry (ICP-MS, Varian,

Darmstadt, Germany) after 7, 14 and 28 days. The quantitative measurement was

carried out against standard solutions (Merck, Darmstadt, Germany) containing defined

concentrations of all ions of interest.

2.1.4 Statistical analysis

Statistical analysis was performed using IBM® SPSS® (v. 19, IBM Corp.; New York;

USA) statistical software. Mann-Whitney test was used to evaluate head-to-head

differences between the aged cement blocks. Statistical significance was set at a value

of P < 0.05.

3. Results

Initially, Ca2KNa(PO4)2 cements (alone and with 3 and 6% silica addition) were reacted

with phytic acid solutions of different concentrations. This was performed in order to

determine whether addition of 3% or 6% SiO2 would affect both the setting reaction and

the mechanical properties of the set cements. Typical X-ray diffraction patterns (Figure

1A) revealed a strong decrease of the diffraction peak intensity of the cement raw

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powder after reaction with phytic acid solutions as well as the appearance of a broad

amorphous background in a 2Theta range of 25-35°. FT-IR spectra (Figure 1B) showed

the valence vibration ν(P-OH) of pure PA at a wave number of 2770 cm-1, which

disappeared after cement setting and hence proofed the chelation reaction with Ca2+

ions released from the cement powder. It was further observed that the Ca2KNa(PO4)2

powders set with 40% phytic acid had a compressive strength of 2.5 ± 0.3 MPa (n=6),

while those powders with 3% and 6% silica addition had compressive strengths after

setting with 40% phytic acid of 2.5 ± 0.6 and 7.7 ± 0.2 MPa respectively (n=6, Table 1).

Higher strength of the silica modified cements is likely attributed to a slower setting

reaction such that cement samples with less macroscopically visible flaws can be

produced. Based on these results it was decided to use Ca2KNa(PO4)2 powders

modified with 6% silica for all further experiments.

Table 1: Characterisation of cements produced by setting of several calcium alkali phosphates with different concentrations of phytic acid.

Cement raw material

Concentration of phytic acid

Total relative porosity (%)

SSA (m

2/g)

Density (g/cm

3)

Compressive Strength (MPa)

Ca2KNa(PO4)2

40% 2.5 ± 0.3

Ca2KNa(PO4)2 + 3% SiO2

40% 2.5 ± 0.6

Ca2KNa(PO4)2 + 6% SiO2

20 % 35.3 ± 1.4 5.08 ± 0.07 2.14 ± 0.04 22.0 ± 5.3 30 % 43.8 ± 1.8 4.51 ± 0.08 2.18 ± 0.05 8.5 ± 1.6 40 % 53.3 ± 1.3 3.93 ± 0.41 2.19 ± 0.04 7.9 ± 0.2 50 % 61.0 ± 1.0 2.18 ± 0.40 2.20 ± 0.06 4.0 ± 1.0

Values are presented as mean ± standard deviation (n=5). SSA- specific surface area

After cements were set using 20, 30, 40 and 50% concentration phytic acid,

compressive strengths were measured before and after in vitro ageing in PBS (Figure

2A). The cements set with 20% phytic acid had the highest recorded compressive

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strengths (22.0 ± 5.3), while cements set with higher concentrations of phytic acid (30,

40 and 50%) had 2, 3 and 5 times less compressive strengths noted respectively than

the 20% phytic acid cement (Figure 2A). Upon ageing in PBS for 1 week the

compressive strength of the Ca2KNa(PO4)2 cements set with 20% phytic acid reduced to

~50% of the original strength observed. The higher concentration phytic acid set

cements after 4 weeks of ageing had significantly greater decrease in compressive

strengths observed (Figure 2A).

The concentration of phytic acid used to set cements had an inverse effect on total

porosity (Figure 2B). Cements set with 20% phytic acid had ~35% initial porosity which

increased to ~52% after 4 weeks in PBS. Increasing the phytic acid concentration both

increased the initial porosity to 44% - 61% which was further increased to 60% - 72%

after 4 weeks in PBS. The mechanical properties and porosity of cements had a direct

effect upon the mass loss observed after ageing in PBS. As would be expected, the

higher compressive strength and lower porosity cement (set with 20% phytic acid) lost

the least amount of mass after 4 weeks in PBS ~29%, while the 30, 40 and 50% phytic

acid samples lost ~45, 55 and 62% mass respectively after 4 weeks of ageing in vitro

(Figure 2C). The lower phytic acid concentration cements had higher specific surface

area (SSA) in comparison with the higher concentration prepared samples. After ageing

in PBS it was observed that the highest increase in SSA was recorded for the 50%

phytic acid cements (from ~2 to ~56 m2/g) (Figure 2D). The SSA increased significantly

from the initial recorded values for all other cements after 4 weeks in PBS as well

(Figure 2D).

