bioactive glass: A study by design of experiments

Accepted Manuscript Title: Isothermal phase transformations of bovine-derived hydroxyapatite/bioactive glass: a study by design of experiments Authors: July Andrea Rinc´on-L´opez, Jennifer Andrea Hermann-Mu˜noz, David Andr´es Fern´andez-Benavides, Astrid Lorena Giraldo-Betancur, Juan Manuel Alvarado-Orozco, Juan Mu˜noz-Salda˜na PII: DOI: Reference:

S0955-2219(18)30686-1 https://doi.org/10.1016/j.jeurceramsoc.2018.11.021 JECS 12173

To appear in:

Journal of the European Ceramic Society

Received date: Revised date: Accepted date:

8 September 2018 7 November 2018 11 November 2018

Please cite this article as: Rinc´on-L´opez JA, Hermann-Mu˜noz JA, Fern´andezBenavides DA, Giraldo-Betancur AL, Alvarado-Orozco JM, Mu˜noz-Salda˜na J, Isothermal phase transformations of bovine-derived hydroxyapatite/bioactive glass: a study by design of experiments, Journal of the European Ceramic Society (2018), https://doi.org/10.1016/j.jeurceramsoc.2018.11.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Isothermal phase transformations of bovine-derived hydroxyapatite/bioactive glass: a study by design of experiments

July Andrea Rincón-López 1, Jennifer Andrea Hermann-Muñoz 1, David Andrés Fernández1

,

Astrid Lorena Giraldo-Betancur 2, Juan Manuel Alvarado-Orozco 3, Juan

SC RI PT

Benavides

Muñoz-Saldaña 1*

1

Centro de Investigación y de Estudios Avanzados del IPN, Unidad Querétaro, Libramiento Norponiente #2000 C.P. 76230, Querétaro, México.

CONACyT-Centro de Investigación y de Estudios Avanzados del IPN, Unidad Querétaro,

U

2

Libramiento Norponiente #2000 C.P. 76230, Querétaro, México. Centro de Ingeniería y Desarrollo Industrial, Av. Playa Pie de la Cuesta No. 702, Desarrollo

M

A

San Pablo, C.P. 76125, Querétaro, México.

N

3

Abstract

TE

D

*Corresponding author: [email protected]

EP

In this work, a systematic methodology to obtain third-generation bioceramics within the Ca2SiO4-Ca3(PO4)-NaCaPO4 ternary system is proposed. The synthesis of Silicocarnotite (Ca5-x(PO4)2+x(SiO4)1-x; x  0.3) (SC) and Nagelschmidtite (Ca7-xNax(PO4)2+x(SiO4)2-x; x  2)

CC

(Nagel) single phases, from mixtures of bovine-derived HAp (BHAp) and 45S5-bioactive glass (BG) as precursors was developed using design of experiments (DoE). A combination

A

of milling and sintering processing parameters was established by statistical analysis to optimize the microstructural and mechanical properties of BHAp/BG ceramics. The optimized sintering temperature obtained with simultaneous responses was 1220 °C. Variations in BHAp/BG ratios lead to isothermal phase transformation to single crystalline phases, where 85/15 and 70/30 vol:vol ratios transformed to SC and Nagel, respectively. Finally, the proposed methodology allows a feasible composition control depending only of

1

BHAp/BG ratio to obtain non-stoichiometric SC and Nagel phases with potential applications in bone repair.

Keywords: Bioceramics, Design of experiments, Nagelschmidtite, Silicocarnotite, Bovine-

SC RI PT

derived Hydroxyapatite.

1. Introduction

The current trend in bioceramics is focused on substituting replacement tissues by

regenerating tissues, which are known as third-generation biomaterials [1–3]. This category

U

implies a convergence between the bioactive and resorbable material behavior to stimulate

at the molecular level specific cellular responses [1,4]. Hydroxyapatite (HAp) is the most

N

known calcium phosphate and is recognized to be biologically active and osteoconductive

A

due to its similarity to natural bone mineral [5]. Within the main drawbacks of HAp are its

M

lack of mechanical strength, biodegradability after implantation and osteoinductivity [5–7]. The addition of ions to HAp such as silicates, magnesium, sodium, carbonates, among others has evidenced improvement in the mechanical and biological performance of these new

D

materials leading to the development of third-generation ceramics [4,8]. According to several

TE

authors, the presence of these ions in the physiological environment can stimulate cellular responses enhancing bone mineralization, calcification and metabolism processes [9–11].

EP

Ceramics obtained from mixtures of the ternary system formed by Ca2SiO4, Ca3(PO4)2, and NaCaPO4 have shown promising characteristics such as an improved apatite formation, cell

CC

infiltration, and osteogenic differentiation also observed in silicate-based biomaterials and Si-doped hydroxyapatite [2,12]. Within the main phases of this complex system are the Silicocarnotite (Ca5-x(PO4)2+x(SiO4)1-x; x  0.3) (SC), Nagelschmidtite structure (Ca7-

A

xNax(PO4)2+x(SiO4)2-x;

x  2) (Nagel), Nurse’s Ass-phase (2Ca2SiO4·Ca3(PO4)2),

Silicorhenatite (Ca4Na2(PO4)2(SiO4)) and Rhenanite (CaNa(PO4)) [13–17]. The study of these systems has been reported for several authors without a total agreement regarding the formation and identification of phases with similar crystalline structures and different stoichiometry due to the presence of isomorphism in this kind of compounds [14,16,18,19]. On the other hand, the presence of temperature-dependent polymorphism, extended solid 2

solutions, super and substructures between the phases of the ternary system difficult the correct indexing of their crystal structure [15]. The synthesis of these biocompatible phases has been reported using several precursors and techniques such as sol-gel, solid-state reaction, mechanochemical synthesis, among others. In the SC case, Bulina et al. and Gomes et al., suggested a synthesis route from silicon-substituted apatite using mechanochemical

SC RI PT

and aqueous precipitation methods, respectively [20,21]. Moreover, Duan et al., reported the synthesis of stoichiometric SC by a sol-gel method using tetraethoxysilane, triethyl

phosphate, and calcium nitrate tetrahydrated, as precursors [22] and Serena et al., used solid-

state reaction of pre-synthesized tricalcium phosphate and dicalcium silicate [23]. On the other hand, Zhou et al., synthesized stoichiometric Nagel powders (Ca7Si2P2O16) by the sol– gel process using tetraethyl orthosilicate, triethyl phosphate and calcium-nitrate tetrahydrate

U

[2]. Celotti et. al., reported the wet chemical synthesis of Nagel-type structure

N

(Ca5Na2(PO4)4) with a considerable amount of CaO and a small residual of HAp, using as

ammonium or sodium bicarbonate [16].

A

precursors calcium nitrate tetrahydrated, ammonium or sodium phosphate dibasic and

M

Demirkiran et. al., reported that additions of Bioglass® 45S5 to HAp ceramics result in the formation of a SC phase and sodium-calcium phosphate (Na3Ca6(PO4)5) in an amorphous

D

silicate matrix, with 10 and 25 wt.% bioactive glass (BG) contents, respectively [24].

