TCP ratio in biphasic calcium phosphate

Available online at www.sciencedirect.com

ScienceDirect Materials Today: Proceedings 16 (2019) 1796–1803

www.materialstoday.com/proceedings

Bio-CAM 2017

The influence of silicon addition in modulation of HA/TCP ratio in biphasic calcium phosphate Nor Shahida Kader Bashaha,b, Ahmad Fauzi Mohd Noorb* a

SIRIM Industrial Research, SIRIM Berhad, Lot 34, Jalan Hi-Tech 2/3, Kulim Hi-Tech Park, 09000 Kulim, Kedah Darul Aman, Malaysia b School of Mineral and Materials Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal, Pulau Pinang, Malaysia

Abstract Biphasic calcium phosphate (BCP) granules were prepared using calcium hydroxide (Ca(OH)2) and orthophosphoric acid (H3PO4) as chemical precursors at Ca/P ratio 1.60 via wet chemical technique. Apart from that, silica (SiO2) was also added in the calcium phosphate material as it enhance surface chemical structure, mechanical structure and bioactivity. In this study, 3 wt% silica was added during synthesis to form slurry, before proceeding with spray drying. Granules obtained after the spray drying were calcined at different temperatures (900 – 1300 oC) to study on modulation of HA and TCP phase in BCP. Studies were also conducted on samples’ morphology resulted from spray drying and calcination process. Bioactivity study was also conducted in simulated body fluid (SBF) as a preliminary observation of sample behaviour in-vivo. Addition of silica was found to favour TCP formation and also resulted in faster dissolution process in-vitro. © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of Biomedical and Advanced Materials (Biocam 2017). Keywords: biphasic calcium phosphate; silica; spray drying

1. Introduction Biphasic calcium phosphates (BCPs) have attracted considerable attention as bone graft substitute in dental and orthopaedic reconstructive medicine [1-3]. Generally, BCPs are bioceramic which consist of biocompatible hydroxyapatite (HA) and biodegradable β-tricalcium phosphate (β-TCP). HA was found to be more stable in the body environment while TCP was more soluble. Thus, with the combination of a balance rate between a more stable phase and more soluble phase, it is possible to formulate better bio-resorbability and osseointegration material. Efforts to improve the biological response of BCPs have recently led to studies of various chemical compositions, * Corresponding author. Tel.: +604-4017100 E-mail address: [email protected], [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of Biomedical and Advanced Materials (Biocam 2017).

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

1797

and microstructures with control of the crystal phase. The Ca/P ratio of BCP plays important roles in determining its bioactivity and performance in vitro. The resorption kinetics of BCP can be controlled by varying the CA/P molar ratio between 1.5 (β-TCP) and 1.67 (HA), results in the formation of calcium-deficient apatite, CDA. The molar composition of resulting biphasic powder mixture depends on the degree of Ca-deficiency, x, upon calcination according to following equation (1):

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

3xCa3(PO4)2 + (1-x)Ca10(PO4)6x(OH)2+ xH2O

(1)