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SEM images revealed changes in microstructural appearance of cements upon ageing.

The cements set with higher concentrations of phytic acid had a more porous

appearance than the ones set with lower concentration acid (Figures 3 a, b, c and d).

Upon ageing for 7 days and 28 days, the 20 and 30% phytic acid cements showed

cracks in its structure (Figures 3 e, f, I and j). The 40 and 50% phytic acid cements

became progressively more porous upon ageing and disintegrated (Figures 3 g, h, k

and l).

X-ray diffraction data revealed the phase composition of set cements before and after

ageing (Figure 4). All cements showed peaks representing cement raw material and it

was observed that phytic acid concentration inversely affected peak intensity. The

amorphous broad peak (at 25-35 2ϴ) observed prior to ageing disappeared after 28

days in PBS, but no phase transformation into hydroxyapatite was observed for any

cement group. Ion release in PBS buffer (Figure 5) showed a different behavior for the

various ions. Phosphate was initially released at concentrations of 450-770 mg/l*d

(calculated as pure phosphorus) with a higher release with increased PA concentration,

however after 14d or later release (370-420 mg/l*d) was practically independent of PA

concentration. Calcium was released to a much lower extent (initially 31-85 mg/l*d),

which decreased to 28-31 mg/l*d after 28days in PBS. Silicon release for cements

formed with 20% PA was practically independent of the time point (2.4-3.2 mg/l*d), but

was increasing with immersion time for all other cements to a maximum of 5 mg/l*d for

50% PA cement.

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4. Discussion

It was previously demonstrated that crystalline Ca2KNa(PO4)2 is non-reactive in water

and needs prolonged grinding to induce partial amorphization and to produce self-

setting cements [24]. During setting of such cements, nanocrystalline hydroxyapatite is

formed in a two step reaction via an alkali ion containing apatite intermediate [31]:

5Ca2KNa(PO4)2 + 4H2O → 2[Ca5K2Na2(HPO4)(PO4)4(H2O)] + Na+ + K+ +2OH- (1)

18[Ca2K2Na2(HPO4)(PO4)4·(H2O)] → 10Ca9(PO4)5(HPO4)(OH) + 18(K,Na)2HPO4 + 12(K,

Na)3PO4 + 8H2O (2)

The formed cements have been shown to possess antimicrobial properties due to both

the cations (Ca2+, Na2+ and K+) and basic tertiary phosphates present in the biomaterial

that are released [32,33]. Ions are released from the surface of these cements which

result in a strong alkaline pH value and an elevated osmotic pressure locally influencing

the viability of microorganisms in the immediate environment [34,35]. However, due to the

formation of low-soluble hydroxyapatite as the end product of setting, such cements

have to be considered to be non-degradable under in vivo conditions.

The current study aimed to prevent HA formation during setting of Ca2KNa(PO4)2 by

applying a chelate setting mechanism recently demonstrated for the reaction of

tricalcium phosphate with phytic acid [36,37]. The same appears for Ca2KNa(PO4)2 as

proofed by both FT-IR and XRD measurements, whereas the addition of 3% or 6% SiO2

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to the material before synthesis had no effect on the setting reaction, but strongly

increased the mechanical performance of the set matrix. Hydroxyapatite formation was

not detected for any of the investigated compositions, neither directly after setting, nor

after prolonged in vitro ageing for one month in PBS buffer solution. The setting process

is thought to produce both Ca2+- phytic acid complexes as well as primary alkali

phosphates according to Equation (3):

3Ca2KNa(PO4)2 + C6H18O24P6 → C6H6O24P6Ca6 + 3NaH2PO4 + 3KH2PO4 (3)

The primary alkali phosphates are highly soluble and are readily released after setting

into an aqueous environment, which explains the high phosphate release during PBS

immersion over 28 days (Figure 5). A reaction of the primary phosphates with residual

cement raw material to form secondary calcium phosphate compounds such as brushite

(CaHPO4·2H2O) or monetite (CaHPO4) could not be detected in X-ray diffraction

analysis (Figure 1).