TE

Nevertheless, in a previous study, Rivenet et al., claimed that the phosphate described as Na3Ca6(PO4)5 cannot exist and should be actually Na2Ca5(PO4)4 based on a double

EP

substitution mechanism which defines the silicophosphate as a solid solution with the following stoichiometry Na2-xCa5+x(PO4)4-x(SiO4)x [17]. Later, Widmer et. al., synthesized

CC

single crystals of Nagel structure starting from stoichiometric mixtures of SiO2 (quartz), Ca3(PO4)2, CaCO3, and NaHCO3 and using several thermal treatments. The authors showed that Nagel structure exist as Ca7-xNax(PO4)2+x(SiO4)2-x solid solution with x ≤ 2 including:

A

Na2Ca5(PO4)4 (x=0), NaCa6(PO4)3SiO4 (x=1) and Ca7(PO4)2(SiO4)2 (x=2). Likewise, the crystallographic information of Na2Ca5(PO4)4 and Ca7(PO4)2(SiO4)2 phases has been reported elsewhere [15]. A conventional processing route for dense ceramics includes several milling and sintering parameters, such as particle size distribution, morphology and sintering temperature, among others [25]. These process parameters play a fundamental role in ceramic densification 3

correlated with the microstructural characteristics and mechanical properties. Considering the great number of variables involved in ceramics processing, the use of methodologies to systematically analyze the effect of input parameters on ceramics characteristics is necessary, leading to an optimized processing route. Because of its effectiveness and versatility, the family of factorial design is considered as one of the high impact tools processes

SC RI PT

optimizations [26–31]. Based on this technique, the influence of the studied factors and their interactions with the responses are calculated by statistical analysis and subsequently, compared with an accepted value of significance. In this work, a combination of milling and sintering processing parameters was established by statistical analysis to improve the

microstructural and mechanical properties of bovine-derived HAp/bioactive glass Vitryxx®

(BHAp)/BG) ceramics. Isothermal phase transformations were studied by a compositional

U

mapping varying the BHAp/BG ratios from 100/0 to 70/30 vol:vol. Finally, a methodology

N

for the synthesis of single-phased materials of SC and Nagel from BHAp and BG mixtures

A

using DoE methodologies is presented and discussed.

M

2. Materials and methods

2.1. Definition of processing parameters using DoE

D

2.1.1. Milling

TE

BHAp powder prepared at Cinvestav, México was used to optimize the milling parameters by a statistical analysis. A detailed description of the preparation and characterization of

EP

BHAp powder has been reported elsewhere [32]. Bioactive glass in composition 45S5 was acquired from Vitryxx® (Schott, Germany) [33].

CC

The process to establish the milling parameters was achieved through a 23 factorial design (3 factors with 2 levels each) and two replicates. The selected factors and levels were: milling time (5 – 15 min), powder mass of BHAp (2 – 5 g) and mass to ball ratio (1:5 – 1:10). The

A

milling process was carried out by a high energy ball mill 8000D Mixer/Mill® (SPEX, USA) and yttria-stabilized zirconia (YSZ) balls of 10 mm in diameter. The studied responses were the maximum frequency (mode) and the percentage of particles between 5 and 15 μm. The milling parameters to prepare mixtures with different BHAp and BG ratios from 0 to 30 vol. % BG, were established considering that the hardness ratio of HAp/BG is around 1, and both HAp/YSZ(balls) and BG/YSZ(balls) is around 1/3 [34–36]. 4

2.1.2. Sintering process To evaluate the effect of temperature and BG content on the physicochemical properties of the samples, a 22 factorial design (2 factors with 2 levels each) with three replicates was carried out. The mixed powders were uniaxially pressed at 35 kg/cm2 to form disk-shaped green samples using a die of 10 mm in diameter and 0.25 g of powder per sample. The

SC RI PT

selected factors and levels were temperature (1160 – 1220 ºC) and BG content (2.5 – 30 % vol.). The studied responses were the phase fraction content, porosity, grain size, and

compressive strength. The sintering process was carried out for 4 h with a heating rate of 5 °C/min and a cooling rate of 10 °C/min in a chamber furnace (Thermolyne 46100, Thermofisher Scientific).

U

2.2. Processing of BHAp/BG ceramics

N

Based on the established processing parameters defined in section 2.1, the effect of BG content on the physical and chemical properties of the BHAp/BG mixtures was

A

systematically studied from 0 to 30 % vol., in steps of 3.75 %, by mixtures design using

M

Minitab®. As mentioned before, the samples were ball milled by adding 3.5 g of precursors for 15 min and 1:10 powder-to-ball weight ratio. Disk-shaped green samples were

D

subsequently sintered at 1220 ºC for 4 h in a chamber furnace. The content of elements used

TE

for each BHAp/BG ratio was calculated and reported in table 1. 2.3. Sample characterization

EP

2.3.1. Powder size distribution

Particle size distribution of powder mixtures was measured by triplicate using a laser

CC

diffractometer (HELOS/BR, Sympatec GmbH, Germany). A RODOS technique for dry powder was used for the measurements, where samples were placed in the powder feeder and

A

0.2 bar pressurized air. 2.3.2. Ceramics microstructure and composition Microstructural characterization of sintered bodies was performed by scanning electron microscopy (SEM) using an environmental scanning electron microscope Philips XL30 ESEM at 10 kV electron acceleration voltage and a backscattering electron detector. Grain size was determined according to the ASTM E112 standard from at least four micrographs

5

recorded at different magnifications (500 – 1000X). Porosity quantification was carried out using ImageJ® filtering micrograph darkest pixels and measuring the corresponding area fraction. Moreover, the chemical composition of sintered ceramics was measured using an Energy Dispersive Spectroscopy Analyzer (Bruker) coupled to the SEM.

SC RI PT

2.3.3. Structure Structural characterization of the sintered ceramics was performed using a Rigaku DMax

2100 diffractometer with a CuK radiation ( = 1.5406 Å) operating at 30 kV and 20 mA.

The XRD patterns were measured over a 2θ range from 20 to 70º in 0.02 º steps and 1 s integration time using Bragg-Brentano geometry at a fixed angle of 5 º. To determine the phase fraction and crystallographic parameters in each sample, Rietveld analysis of the XRD

U

patterns was performed using GSAS® using a method previously described in detail [32].

N

The crystallographic structures for HAp and SC phases were taken from [37] and [38], respectively. Within the initial structural HAp parameters are: space group P63/m and lattice

A

parameters a = b = 9.432 Å, c = 6.881 Å. For SC: space group Pnma and lattice parameters

M

a = 6.737 Å, b = 15.5080 Å, c = 10.132 Å. On the other hand, the Nagel crystallographic structure was taken from Ca5Na2P4O16 stoichiometry described in [15], based on the atom

D

coordinates, occupancies and displacement parameters for the N3q sample with space group

TE

P61 and lattice parameters a = b = 10.6336(1) Å, c = 21.6422(3) Å. Moreover, the vibrational modes of each sample were obtained by Bruker SENTERRA confocal Raman microscope using an excitation source of 532 nm measured at room temperature. The spectral resolution

EP

was 0.5 cm-1 with an integration time of 40 s. All samples were measured in two points and the spectrum in each point was recorded in triplicate. The spectra were recorded in the 200-

CC

3700 cm-1 frequency range to establish the silicates, phosphates, and hydroxyl Raman active vibrations.