For x = 0, single phase HA would formed, while single phase β-TCP is obtained for x = 1. Equal molar of both phases requires x = 0.25. The resulted final product is typically affected by the physical properties of powders such as particle morphology, specific surface area, mean particle size and particle size distribution. Besides that, the calcination temperature also effects the BCP powders in terms of powder characteristics. For this study, Ca/P ratio of the chemical precursors was set to 1.60 as previously used in by Kim et al. [4] in order to obtain 60:40 of HA: βTCP after calcination process. HA: β-TCP ratio of 60:40 was reported to be optimum combination of HA and β-TCP in terms of bioresorption and dissolution of the material in-vivo [5]. Additionally, several literatures [4-8] reported on silica-based bioactive glasses which have superior bioactivity than calcium phosphate. Therefore, this material is used to be incorporated into calcium phosphate material in order to increase its bioactivity performance. 2. Experimental 2.1. Materials Chemical precursors for synthesis of calcium phosphates were calcium hydroxide (Sigma-Aldrich, Germany) and phosphoric acid (Avantor Material Performance, Thailand). Colloidal SiO2 (Provier Pharma, India) was used as a Si source to be added into calcium phosphate slurry. All chemicals were analytical grade used as received. Double distilled deionized water (DDI H2O) was used throughout the experiment. 2.2. Synthesis of calcium phosphate slurry Calcium phosphate slurry was prepared via wet synthesis technique. The mixture precursors were based on 1.60 Ca/P ratio. Phosphoric acid was added gradually into calcium hydroxide solution under stirring at 300 rotation per minute (rpm) using mechanical stirrer. The temperature during the reaction of the mixture was controlled at 80oC. pH of the suspension was measured before and after complete addition of acid. 3wt% of silicon dioxide (SiO2) was added into apatite slurry at 3 wt% after complete acid addition and continue with stirring process. The white gelatinous apatite slurry was obtained at the end of the process. 2.3. Fabrication of granules using spray dryer This gelatinous apatite slurry was then spray dried to obtain apatite granules. Fig. 1 illustrates the spray drying process. Prior to spraying, the spray dryer inlet temperature was set to 280 °C and an outlet temperature was less than 120 °C. The compressed air was turned on to 0.05 MPa to 0.3 MPa (0.5-3 bar) and 50-70% air- on the flow meter. This was followed by turning on the feed pump (Brand Watson Marlow 505s) in the range of 20-45 rpm. To spray dry, DDI water was slowly fed first into the atomizer until the required outlet temperature was stable at 95 °C. Then the feed pump was switched on from distilled water to the gelatinous apatite slurry. The air pressure was kept constant in - order to obtain homogenous atomization. Once the spray drying process was completed, the spray dried apatite powder was collected from the glass-jar collector at the bottom of the spray dryer.

1798

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

Fig. 1: Schematic diagram of spray drying process (Alenjandro et al., 2015)

2.4 Calcination process Granules derived from the spray drying process were calcined at different temperature ranging from 900 to 1300 C. This process was conducted to obtain biphasic calcium phophate phase granules. The sample was placed in alumina crucible and calcined in atmospheric furnace for 1 hour with 5 oC/min of heating rate.

o

2.5 Bioactivity study in SBF The evaluation of bioactivity was carried out in Kokubo’s simulated body fluid (SBF) [9]. The observation was conducted on samples’ surface morphology prior to immersion process. The samples were immersed at predetermined times (3, 7, 14, 21 and 28 days) into the solution which was kept at 37 oC in the incubator. After the samples were taken out from the solution and dried out at room temperature, pH of the solutions was measured. 2.5. Characterization The phase analysis of silica-BCP and BCP were carried out using AXS Bruker D8 Advanced x-ray diffractometer having Cu Kα radiation at 40kV and 40mA at room temperature. The data were collected at 2ϴ range 20-50o at a step size of 0.5o/s. The relative intensity ratio (RIR) corresponding to the major phases observed in the XRD spectra; HA (2 1 1) and β-TCP (0 2 11) were computed using the relationship given in equation (2) [10]:

(2) Ʃ The equation related to HA and β-TCP % ratio based on their major peaks observed in XRD spectra. Characterization of surface area of the samples was determined using Brunauer–Emmett–Teller (BET- Autosorb, Quantachrome Instruments) for both BCP samples after calcination process. The investigation of their microstructural and morphological changes prior to calcination and SBF immersion was conducted by a scanning electron microscope (SEM) LEO 1500 series operated by a typical accelerating voltage of 20 kV. Prior to SEM observation, all samples were sputter-coated with carbon to minimize surface charging effects. Investigation on

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

1799

apatite formation was carried out on both BCP samples (0 wt% and 3 wt% silica) calcined at 1000 oC at different immersion durations. 3. Results and discussion 3.1 X-ray diffraction (XRD) Fig. 2 (a) and (b) shows the XRD pattern of BCP and Si-BCP after calcination at different temperatures. The calcination temperature plays important role in the formation of biphasic phases. The ratio percentage computed for HA and β-TCP are based on formulation (2) presented in Fig. 2 (a) and (b) respectively. (a)

20

30 2-theta scale

40

50

20

30

40

50

2-theta scale

Fig 2. XRD diffraction pattern of (a) BCP without silica addition and (b) BCP with 3wt% silica addition calcined at various temperatures.