Cement strength after setting results from a three-dimensional entanglement of

precipitated cement crystals and strength is commonly affected by several parameters

such as the type of setting product, the degree of conversion [38] or the porosity of the

cement matrix [39]. The latter is formed by the aqueous cement phase which is the

reaction medium for dissolving cement components and precipitating the setting

product. While cements forming hydrated phases (e.g. brushite or struvite) consume

water during setting [11], the chelate setting cements from the current study form non-

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hydrated setting products similar to hydroxyapatite biocements. The amount of water

used for mixing the cement paste is hence mainly responsible for the porosity of the set

cement matrix. Surprisingly, although the same PLR was used throughout the study for

cement mixing, porosity was increasing with an increasing concentration of phytic acid.

This might be a result of the relatively fast setting reaction, which made it hard to

homogeneously mix cement pastes and prepare samples for strength testing at higher

PA concentrations. In fact, most of the samples showed clearly visible macropores

derived by entrapped air bubbles. Porosity is known to commonly show an exponential

relationship to strength of porous ceramics and cements [39,40,41], this was confirmed in

the current study for chelate setting cements (Figure 6).

The cements were proofed to be degradable with a mass loss in the range of 35% (20%

phytic acid) to 63% (50% PA) over 28 days in PBS, which was accompanied by an

increase in cement porosity and a strong decrease of cement strength. Ion release

measurement during degradation showed a phosphate release approx. one order of

magnitude higher than the release of calcium. This likely stems from the above

mentioned release of soluble alkali phosphates produced during setting (Equation 3).

This appears over the whole course of the experiment since even after 28 days in PBS,

the Ca:P ratio of the immersion solution is still < 0.1 for all cements. Calcium release is

thought to predominantly result from dissolution of phytic acid – calcium complexes

rather than from unreacted cement raw powder, since the broad amorphous peak in X-

ray diffraction patterns at 25-35 ° (Figure 1 and 4) characteristic for the set cements

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disappeared while the peaks of the crystalline cement raw powder were still present

after 28 days in PBS.

5. Conclusion

Novel alkali ion substituted calcium phosphate cements were achieved by using calcium

ion complexing phytic acid as cement reactant. Material properties such as mechanical

performance and speed of degradation could be adjusted by varying phytic acid

concentration. An increasing concentration of phytic acid was found to increase mass

loss in physiological solution but simultaneously had an inverse effect on mechanical

properties. All cements showed no phase conversion to low soluble hydroxyapatite

upon in vitro ageing, which indicates their potential to be evaluated further for

orthopaedic and dental bone repair and regeneration applications.

6. Acknowledgements

The authors acknowledge financial support from GPS, Graduate Research Mobility

Award (McGill University) and from the Deutsche Forschungsgemeinschaft (DFG

GB1/15-1)

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18

Figure and Table captions

Figure 1: A) A) X-ray diffraction patterns showing a strong decrease of the diffraction

peak intensity of the cement raw powder after reaction with phytic acid solutions as well

as the appearance of a broad amorphous background in a 2Theta range of 25-35°. B)

FT-IR spectra of phytic acid, cement raw powder and set cements prepared with

(Ca2KNa(PO4)2 powders at PLR of 2.0 g/ml revealed disappearance of the valence

vibration ν(P-OH) of pure PA at a wave number of 2770 cm-1by the chelation reaction

with cement calcium ions.

Figure 2: Basic cement properties after immersion in PBS buffer for 7d and 28d

demonstrated a loss of mass and mechanical performance and an increase of cement

porosity and specific surface area.

Figure 3: SEM images of surface of cements set with varying concentrations of phytic

acid respectively after in vitro ageing at day 0 (a, b, c, d), day 7 (e, f, g, h) and day 28

(i, j, k, h) (Scale bars represent 5µm).

Figure 4: X-ray diffraction patterns of cements formed with either 20 % or 50 % phytic

acid before and after ageing for 28 d in PBS solution showing dissolution of amorphous

calcium chelates (broad peak at 25-35 2ϴ) and the absence of hydroxyapatite formation

within the investigated time period

Figure 5: Ion release rate of calcium, phosphor and silicon during aging in PBS after 7,

14 and 28 days. Values were obtained by ICP-MS analysis showing the mass

concentration of pure elements (Ca, P, Si) in the immersion solutions. The high

phosphate release likely stems from release of soluble alkali phosphates produced

during setting

19

Figure 6: Correlation between ln(compressive strength) and porosity for the chelate

setting cements from this study.