A

2.4. Statistical analysis The experiments were studied by analysis of variance (ANOVA) at a confidence level of 95 % (  = 0.05) using Minitab® as statistical software. The trend to maximize the responses were individually identified through contour plots. Simultaneous feasible regions (white zones) composed of local optima were established by overlapping the contour plots following the methodology of simultaneous optimization of several responses. 6

3. Results The systematic study of the processing route to successfully prepare BHAp/BG ceramics includes the analysis of the two main stages: milling and sintering process. The processing parameters effect on the characteristics of ceramics with different BHAp/BG ratios, its

SC RI PT

interaction and the trend to optimize the particle distribution, grain size, porosity and compressive strength will be discussed as follows. 3.1. Definition of processing parameters using DoE 3.1.1. Milling process

Both BHAp and BG powders (figure 1a) exhibit a monomodal particle size distribution with 𝐵𝐻𝐴𝑝 𝐵𝐺 a 𝑋90 = 27.71 μm and 𝑋90 = 30.33 μm, respectively. Figure 1b shows a schematic

U

representation of the quantified responses. First, the mode (response 1) of the particle size

N

was measured from the highest point of the density distribution curve. The response 2 is the

A

percentage of particles between 5 and 15 m, which was determined by measuring the shaded area from the cumulative distribution curve. This process was first optimized to fix the

M

milling parameters for the optimization of the sintering process. The results are summarized in the contour plots (figures 1c and 1d) for the experimental region at 15 min of milling time,

D

each stripe indicates the area were responses values can be predicted by establishing a

TE

combination of factors. These contour plots also indicate the trend to individually optimize the milling responses. It is worth to notice that the maximum values for the mode (7.13 μm)

EP

and percentage of particles between 5 and 15 μm (33.66 %) are reached at the upper levels of the studied factors.

CC

Further analysis of the optimization procedure will be presented in the discussion section. 3.1.2 Sintering process

A

Figure 2 shows the XRD patterns and micrographs of the 22 factorial design treatments from the sintering process. A phase transformation from HAp to Nagel is evidenced at 70/30 BHAp/BG ratio while the HAp phase remains stable at 97.5/2.5 ratio, both independent of the sintering temperature. On the other hand, both sintered samples showed a clear effect on the grain size and porosity (figure 2b) as a function of BG content and sintering temperature. Based on image analysis, a decrease in porosity of approximately 8 % was calculated from

7

1160 to 1220 °C, while a grain growth of about 3 times was observed from 2.5 to 30 % BG content at 1220 °C. A summary of the measured responses is shown in table 2. The effect of sintering temperature and BG content is observed in the contour plots of figure 3 a-c, which shows the trend to optimize the grain size, porosity and compressive strength of dense ceramics. In all cases, the highest level of the studied factor allows to maximize the

SC RI PT

grain size (4.15 μm) and compressive strength (42.5 MPa) and to minimize the porosity (1.75 %). Further analysis of the optimization procedure of the sintering process will be presented in the discussion section.

Up to here, the processing conditions based on statistical optimization methods were fixed for the processing of ceramics in different BHAp/BG ratios.

U

3.2. Characterization BHAp/BG mixtures

N

XRD patterns and Rietveld refinement results of ceramics fabricated with 100/0 (Fig. 4a and

A

4h), 96.25/3.75 (Fig. 4b and 4i), 88.75/11.25 (Fig. 4c and 4j), 85/15 (Fig. 4d and 4k) 77.75/22.25 (Fig. 4e and 4l) and 70/30 (Fig. 4f and 4m) BHAp/BG ratios are shown in figure

M

4. The Rietveld refinements of each composition were performed using either one, two or three theoretical patterns to determine phase content and lattice parameters. Each refinement

D

is composed by three curves: a) the experimental patterns shown in black (“X”-symbol), b)

TE

the adjusted patterns (red), c) the adjusted background to calculate the phase content and lattice parameters (green) and d) the error line (blue) generated from the difference between

EP

the experimental and adjusted patterns. The phases weight percentage (wt.%) and the refinement quality measured through the

CC

goodness coefficient (χ2) and the structural factor (R) are reported in table 3. In all cases, the obtained values were lower than 2.21 and 0.6%, respectively. The R-value suggests an

A

excellent relationship between the calculated model and the experimental XRD pattern. According to the phase identification, the ceramics with different BHAp/BG ratios can be classified into two groups: Group 1 - Single Phase compositions a) As expected, hydroxyapatite phase was identified in 100/0 ratio (figure 4a), with strains in lattice parameters a and c, due to ionic substitutions in BHAp related to Na+

8

and Mg2+ contents. Further structural details about the BHAp are reported elsewhere [32]. b) Silicocarnotite phase with a fixed BHAp/BG ratio of 85/15 (Fig. 4d) was identified as a single crystalline phase with a contraction of the a, b and c lattice parameters, which suggests substitutions in the atomic positions of Ca2+ for Na1+ or Mg2+, and Si4+ for

SC RI PT

P5+ since it is a non-stoichiometric SC. The SC phase is a calcium silicophosphate with

orthorhombic structure exhibiting a wide range of solid solutions with CaO, SiO2 and P2O5 [21,38,39].

c) Nagelschmidtite phase with fixed BHAp/BG mixtures of 73.75/26.25 and 70/30 (Fig.

4f) was identified, showing expanded a,b and contracted c lattice parameters that suggest partial substitutions of P5+ in the atomic position of Si4+ [15,40]. In the Nagel

U

phase, with a solid solution range described by Ca7xNax(PO4)2+x(SiO4)2-x, where the x

N

mol are replaced by (PO4)3- at the expense of the (SiO4)4- and charges are compensated

A

by the replacement of Ca2+ with x Na+ mol [15,17].

In all single phase compositions, the strains are possibly related with the lattice substitutions

M

to form non-stoichiometric phases. A summary of structural parameters is shown in table 4.

D

Group 2 – Multiphase compositions

TE

Multiphase ceramics were obtained with 96.25/3.75, 92.5/7.5, 88.75/11.25, 81.25/18.75 and 77.5/22.5 BHAp/BG ratios. Representative patterns of the multiphase compositions are

EP

shown in figures 4b, 4c and 4e.

Moreover, in figures 4h-m the main peaks of the phases formed between 31 and 34.2 º are observed. There is a clear phase transition starting from HAp (figure 4h) with 100/0 ratio,

CC

going through SC at 85/15 ratio (figure 4k) and ending with a Nagel structure (figure 4m) in ceramics with 70/30 BHAp/BG ratio. The coexistence of two or three phases was observed

A

on ceramics with the following BHAp/BG ratios: at the 96.25/3.75 composition, HAp and Nagel phases are present (figure 4i), at 88.75/11.25 (figure 4j), HAp, SC and Nagel are observed. Finally, at the 77.75/22.25 ratio (figure 4l), the coexistence of SC and Nagel is detected. Representative micrographs of sintered ceramics with different BG contents are shown in figure 5 a-f. A homogeneous grain size of 1.57 ± 0.21 μm with the presence of some pores 9

was observed for pure BHAp ceramics (figure 5a). The chemical analysis by EDS showed traces of Na (1.136 ± 0.0138) and Mg (0.649 ± 0.049) on BHAp ceramics related to the bovine source [32]. The ceramic with 96.25/3.75 BHAp/BG content showed three different kinds of microstructural features as shown in figure 5b. This micrograph shows the presence of 4-10 m precipitates (zone 1) surrounded by a coarse-grained microstructure having grains

SC RI PT

3-5 m in size (zone 2). The microstructures observed in zones 1 and 2 can be related with

the formation of a second phase corresponding to Nagel as demonstrated by XRD. Finally,

zone 3 corresponds to a fine-grained microstructure. EDS point analysis confirms the chemical nature of these zones, where zone 1 corresponds to Si-rich precipitates surrounded

by Na-rich grains and the fine-grained matrix with a composition and microstructure similar to that of BHAp. A summary of EDS measurements is presented in table 5.