From the results, both samples were found to exhibit formation of both HA and β-TCP phases at all calcination temperature. However, sample with silica displayed slightly amorphous phase compared to the sample without silica addition. This is observed on the XRD peaks which are slightly broader, compared to much sharper peaks of the other sample. Amorphous phase may result in faster dissolution rate in-vitro [11]. Beside the phase formation, the calcination process of calcium phosphate materials effected the specific surface area, crystallinity and sample morphology. HA

(b)

β-TCP

100

Percentage (%)

Percentage (%)

(a)

50 0 900

1000

1100

1200

Calcination temperature

(oC)

1300

HA

β-TCP

100 50 0 900 1000 1100 1200 1300 Calcination temperature (oC)

Fig 3. The percentage of HA and β-TCP calculated for (a) BCP without silica addition and (b) BCP with 3 wt% silica addition prior to calcination process

1800

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

The percentage of HA and β-TCP plotted is shown in Fig. 3 calculated using Equation (2). Different percentage of HA and β-TCP was observed at different calcination temperature The results show silica addition was found to favour β-TCP formation as compared to the sample without silica addition as the temperature increased. The formation of more soluble phase (β-TCP) may also affect the sample bioactivity performance. The efficacy of BCP was based on the preferential dissolution of the β-TCP compared to HA, allowing the manipulation of bioactivity or biodegradation by manipulating the HA/ β-TCP ratio. The resorbability (in-vivo dissolution) of BCP depends on the HA/ β-TCP ratios, the higher the ratio the greater the reactivity [1]. 3.2 Specific surface area (BET) Specific surface area (BET) for BCP samples after the calcination at different temperatures are shown in Fig. 4. Generally, increasing of calcination temperature resulted in decreasing of surface area displayed by both samples. However, surface area for sample with silica addition significant decrease as compared to sample without silica. Calcination at high temperature resulted in larger crystal size. As previously reported in the literature, Si substitution would affect the material in terms of grain size, protein conformity and surface topography. The morphological changes could be related to the reduction in grain size and presence of Si in Sisubstituted HA is known to have higher specific surface area as studied by Manchon et al.[12].

25

Specific surface area (m2/g)

0 wt% Si

3wt% Si

20

15

10

5

0 800

900

1000

1100

Calcination temperature

1200

1300

(oC)

Fig.4: Specific surface area of BCP samples calcined at different temperatures

3.3 SEM image Fig. 5 shows BCP samples (0 and 3 wt% silica) before and after calcination at 1000 oC. Granules were obtained from spray drying process. Observation was carried out at the granules’ surface. From the SEM images, the grain size of BCP granules with 3 wt% silica after calcination was smaller in size ranging from 20-80 μm with more porous surface compared to BCP without silica where the granule size ranging from 30-150 μm with the less porous surface. The pores of 3 wt% silica BCP sample found to be prominent and thus affect its surface area. Presence of silicon was found to result in morphological changes of BCP which caused reduction in grain size [12].

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

(a)

100μm

(c)

100μm

1801

(b)

0.5μm

(d)

(b1) 0.5μm

Fig. 5: SEM images of BCP granules and their surface after calcination (a) 0 wt% silica at 100x magnification and (b) 0 wt% silica at 25kx magnification and (c) 3 wt% silica at 100x magnification and (d) 3 wt% silica at 25kx magnification

3.4 Bioactivity study (SBF immersion) SEM images of sample immersed in SBF are shown in Fig.6. The sample was previously calcined at 1000oC and consisted both HA and β-TCP phase.