Table 1: Characterisation of cements produced by setting of several calcium alkali

phosphates with different concentrations of phytic acid.

20

20 25 30 35 40

2Theta [°]

cement raw powder

cement set with 50% phytic acid

cement set with 20% phytic acid

intensity [a.u.]

A

4000 3500 3000 2500 2000 1500 1000

50% phytic acid

Ca2KNa(PO

4)2 + 6%SiO

2 + 50% phytic acid

wave number (cm-1)

Ca2KNa(PO

4)2 + 6%SiO

2

intensity [a.u.]

ν(P-OH)

B

Figure 1: A) X-ray diffraction patterns showing a strong decrease of the diffraction peak

intensity of the cement raw powder after reaction with phytic acid solutions as well as the

appearance of a broad amorphous background in a 2Theta range of 25-35°. B) FT-IR spectra of

phytic acid, cement raw powder and set cements prepared with (Ca2KNa(PO4)2 powders at PLR

of 2.0 g/ml revealed disappearance of the valence vibration ν(P-OH) of pure PA at a wave

number of 2770 cm-1by chelation reaction with cement calcium ions.

21

0

5

10

15

20

25

30com

pre

ssiv

e s

trength

[M

Pa]

20% phytic acid

30% phytic acid

40% phytic acid

50% phytic acid

initially 7d 28d

A

0

20

40

60

80

100

rem

ain

ing m

ass [

%]

20% phytic acid

30% phytic acid

40% phytic acid

50% phytic acid

7d 28d

0

10

20

30

40

50

60

70

80

90

poro

sity [

%]

20% phytic acid

30% phytic acid

40% phytic acid

50% phytic acid

initially 7d 28d

B

C

0

10

20

30

40

50

60

70

specific

surf

ace a

rea [m

2/g

]

20% phytic acid

30% phytic acid

40% phytic acid

50% phytic acid

initially 7d 28d

D

Figure 2: Basic cement properties after immersion in PBS buffer for 7d and 28d demonstrated

a loss of mass and mechanical performance and an increase of cement porosity and specific

surface area.

Figure 3: SEM images of surface of cements set with varying concentrations of phytic

acid respectively after in vitro

(i, j, k, h) (Scale bars represent 5µm).

22

SEM images of surface of cements set with varying concentrations of phytic

ageing at day 0 (a, b, c, d), day 7 (e, f, g, h)

(Scale bars represent 5µm).

SEM images of surface of cements set with varying concentrations of phytic

(e, f, g, h) and day 28

23

20 25 30 35 4020 25 30 35 40

2Theta [°]

before ageing

28 d in PBS

intensity [a.u.]

50% PA

2Theta [°]

20% PA

intensity [a.u.]

before ageing

28 d in PBS

Figure 4: X-ray diffraction patterns of cements formed with either 20 % or 50 % phytic

acid before and after ageing for 28 d in PBS solution showing dissolution of amorphous

calcium chelates (broad peak at 25-35 2ϴ) and the absence of hydroxyapatite formation

within the investigated time period

24

0

2

4

6

8sili

co

n r

ele

ase

[m

g/l*d

]

7d 14d 28d

silicon

0

200

400

600

800

1000

pho

sp

hor

rele

ase

[m

g/l*d

]

7d 14d 28d

phosphorus

0

20

40

60

80

100

ca

lciu

m r

ele

ase [

mg

/l*d

] 20% phytic acid

30% phytic acid

40% phytic acid

50% phytic acid

7d 14d 28d

calcium

Figure 5: Ion release rate of calcium, phosphor and silicon during aging in PBS after 7,

14 and 28 days. Values were obtained by ICP-MS analysis showing the mass

concentration of pure elements (Ca, P, Si) in the immersion solutions. The high

phosphate release likely stems from release of soluble alkali phosphates produced

during setting

25

30 40 50 60 70

-1

0

1

2

3

4

5ln

CS

porosity [%]

y=5.844 - 0.0798*x

R2 = 0.8698

Figure 6: Correlation between ln(compressive strength) and porosity for the chelate

setting cements from this study.