U

In ceramics fabricated with 88.75/11.25 and 77.75/22.25 BHAp/BG ratios, two types of

N

microstructures are shown in figures 5c and 5e. For zones 1, 3-5 m heterogeneous Na-rich

A

grains (Nagel structure) having homogeneously distributed microcracks were observed,

M

while in zones 2 Si-rich areas (SC structure) some protuberances and 1-2 m pores were detected. Likewise, for these compositions, three (HAp, SC and Nagel) and two (Nagel and

D

SC) phases were respectively determined by Rietveld refinements. Further on, the sample with 85/15 BHAp/BG ratio exhibits a faint microstructure with

TE

“blurred” grain boundaries and again some microcracks (figure 5d). In this zone, Si was homogeneously distributed. Finally, the ceramics with 70/30 BHAp/BG ratio show a well-

EP

defined microstructure with an average grain size of 4.67 ± 0.86 μm (figure 5f). Figure 6 shows the single HAp, SC and Nagel crystalline phases (Group 1) confirmed by

CC

Raman spectroscopy measurements and SEM micrographs. Multiphase samples (Group 2) are presented in figure 7. The characteristic four vibrational modes associated to the

A

phosphate tetrahedral (PO43-) were observed for all samples in the spectral range between 200 to 1250 cm-1 as shown in figures 6 (d,e,f)) and 7 (d,e,f)). The 𝛖1, 𝛖2, 𝛖3 and 𝛖4 modes correspond to a) P-O symmetric stretching bond around 962 cm-1, b) O-P-O symmetric bending bond around 431 cm-1, c) P-O antisymmetric stretching bond from 1042 to 1077 cm1

and d) O-P-O antisymmetric bending bond around 590 cm-1 [21,23,41], respectively.

10

The transformation of HAp to SC and Nagel phases as a function of BG content is evidenced. Additionally, a decrease and even the complete disappearance of the OH- band for the case of single phases where the complete HAp phase transformation is confirmed. The analysis of Raman spectra from the mentioned groups is discussed as follows:

SC RI PT

Group 1: Single phases: The differences among the three samples with single crystalline phase are observed between

920 to 1000 cm-1. Figure 6g shows the main zone of 𝛖1–PO43-band [21] for HAp and

deconvolution of the overlapped bands of SC and Nagel for 85/15 and 70/30 BHAp/BG ratios, respectively. HAp phase was identified by the characteristic phosphate tetrahedral

vibrational modes with only an associated band with symmetric stretching of the P-O bonds

U

and the O-H stretching peak [21] at 3572 cm-1 (See right inset in figure 6d). Moreover, a

N

contribution between 1019 – 1088 cm-1 (See left inset in figure 6d) is apparently associated

A

with B-type carbonate vibration (𝛖1 CO32-) [21] due to the BHAp source. For the SC phase, the deconvoluted peaks for the most intense band in the 920-1000 cm-1

M

range, showed the phosphate and silicate combined contribution at 962 cm-1 with shoulders at 958 and 982 cm-1 (figure 6g). The most representative silicate bands are observed at

D

856 cm-1 and 587 cm-1 associated with the 𝛖1 and 𝛖2 vibrational modes of the SiO44- [23],

TE

respectively. Finally, a remaining HAp aggregate is evidenced by a low contribution of the OH- vibration in the right inset of figure 6e [21].

EP

Despite the absence of Raman reports for the Nagel phase, the reported Nurse’s Ass-phase with a stoichiometry Ca7Si2P2O1 can be properly used as reference [41]. Figure 6f shows the recorded Raman spectrum in a typical microstructure of the mentioned phase, where the

CC

PO43- for 𝛖1, 𝛖2, 𝛖3 and 𝛖4 modes are observed. The contributions are observed at 866, 638 and 589 cm-1 and are related to 𝛖1, 𝛖2, and 𝛖4 vibrational modes of the SiO44-, respectively

A

[41] (See left inset in figure 6f). Furthermore, the OH- vibration disappears (See right inset in figure 6f) due to the complete phase transformation as a consequence of the BG content addition [41]. Group 2: Phase coexistence

11

A set of compositions evidenced the coexistence of phases (figure 7) belonging to the transition among single HAp, SC, and Nagel. These phases showed an overlapping of PO43and SiO44- [23] bands, which difficult the interpretation of Raman spectra. The most intense band in the 900-1000 cm-1 range, showed a combined contribution of phosphate and silicate

SC RI PT

between 950 and 965 cm-1 (figure 7g). The 96.25/3.75 BHAp/BG sample with the lowest BG content, showed the typical vibrational

modes of single HAp. In the spectrum labeled as (1) (figure 7d), a slight contribution of a

silicate is observed in 587, 615 and 853 cm-1 and corresponds to the characteristic SiO44vibrations [41], which are associated to the Nagel phase. For 88.75/11.25 and 77.75/22.25 BHAp/BG samples, both the phosphate and silicate contributions of HAp, SC and Nagel phases are respectively shown in the Raman spectra recorded in 3-4 zones in figure 7e and

U

5-6 zone in figure 7f. Table 6 summarizes the Raman modes of prepared samples.

N

These results evidenced the coexistence of HAp, SC and Nagel phases and are in agreement

A

with the obtained XRD and SEM micrographs results.

M

4. Discussion

The successful synthesis of either SC or Nagel phases using BHAp and BG powder mixtures

D

strongly depend on the optimization of the processing route. In order to determine the optimal

TE

experimental region, a systematic study based on DoE was performed. Details of the simultaneous responses optimization and the effect on BHAp-BG phase

EP

transformations are discussed as follows. 4.1. Processing parameters

CC

Figures 8a and 8b show typical overlaid contour plots, where the white regions represent feasible zones of local optima in which responses are simultaneously analyzed for milling

A

(8a) and sintering processes (8b), respectively. Both processes were optimized in the following narrow ranges: i) milling: mode (6.5-7 μm), the percentage of particles between 5 and 15 μm (31-32.5 %) and ii) sintering: grain size (3-4 μm), porosity (0-4 %) and compressive strength (38-42 MPa). Therefore, the local optima of processing parameters to obtain BHAp/BG ceramics with desired responses are the following: 3.5 g of powder-to-vial volume, 1:10 ball-to-powder weight ratio, 15 min for milling and 1220 ºC for sintering.

12

Additionally, figure 8c shows a similar particle size distribution of the BHAp powder and BHAp/BG mixtures with 97.5/2.5 and 70/30 ratios using the mentioned optimized milling parameters. As result, all the milled powders exhibit a trimodal distribution with modes at 1.42, 6.85 and 27.89 m with a X10 = 0.7, X50 = 5.5 and X90 = 27.5 m. It is well known that a non-monomodal distribution favors the packing density of a green body by filling the

SC RI PT

interstitial holes with a number of fine particles [25], unlike the monomodal distributions which result to crack-like voids and porosity [25,42]. Based on the current results, a

remarkable relationship between milling and sintering process to obtain dense BHAp/BG ceramics with desired microstructural and mechanical properties is observed.

An optimized milling process naturally leads to homogeneous particle size distribution and

reproducibility of prepared BHAp/BG mixtures. It is worth to notice that the particle size

U

distribution of milled samples in optimized conditions is independent of the BG content

N

between 2.5 to 30 vol. %. Additionally, the optimized sintering process not only allows the

A

densification of BHAp/BG samples but favors the required chemical reactions to promote

M

the phase transformations between the different mixtures. This is only possible by using systematic methodologies such as DoE, that can be used not

D

only to establish the relationships between multifactor processes but also to contribute to the

TE

general understanding of the synthesis process and materials properties [27,29–31]. 4.1. Phase transformations in BHAp/BG mixtures

EP

The synthesis of SC or Nagel phases has been reported elsewhere using tetraethoxysilane, triethyl phosphate, calcium nitrate tetrahydrated, etc., as precursors and by several methods such as sol-gel, solid-state reaction, mechanochemical synthesis, among others [2,22,43].