(a)

0.5μm

(c)

(b)

Apatite formation

0.5μm

(d)

Macropores

Micropores

0.5μm

0.5μm

Fig. 6: SEM images of BCP granule surface without silica after immersion in SBF at (a) 7 days and (b) 21 days and BCP sample with 3wt% silica at (c) 7 days and (d) 21 days respectively.

1802

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

Calcined at 1000 oC, the percentage of HA: β-TCP of sample without silica addition was calculated to be 58:42, while for BCP sample with 3 wt% silica the percentage was 61:39. Thus, the sample with silica addition would have more soluble phase as compared to sample without silica addition The immersion duration were carried out at 7 and 21 days respectively. The characterization conducted in order to preliminary predict the samples, behavior in-vivo. From the result, apatite formation started to form at day 7 for sample with 3 wt% silica, while no apatite formation observed for sample without silica at the same immersion duration. However, at day 21 apatite formation observed on both samples, indicating that both samples exhibited bioactivity. Besides that, macro and microporosities were also observed on the surface of both samples. This indicated that Si changes grain size, topography, chemical composition, crystallinity and surface charges that enhances the formation of apatite on the material surface [15]. Apart from SEM observation, the pH of the SBF solution was measured during the immersion period. pH changes during immersion period were plotted as shown in Fig. 7.The changes of pH was affected by the release of the high concentration of Ca2+ to the microenvironment which may promote a mild inflammatory response and the ability to favor fibrous tissue formation [3].

7.9 7.8

PH

7.7 7.6 7.5 7.4

0% silica 3% silica

7.3 7.2 0d

3d

7d 14 d Immersion duration (day)

21 d

30 d

Fig. 7: pH changes of SBF solutions measured in stipulated time frame.

From the result, the pH of the solutions was found to increase steadily over time. However, the solution contained BCP sample with 3 wt% silica displayed higher pH value compared to BCP sample without silica addition. Both samples exhibited the highest pH value at day 21 and started to decrease to the initial pH. Properties of BCP sample with 3 wt% silica with the higher surface area, slightly amorphous structure and greater HA: -TCP ratio may have contributed to the higher dissolution rate which caused the higher concentration of Ca2+ into the solution. Ca2+ and H+ ion exchanged upon dissolution process may result in accumulation of OH- on the sample surface which also promote higher pH value [12]. 4. Conclusion BCP granules were successfully produced using the wet synthesis technique with the presence of biphasic phase confirmed by XRD results. The ratio of HA:β-TCP was found to modulate at different calcination temperature. Modulation of HA: β-TCP ratio of BCP with silica addition was found to favour β-TCP (more soluble phase) formation as calcination temperature increased compared to BCP without silica addition which favour HA formation. Silica addition to BCP material was found to alter the structure of the sample by decreasing the crystallinity and also increased the surface area. These two factors plus HA: β-TCP ratio contributed to higher dissolution of silica-BCP sample and faster formation of an apatite layer in-vitro.

Nor Shahida Kader Bashah and Ahmad Fauzi Mohd Noor / Materials Today: Proceedings 16 (2019) 1796–1803