CC

Moreover, there are few reports, where HAp and Bioactive glass 45S5 are also used to obtain these phases, being however unavoidable the presence of secondary phases [24,44].

A

In this section, details of phase transformation leading to either SC or Nagel phases from specific compositions BHAp/BG mixtures are presented below. On the synthesis of Silicocarnotite (SC) Demirkiran et al. reported the formation of SC and Sodium Calcium Phosphate (Na3Ca6(PO4)5) using HAp/BG mixtures with 90/10 and 75/25 wt.% ratios, respectively [24].

13

The reported processing route includes a ball milling step for 30 h with acetone and sintering at 1200 °C for 4 h. The SC phase was reported to coexist with the-TCP as a secondary phase, both embedded in a glassy silicate matrix. In the current contribution, a single SC phase was obtained with 15 vol.% (13.10 wt.%) of BG added to BHAp, without amorphous silicate phases. The stoichiometric SC requires 5.822 wt.% of Si and 12.841 wt.% of P (table

SC RI PT

5). Comparing these with the EDS measured values for 85/15 BHAp/BG mixture, it has 2.148 wt.% of Si and 17.412 wt.% of P, evidencing a lack of Si (3.674 wt.%) and a P

excess (4.571 wt.%). According to this analysis, the simultaneous formation of SC on an amorphous silicon matrix should not occur since the silicon content is insufficient even for

SC formation. On the other hand, it is well known that the SC formation involves the reaction

between BG and -TCP from decomposed HAp during the sintering [21]. The remaining -

U

TCP secondary phase [24,44] can be related to an inhomogeneous distribution of BG in the

N

mixture as a result of a non-optimized milling process, which is not present in the current

A

contribution. Such a fact enhances the importance of DoE and statistical analysis to optimize the milling and sintering processes, promoting a complete reaction between BG and BHAp,

M

that leads to the formation of SC phase.

D

On the synthesis of Nagelschmidtite

According to the possible atomic substitution 0 ≤ x ≤ 2 in the general stoichiometry, Ca7the lattice parameters for the end-member compositions should be

TE

xNax(PO4)2+x(SiO4)2–x,

between the ranges 10.6336 Å < a, b < 10.7754 Å and 21.4166 Å < c < 21.6422 Å, allowing

EP

a maximal strain of 1.333 % and 1.042 %, respectively. The calculated strains of synthesized Nagel, using as reference the Ca5Na2(PO4)4 lattice parameters, are within the

CC

permissible range for the solid solution with the following values: a = b = 0.409 % and c = 0.059 %. Additionally, the a, b expansion and c contraction (table 4) could be related to

A

the lower phosphorus and higher silicon contents of the synthesized Nagel compared to the Ca5Na2(PO4)4 contents, as shown in table 5. Demirkiran et al., identified the Na3Ca6(PO4)5 phase in a silicate amorphous matrix in HAp/BG: 75/25 wt.% mixtures [24]. Moreover, considering the elements of the studied system (Na2O, CaO, P2O5, SiO2) and according to the double substitution mechanism proposed by Rivenet et.al., Na+ + P5+  Ca2+ + Si4+, the resulting solid solutions should

14

follow the Ca7-xNax(PO4)2+x(SiO4)2–x, (x ≤ 2) stoichiometry [15,17]. Therefore, it has led to a misleading identification of the Na3Ca6(PO4)5 stoichiometry, which should actually be Na2Ca5(PO4)4 [17]. The latter belongs to the solid solutions range resulting in Nagel structure, which was further studied by Widmer et. al., who established its crystallographic information [15]. Similar misleading phase identification of Na3Ca6(PO4)5 phase was reported by Cozza

SC RI PT

et. al., with mixtures of 70 wt.% of BG content with commercial and cuttlefish HAp.

Again, in mixtures with 26.25 and 30 vol.% BG content (23.45 and 26.80 wt.% BG,

respectively) the identified phase in the current contribution corresponds to Nagel structure

with stoichiometric variations in Ca, Na, P, and Si contents according to the double substitution mechanism [17]. The problem in the misleading identifications in some literature

reports is possibly related to isomorphism in this kind of compounds [14,16,18], temperature-

U

dependent polymorphism, extended solid solutions, super and substructures between the

N

phases of the ternary system. All of them difficult the correct indexing of their crystal

A

structure [15]. Consistently, tools such as Rietveld Refinement allowed conducting a precise phase identification through the adjustment of a theoretical pattern to the experimental one,

M

determining later the lattice parameters and phase content, based on well-known

TE

On the phase coexistence

D

mathematical models.

The compositional mapping performed with BG contents from 0 to 30 vol.% is shown in figure 9 and represented as a quasi-ternary P2O5-CaO-(SiO2+Na2O) diagram considering the

EP

theoretical oxide contents from stoichiometric BHAp/BG mixtures. The axes represent the P2O5, CaO and the SiO2+Na2O content in wt.%. (i.e. for 45S5 composition, SiO2 content is

CC

1.84 times Na2O content). As previously mentioned, the mixtures with 0, 15, 26.25 and 30 vol.% of BG content showed the presence of HAp, SC, and Nagel phases, respectively.

A

Moreover, coexistence of HAp-Nagel, HAp-Nagel-SC and SC-Nagel phases was observed in compositions with 3.75, 7.5, 11.25, 18.75 and 22.5 vol.% of BG, respectively. Compositions reported elsewhere are also included for comparative purposes [24,44]. The phase transformations reported in the present contribution are fully consistent to studies in the Ca2SiO4 – Ca3(PO4)2 binary system processed from pure precursors at temperatures around 1200 ºC [14,18,45], were Nagel, SC and TCP phases are shown in specific 15

composition ranges. Furthermore, the current results of crystalline structures and stoichiometry are in agreement with the Ca2SiO4 – Ca3(PO4)2 – NaCaPO4 ternary diagram, recently reported by Widmer et al. [15]. Summarizing, the present contribution discusses the phase transformations in the BHAp-BG

SC RI PT

system as well as its microstructural aspects. As the main result, a systematic methodology for the synthesis of pure SC or Nagel phases using two stable starting materials (BHAp or BG) with a feasible composition control depending only of BHAp/BG ratio is presented.

Previous reports using Nagel phase for 3D plotted scaffolds showed interesting in vitro

properties such as excellent apatite mineralization ability, osteo/cementostimulation and

improved angiogenesis capacity [46]. On the other hand, the properties of SC phase have

U

been also reported as high bioactive phase having good cytocompatibility and osteogenic activity [22,43]. Finally, it is well known that Ca2+, PO43-, SiO44- ions released from the

N

material affect cell attachment, differentiation, apatite formation, among others [12]. The

A

non-stoichiometry and the presence of additional ions such as Mg2+, which is involved in

M

bone remodeling regulation [10,11] are promising characteristics for the biological performance of the phases synthesized in the current work.

D

5. Conclusions

TE

A systematic methodology to obtain SC and Nagel phases was established by DoE, using bovine-derived HAp and 45S5 bioactive glass as starting powders. The formation of non-

EP

stoichiometric SC and Nagel single phases was carried out during the sintering process, in which solid state reactions between the BHAp and BG take place. A homogeneous distribution and specific BG contents assure the formation of stand-alone phases in specific

CC

compositions. The isothermal phase transitions of BHAp-BG system followed the behavior of the Ca2SiO4-Ca3(PO4)2-NaCaPO4 ternary system and were consistently confirmed by SEM

A

micrographs, Raman spectra and Rietveld refinements of XRD patterns. Bioceramics within this system are recognized as third generation biomaterials due to stimulation of specific cellular responses. Among these, the synthesized SC and Nagel phases have potential application in bone repair. Finally, characteristics such as the non-stoichiometry and substitutions in the crystal structure could positively affect their ionic release leading to an improved biological performance.

16

Acknowledgments: The authors thank CONACYT for the financial support. This project was funded by CONACYT Projects 272095, 279738 and 279738 and carried out partially at CENAPROT, LIDTRA, and LANIMFE national laboratories and LISMA. Additionally, the thanks are extended to Dr. Juan Zarate Medina of the Metallurgy Research Institute at the UMSNH for the access to the Raman Spectrometer used in this work and to Jose Eleazar

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Urbina-Alvarez and Adair Jimenez-Nieto for technical support in SEM measurements.

17

References [1]

L.L. Hench, J.M. Polak, Third-generation biomedical materials., Science. 295 (2002) 1014–1017. doi:10.1126/science.1067404.

[2]

Y. Zhou, C. Wu, Y. Xiao, The stimulation of proliferation and differentiation of

SC RI PT

periodontal ligament cells by the ionic products from Ca7Si2P2O16 bioceramics, Acta Biomater. 8 (2012) 2307–2316. doi:10.1016/j.actbio.2012.03.012. [3]

M. Vallet-regí, Bioceramics, in: Bioceram. with Clin. Appl., John Wiley & Sons, 2014: p. 484.

[4]

P.N. De Aza, J.E. Mate-Sanchez de Val, C. Baudin, C. Perez Albacete-Martínez, A. Armijo Salto, J.L. Calvo-Guirado, Bone neoformation of a novel porous resorbable

U

Si-Ca-P-based ceramic with osteoconductive properties: physical and mechanical

N

characterization, histological and histomorphometric study, Clin. Oral Implants Res.

M. Vallet-Regí, Ceramics for medical applications, J. Chem. Soc. Dalt. Trans. (2001)

M

[5]

A

27 (2016) 1368–1375. doi:10.1111/clr.12745.

97–108. doi:10.1039/b007852m.

J. Lu, M. Descamps, J. Dejou, G. Koubi, P. Hardouin, J. Lemaitre, J.P. Proust, The

D

[6]

biodegradation mechanism of calcium phosphate biomaterials in bone, J. Biomed.

[7]

TE

Mater. Res. 63 (2002) 408–412. doi:10.1002/jbm.10259. S.R. Radin, P. Ducheyne, The effect of calcium phosphate ceramic composition and

EP

structure on in vitro behavior. II. Precipitation, J. Biomed. Mater. Res. 27 (1993) 35–

CC

45. doi:10.1002/jbm.820270106. [8]

H. Yu, K. Liu, F. Zhang, W. Wei, C. Chen, Q. Huang, Microstructure and in vitro

A

Bioactivity of Silicon-Substituted Hydroxyapatite, Silicon. 9 (2017) 543–553.

[9]

doi:10.1007/s12633-015-9298-3. C. Wu, J. Chang, A review of bioactive silicate ceramics, Biomed. Mater. 8 (2013). doi:10.1088/1748-6041/8/3/032001.

[10] A. Hoppe, N.S. Güldal, A.R. Boccaccini, A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics, Biomaterials. 32

18

(2011) 2757–2774. doi:10.1016/j.biomaterials.2011.01.004. [11] M. Zhang, X. Chen, X. Pu, X. Liao, Z. Huang, G. Yin, Dissolution behavior of CaOMgO-SiO2-based multiphase bioceramic powders and effects of the released ions on osteogenesis, J. Biomed. Mater. Res. - Part A. 105 (2017) 3159–3168.

SC RI PT

doi:10.1002/jbm.a.36154. [12] M. Sepantafar, H. Mohammadi, R. Maheronnaghsh, L. Tayebi, H. Baharvand, Single

phased silicate-containing calcium phosphate bioceramics: Promising biomaterials for periodontal

repair,

Ceram.

Int.

44

doi:10.1016/j.ceramint.2018.03.050.

(2018)

11003–11012.

[13] G. Nagelschmidt, Nagelschmzdt : A New Calcium Silicophosphate, J. Ceram. Soc.

U

(1937) 865–867. doi:10.1039/JR9370000865.

N

[14] R.W. Nurse, J.H. Welch, W. Gutt, High-temperature phase equilibria in the system

A

dicalcium silicate–tricalcium phosphate, J. Chem. Soc. (1959) 1077–1083.

M

doi:10.1039/JR9590001077.

[15] R. Widmer, F. Gfeller, T. Armbruster, Structural and Crystal Chemical Investigation of Intermediate Phases in the System Ca2SiO4-Ca3(PO4)2-CaNaPO4, J. Am. Ceram.

D

Soc. 98 (2015) 3956–3965. doi:10.1111/jace.13850.

TE

[16] G. Celotti, E. Landi, A misunderstood member of the nagelschmidtite family unveiled: structure of Ca5Na2(PO4)4 from X-ray powder diffraction data, J. Eur. Ceram. Soc. 23

EP

(2003) 851–858. doi:10.1016/S0955-2219(02)00211-X. [17] M. Rivenet, O. Cousin, J.C. Boivin, F. Abraham, N. Ruchaud, P. Hubert, Study of the

CC

Na2O-CaO-P2O5-SiO2 system with respect to the behaviour of phosphate bonded basic refractories at high temperature, J. Eur. Ceram. Soc. 20 (2000) 1169–1178.

A

doi:10.1016/S0955-2219(99)00276-9.

[18] R.P. Gunawardane, F.P. Glasser, Reaction of chlorapatite, Ca5(PO4)3(Cl, F) with sodium

carbonate

and

silica,

J.

Mater.

Sci.

14

(1979)

2797–2810.

doi:10.1007/BF00611458. [19] M.A. Bredig, Isomorphism and Allotropy in Compounds of the Type A2XO4, J. Phys. Chem. 46 (1942) 747–764. doi:10.1021/j150421a009. 19

[20] N.V.N. V. Bulina, M.V.M. V Chaikina, K.B. Gerasimov, A.V.A. V Ishchenko, D. V Dudina, K.B. Gerasimo, A.V.A. V Ishchenko, D. V Dudina, D. V., A novel approach to

the

synthesis

of

silicocarnotite,

Mater.

Lett.

164

(2016)

255–259.

doi:10.1016/j.matlet.2015.10.047.

SC RI PT

[21] S. Gomes, J.M. Nedelec, E. Jallot, D. Sheptyakov, G. Renaudin, Silicon location in silicate-substituted calcium phosphate ceramics determined by neutron diffraction, Cryst. Growth Des. 11 (2011) 4017–4026. doi:10.1021/cg200587s.

[22] W. Duan, C. Ning, T. Tang, Cytocompatibility and osteogenic activity of a novel

calcium phosphate silicate bioceramic: Silicocarnotite, J. Biomed. Mater. Res. - Part A. 101 A (2013) 1955–1961. doi:10.1002/jbm.a.34497.

Ca3(PO4)2-Ca2SiO4,

Ceram.

40

(2014)

8245–8252.

A

doi:10.1016/j.ceramint.2014.01.022.

Int.

N

subsystem

U

[23] S. Serena, M.A. Sainz, A. Caballero, Single-phase silicocarnotite synthesis in the

[24] H. Demirkiran, A. Mohandas, M. Dohi, A. Fuentes, K. Nguyen, P. Aswath, Bioactivity

M

and mineralization of hydroxyapatite with bioglass as sintering aid and bioceramics with Na3Ca6(PO4)5 and Ca5(PO4)2SiO4 in a silicate matrix, Mater. Sci. Eng. C. 30

D

(2010) 263–272. doi:10.1016/j.msec.2009.10.011.