1803

Acknowledgement The authors wish to acknowledge full gratitude to the Ministry of Science and Technology (MOSTI) for funding this work (Grant no: 03-03-02-SF0261) References [1] G. Daculsi, O. Laboux, O. Malard, P. Weiss, Journal of Material Science: Material in medicine 14 (2003) 195-200. [2] M. Bohner, Biomaterials 30 (2009) 6403-6406. [3] S. E. Lobo and T. L., Materials 3 (2010) 815-826 [4] Dong-Hyun Kim, Ho Hwan Chun, Ju Dong Lee, Seog Young Yoon, Ceramic International 40(4) (2013) 5145-5155. [5] E.S. Thian, J. Huang, S.M. Best, Z.H. Barber, W. Bonfield, Materials Science and Engineering C 27 (2007) 251-256. [6] N. Shadjou and M. Hasanzadeh, Materials Science and Engineering C 55 (2015) 401-409. [7] T. Tian, D. Jiang, J. Zhang, Q. Lin, Materials science and Engineering C 28 (2008) 57-63. [8] S. Ibrahim, S. Sabudin, S. Sahid, M.A. Marzuke, Z.H. Husin, N.S.K. Bashah, K. Jamuna Thevi, Saudi Journal of Biological Sciences 23(1) (2016) S56-S63. [9] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi and T. Yamamuro, J. Biomed. Mater. Res.24 (1990) 721-734. [10] K.P. Deepak, D. rajalaxmi, R.C. Prasad, B.T. Rao, T.R. Rama Mohan, Materials Science and Engineering C 27 (4) 2007, 684-690. [11] C. Combes and C. Rey, Acta Biomater. 6(9) (2010) 3362-3378. [12] ] A. Manchon, M. Alkhraisat, C. Rueda-Rodriguez, J. Torres, J.C. Prados-Frutos, A. Ewald, U. Gbureck, J. Cabrejos-Azama, A. Rodriguez González, E. López-Cabarcos, J Biomed Mater Res A. 103(2) (2015) 479-488. [13] L.J. Fuh, Y.J. Huang, W.C. Chen, D.J. Lin, Mater Sci Eng C Mater Biol Appl. 75 (2017) 798-806 [14] I.R. Gibson, S.M. Best, W. Bonfield, Journal of American Ceramic Society 85 (2002) 2271-2227 [16] G. Tomoaia, A. Mocanu, I. Vida-Simiti, N. Jumate, L.D. Bobos, O. Soritau, M. Tomoaia-Cotisel, Mater Sci Eng C Mater Biol Appl. 1 (37) (2014) 37-47. [17] A. Sosnik and K. Seremeta, Advances in Colloid and Interface Science 223 (2015) 40-54. [18] E. Caroline Victoria and F.D. Ghanam, Trends Biomater. Artif. Organs. 16(1) (2002) 12-14. [19] M. Tamai, M. Nakamura, T. Isshiki, K. Nishio, H. Endoh, A. Nakahira, J. Mater. Sci. Mater. Med. 14 (2003) 617-622. [20] F. Xin, C. Jian, J. Zou, Transactions of Nonferrous Metals Society of China 19 (2009) 347-352. [21] M.A. Jyoti, V.V. Thai, Y.K. Min, B.T. Lee, H.Y. Song, Applied Surface Science. 257 (2010) 1533-1539. [22] E. Champion, Acta Biomaterialia 9 (2013) 5855-5875. [23] G. Daculsi, Biomaterials 19 (1998) 1473-1478. [24] J. Marchi, International Journal of Applied Ceramic Technology 6(1) (2009) 60-71. [25] J.-M. Bouler, R.Z. LeGeros, G. Daculsi, Journal of Biomedical Materials Research 51(4) (2000) 680-684. [26] M. Aslanidou, V. Tiverios, A. Mitsionis, C. Trapalis, Ceramics International 39 (2013) 539-546. [27] M.-C. Wang, W.-J. Shih, I.-M. Hung, H.-T. Chen, M.-H. Hon, H.-H. Huang, Ceramics International 41 (2015) 1223-1233. [28] N. Koga, K. Ishikawa, K. Tsuru, I. Takahashi, Ceramics International 42 (2016) 7912-7917. [29] G.M.L. Dalmônico, D.F. Silvaa, P.F. Franczaka, N.H.A. Camargoa, M.A. Rodríguez, Bol. Soc. Esp. Ceram. Vidr. 54(1) (2015) 37–43. [30] M. Ebrahimi and M. Botelho, Data in Brief 10 (2017) 93–97.