TE

[25] M.N. Rahaman, Ceramic Processing and Sintering, 2nd ed., CRC Press, 2003. [26] H.G. Pulido, H. Gutiérrez Pulido, R. De La Vara Salazar, Análisis y Diseño de

EP

Experimentos, 2nd ed., MCGRAW-HILL, 2008. [27] V. Cannillo, L. Lusvarghi, A. Sola, Design of experiments (DOE) for the optimization

CC

of titania-hydroxyapatite functionally graded coatings, Int. J. Appl. Ceram. Technol. 6 (2009) 537–550. doi:10.1111/j.1744-7402.2008.02298.x.

A

[28] B. Guillaume, F. Boschini, I. Garcia-Cano, A. Rulmont, R. Cloots, M. Ausloos, Optimization of BaZrO3sintering by control of the initial powder size distribution; a factorial design statistical analysis, J. Eur. Ceram. Soc. 25 (2005) 3593–3604. doi:10.1016/j.jeurceramsoc.2004.09.022. [29] S. Kehoe, M. Ardhaoui, J. Stokes, Design of experiments study of hydroxyapatite synthesis for orthopaedic application using fractional factorial design, J. Mater. Eng. 20

Perform. 20 (2011) 1423–1437. doi:10.1007/s11665-010-9704-3. [30] A. Lucas-Girot, P. Langlois, J.C. Sangleboeuf, A. Ouammou, T. Rouxel, J. Gaude, A synthetic aragonite-based bioceramic: influence of process parameters on porosity and compressive strength, Biomaterials. 23 (2002) 503–510. doi:10.1016/S0142-

SC RI PT

9612(01)00132-6. [31] M. Sadat-Shojai, M. Atai, A. Nodehi, Design of experiments (DOE) for the optimization of hydrothermal synthesis of hydroxyapatite nanoparticles, J. Braz. Chem. Soc. 22 (2011) 571–582. doi:10.1590/S0103-50532011000300023.

[32] J.A. Rincón-López, J.A. Hermann-Muñoz, A.L. Giraldo-Betancur, A. De VizcayaRuiz, J.M. Alvarado-Orozco, J. Muñoz-Saldaña, Synthesis, Characterization and In

U

Vitro Study of Synthetic and Bovine-Derived Hydroxyapatite Ceramics: A

N

Comparison, Mater. 11 (2018). doi:10.3390/ma11020333.

A

[33] Schott AG. Bioactive - Products - Cosmetics | SCHOTT AG, (2018). https://www.schott.com/epackaging/english/bioactive/products/cosmetics.html?so=u

M

k%26lang=english.

[34] L.L. Hench, Bioceramics, J. Am. Ceram. Soc. 81 (1998) 1705–1728.

D

doi:10.1111/j.1151-2916.1998.tb02540.x.

TE

[35] Chemco, Data Sheet – CHEMCO Type YTZP Ceramic Beads, (2018) 2. www.chemcobeads.com.

EP

[36] D.C. Tancred, A.J. Carr, B.A. McCormack, The sintering and mechanical behavior of

CC

hydroxyapatite with biogla, J. Mater. Sci. Med. 12 (2001) 81–93. [37] K. Sudarsanan, R.A. Young, Significant precision in crystal structural details. Holly

A

Springs hydroxyapatite, Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 25 (1969) 1534–1543. doi:10.1107/S0567740869004298.

[38] B. Dickens, W.E. Brown, The crystal structure of Ca5(PO4)2SiO4 (Silico-Carnotite), Tschermaks

Mineral.

Und

Petrogr.

Mitteilungen.

16

(1971)

1–27.

doi:10.1007/BF01099075. [39] Y. Pliego-Cuervo, F.P. Glasser, Phase relations and crystal chemistry of apatite and

21

silicocarnotite solid solutions, Cem. Concr. Res. 8 (1978) 519–523. doi:10.1016/00088846(78)90033-9. [40] M.W. Barnes, M. Klimkiewicz, P.W. Brown, Hydration in the System Ca2SiO4– Ca3(PO4)2 at 90°C, Chem. Thermodyn. 75 (1992) 1423–1429. doi:10.1111/j.1151-

SC RI PT

2916.1992.tb04204.x. [41] R. Rabadan-Ros, P. Mazón, S. Serena, M.A. Sainz, L. Meseguer-Olmo, P.N. De Aza, In vitro behaviour of Nurse’s Ass-phase: A new calcium silicophosphate ceramic, J. Eur. Ceram. Soc. 37 (2017) 2943–2952. doi:10.1016/j.jeurceramsoc.2017.03.014.

[42] R.K. McGeary, Mechanical Packing of Spherical Particles, J. Am. Ceram. Soc. 44 (1961) 513–522. doi:10.1111/j.1151-2916.1961.tb13716.x.

U

[43] S. Serena, A. Caballero, P.N. De Aza, M.A. Sainz, New evaluation of the in vitro

A

doi:10.1016/j.ceramint.2015.03.319.

N

response of silicocarnotite monophasic material, Ceram. Int. 41 (2015) 9412–9419.

M

[44] N. Cozza, F. Monte, W. Bonani, P. Aswath, A. Motta, C. Migliaresi, Bioactivity and mineralization of natural hydroxyapatite from cuttlefish bone and Bioglass® cosintered bioceramics, J. Tissue Eng. Regen. Med. 12 (2018) e1131–e1142.

D

doi:10.1002/term.2448.

TE

[45] P. Ros-Tárraga, P. Mazón, L. Meseguer-Olmo, P.N. De Aza, Revising the subsystem Nurse’s A-phase-silicocarnotite within the system Ca3(PO4)2-Ca2SiO4, Materials

EP

(Basel). 9 (2016). doi:10.3390/ma9050322. [46] M. Xu, D. Zhai, J. Chang, C. Wu, In vitro assessment of three-dimensionally plotted

CC

nagelschmidtite bioceramic scaffolds with varied macropore morphologies, Acta

A

Biomater. 10 (2014) 463–476. doi:10.1016/j.actbio.2013.09.011.

22

FIGURE CAPTION Fig 1. a) Particle size distribution of starting powders (BHAp and BG); b) schematic representation of percentage of particles between 5 – 15 um (shaded area) and particle size with the maximum frequency (Mode = 7.2 m in this case). Both selected as milling process

SC RI PT

responses and analyzed in contour plots for c) particles percentage between 5 – 15 m and d) mode of particle size, by varying the mass weight (g), mass to balls ratio (g) and time

EP

TE

D

M

A

N

U

(min).

Fig 2. a) X-ray diffraction patterns and b) microstructure for mixtures with 2.5 and 30 % BG

A

CC

content both sintered at 1160 ºC and 1220 ºC.

23

SC RI PT U N

A

Fig 3. Contour plots of properties after sintering for a) grain size, b) porosity and c)

A

CC

EP

TE

D

M

compression strength of mixtures.

24

Fig 4. X-ray diffraction patterns (31º – 34.2º) and their correspondent Rietveld refinements showing the phase transformations for mixtures with BHAp/BG ratios of a,h) 100/0 b,i)

M

A

N

U

SC RI PT

96.25/3.75 c,j) 88.75/11.25 d,k) 85/15 e,l) 77.75/22.25 f,m) 70/30.

D

Fig 5. Typical SEM micrographs (2500x) of BHAp/BG mixtures in a) 100/0 b) 96.25/3.75

A

CC

EP

TE

c) 88.75/11.25 d) 85/15 e) 77.75/22.25 f) 70/30 ratios.

25

SC RI PT U N

A

Fig 6. Micrographs at 5000x of mixtures with a single crystalline phase and their respective

M

Raman spectra a,d) HAp (100/0) b,e) Silicocarnotite (85/15) and c,f) Nagelschmidtite

A

CC

EP

TE

D

(70/30).

26

Fig 7. Micrographs at 5000x of mixtures with phase coexistence and their respective Raman

M

A

N

U

SC RI PT

spectra at different areas a,d) 96.25/3.75 b,e) 88.75/11.25 and c,f) 77.5/22.5.

Fig 8. Overlaid contour plots evidencing the feasible simultaneous region (white zone) to

D

maximize responses simultaneously for a) milling, b) sintering and the c) particle size

TE

distribution of BHAp and BHAp/BG mixtures, successfully milled under the stablished

A

CC

EP

optimized conditions from the milling process.

27

SC RI PT U N

A

Fig 9. Quasi-ternary P2O5-CaO-(SiO2+Na2O) phase diagram considering the theoretical

M

oxide contents in wt.% from stoichiometric BHAp/BG mixtures. Literature experimental

A

CC

EP

TE

D

points are also shown for comparative purposes.

28

TABLE CAPTION Table 1. Chemical composition of initial mixtures according to the theoretical values, calculated from Hydroxyapatite and bioactive glass Vitryxx®

Theoretical value (wt. %) Na

Hydroxyapatite (Ca10(PO4)6(OH)2)

Ca

Mg

39.895

Vitryxx®

18.175

18.499

17.51

2.618

39.895 39.173

92.5/7.5

1.178

38.444

88.75/11.25

1.776

37.707

85/15

2.381

36.962

81.25/18.75

2.994

77.7/22.5

3.613

73.75/26.25

40.661 41.407

0.678

41.383

17.470

1.363

41.359

16.947

2.056

41.334

16.419

2.756

41.309

15.884

3.464

41.284

35.445

15.343

4.181

41.259

4.263

34.645

14.775

4.933

41.232

33.895

14.243

5.638

41.207

A 36.208

M

70/30

21.034

17.988

U

0.586

O

41.407

18.499

N

96.25/3.75

Si

D

100/0

P

SC RI PT

Sample

4.872

TE

Table 2. Microstructural and mechanical properties of the samples fabricated for the DoE of

EP

the sintering process

A

CC

Sintering temperature (°C)

BHAp/BG content

Grain size (µm)

Porosity (%)

Compressive Strenght (MPa)

97.5/2.5

0.976 ± 0.066

12.017 ± 0.202

31.723

70/30

2.498 ± 0.401

10.244 ± 0.980

32.144

97.5/2.5

1.301 ± 0.166

3.76 ± 0.858

35.703

1160

1220

29

70/30

4.155 ± 0.967

1.652 ± 0.324

42.443

Table 3. Phase contents and goodness coefficients determined by Rietveld refinements of XRD patterns for each sample

0

1.56

0.048

32.742

1.44

0.054

1.83

0.047

30.845

2.20

0.026

100

0

1.97

0.057

52.472

47.528

1.91

0.039

0

35.248

64.751

1.84

0.037

0

0

100

1.81

0.059

0

0

100

1.89

0.052

67.258

0

92.5/7.5

36.654

6.799

88.75/11.25

19.757

49.398

85/15

0

81.25/18.75

0

M D

TE

EP

CC

70/30

56.546

N

96.25/3.75

A

0

U

F

100

73.75/26.25

2

χ

100/0

77.7/22.5

SC RI PT

% % % BHAp/BG Hydroxyapatite Silicocarnotite Nagelschmidtite content [36] [37] [15]

A

Table 4. Lattice parameters and strains from Rietveld Refinement analysis

Phase

Theoretical value (Å)

Experimental value (Å)

Lattice parameters strain (%)

30

a HAp Ca10(PO4)6(OH)

c

a

b

∆a/a ∆b/b

c

∆c/c -

9.432 9.432 6.881 9.420 9.420 6.888 0.128 0.128 0.10 4

SC RI PT

2

b

SC 15.50 10.13 15.40 10.10 6.737 6.705 0.471 0.687 0.259 ( ) 8 2 1 6 Ca5 PO4 2SiO4

U

Nagel Ca5Na2(PO4)4 10.63 10.63 21.64 10.67 10.67 21.63 0.40 0.40 0.059 6 6 2 9 9 0 9 9

Table 5. Theoretical values of stoichiometric phases and EDS measurements of sintered

A

N

ceramic with different BG contents

Theoretical values (wt. %)

Na

CC

EP

Na- rich Nagelschmidtite Ca5Na2P4O16

TE

Si- rich Nagelschmidtite Ca7Si2P2O16

A

7.342

Zone

P

Si

O

41.539

12.841

5.822

39.798

42.854

9.463

8.580

39.103

31.998

19.783

40.876

EDS Measurements (wt. %) 1.136 ± 0.138

37.057 ± 0.961

0.649 ± 0.049

17.971 ± 0.065

1

2.172

43.536

0.300

12.487

4.982

36.520

2

6.450

32.155

0.239

20.031

0.135

40.988

3

0.382

35.148

0.266

17.507

0.011

46.683

1

4.653

35.666

0.135

20.337

0.462

38.744

2

3.365

33.249

0.092

16.696

2.502

44.094

100/0

96.25/3.75

Mg

D

Silicocarnotite Ca5(PO4)2SiO4

Ca

M

Sample

43.030 ± 0.808

88.75/11.25

31

3.388 ± 0.058

36.217 ± 0.087

0.195 ± 0.023

17.412 ± 0.050

2.148 ± 0.020

40.608 ± 0.117

1

5.939

29.370

0.637

15.524

2.531

45.997

2

4.946

33.345

0.515

15.376

4.061

41.754

85/15 77.5/22.5

6.259 ± 33.689 ± 0,462 ± 15.111 ± 4.899 ± 39.521 ± 0.072 0.182 0.011 0.003 0.174 0.281 Table 6. Raman band identification for the single crystalline phase and phases coexistence

SC RI PT

70/30

samples

-1

Experimental values (cm ) Single phases Phases coexistance

-1

Reported values (cm )

U

Assignment Ca (PO4) OH Ca SiP O Ca Si P O BHAp - BHAp - SC - SC 10 6 2 5 2 12 7 2 2 16 BHAp SC Nagel Nagel Nagel (HAp) (SC ) (Nagel) (100/0) (85/15) (70/30) Nagel (96.25/3.75) (88.75/11.25) (77.5/22.5) [21] [42] [40] 950-965

950-965

950-965

431

431

431

431

1058-1084

1042–1077

1042– 1077

1042–1077

1042– 1077

---

590

590

590

590

---

---

---

---

856

866

853

853

853

587

589

587

587

587

638

615

615

615

---

---

---

---

450

---

---

1050

1075

𝛖�4 PO4

600

---

B type 2𝛖�1 CO3

1070

---

---

10191088

---

854 (S871)

857

---

---

---

587

---

---

---

642

---

---

---

3572

3-

4-

𝛖�1 SiO4

4-

𝛖�2 SiO4

4-

CC

𝛖�4 SiO4

---

-

3250-3650

3572

A

A

OH

TE

3-

𝛖�3 PO4

EP

3-

𝛖�2 PO4

N

963

D

960

M

962 962 (S958- 960 982)

964 (S945-977)

3𝛖�1 PO4

32