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Fabrication of Monticellite Based Bioactive Porous Scaff olds Obtained from Boron Derivative Waste by Porogen Leaching Technique

Levent Köroğlu, Betül Aydemir, Erhan Ayas

Anadolu University, Faculty of Engineering, Department of Materials Science and Engineering, Eskisehir, Turkey

Abstract

The aim of the study is the fabrication of monticellite based bioactive porous scaffolds from boron derivative waste by porogen leaching technique. The results showed that porous scaffolds were mainly composed of sodalite and monticellite phases, and possessed a pore distribution with pore diameters ranging from a few micrometers to 300 m. The increment of porogen content from 40 to 60 wt.% led to increase in open porosity value from 70.8 to 81.5%. Following to incubation in Lactated Ringer's solution for 28 days, the flake-like calcium carbonate layer was formed on the surface of porous scaffolds. Therefore, monticellite based porous scaffolds can have a potential for being use as bioactive bone graft substitutes and as drug delivery carriers in the field of tissue engineering and regenerative medicine. 1. Introduction

In the field of tissue engineering, the scaffolds have been key components for physical support in new tissue and implanted cells as substrate. Highly open porous scaffolds are required in order to cell growth and transferring of fluids and nutrients. Porogen leaching is one of the most common technique for the preparation of scaffolds with controlled pore structure. That is based upon the dispersion of porogen (salt, sugar, wax, etc.) which acts as temporary placeholder for pores [1-2]. Monticellite (CaMgSiO4) bioactive ceramics have received much attention at last decade as bone graft substitutes due to their high biocompatibility, high bioactivity and enhanced mechanical properties [3-4]. Turkey has 72% of boron reserves in the worldwide with total mass of 3.3 million tons. In K rka Plant of Eti Mine Works General Directorate, approximately 900,000 tons of boron derivative waste are generated throughout the 1 million tons of borax pentahydrate (Na2O·2B2O3·5H2O) production. The

emerged boron derivative waste in large quantities causes environmental pollutions and costly storage problems [5]. In vitro bioactivity and in vitro cytotoxicity of monticellite based ceramic powders obtained from boron derivative waste have been reported before [6-7]. However, it is the first study based on the fabrication of monticellite based bioactive porous scaffolds from boron derivative waste by porogen leaching technique. 2. Experimental Procedure 2.1. Preparation of monticellite based porous scaffolds

Boron derivative waste provided from Eti Mine Works Kirka Borax Plant and NaCl powder (99.5 %, Merck KGaA) were used as starting materials. Boron derivative waste with mean particle size of 0.823 m and NaCl powder with a particle size range of 100 m and 250 m was mixed. The mass ratio of NaCl powder as porogen varied from 40 wt.% to 60 wt.%. Following to uniaxial pressing of prepared powder mixtures, the obtained wafers with dimensions of 12 x 3 mm2 placed onto pure alumina plates. The heat treatment of wafers was carried out at 800°C for 4 hours in an electrically heated furnace (KRC Lab. Eq.) under atmospheric pressure. The heating rate was kept 10°C/min. Next, NaCl was leached out by the immersion of heat-treated wafers into de-ionized water at 100°C for 4 hours. 2.2. Characterization of boron derivative waste and monticellite based porous scaffolds

Monticellite based porous scaffolds (MBPSs) were crushed and ground to below 63 μm. Next, the qualitative phase analysis of boron derivative waste and scaffold was performed by X-Ray Diffractometer (XRD, MiniFlex, Rigaku) with a scan speed of 0.5°/min. The mass densityand open porosity values of fabricated monticellite based

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porous scaffolds were measured using the Archimedes’ Principle [8].

2.3. Assessment of in vitro bioactivity of monticellite based porous scaffolds

For the assessment of in vitro bioactivity, scaffolds were soaked in Lactated Ringer's solution (LRS) for 28 days at 36.5 ± 0.5°C. Lactated Ringer's solution was preferred instead of Simulated Body Fluid (SBF) because the requirement of high number of reagents, precise pH adjustment and long procedure time [9] make the preparation of SBF solutions painful. The ratio of surface area to volume of LRS was kept 0.1 cm2/mL. Scaffolds were taken out after selected incubation times, rinsed with deionized water and dried for 24 h. Microstructural analysis of MBPSs carried out using a scanning electron microscope (SEM, Supra 50VP, Zeiss) equipped with SE and EDS detectors. Scaffolds were coated with a thin layer of gold-palladium by sputter coater (Agar Scientific, UK) to prevent the accumulation of charge.

3. Results and Discussion 3.1. Phase evolution

The XRD patterns of monticellite based porous scaffolds compared to boron derivative waste are illustrated in Fig. 1. Boron derivative waste included dolomite (CaMg(CO3)2, ICDD 36-0426), calcite (CaCO3, ICDD 05-0586), quartz (SiO2, ICDD 87-2096), borax pentahydrate (tincalconite, Na2B4O7-5H2O, ICDD 07-0277) and kaolinite (Al2Si2O5(OH)4, ICDD 29-1488) crystalline phases. Major crystalline phases were dolomite and calcite. XRD patterns of monticellite based porous scaffolds were quite similar and all scaffolds were composed of monticellite (CaMgSiO4, ICDD 76-0727), akermanite (CaMgSiO7, ICDD 83-1815), calcium magnesium borate (CaMgB2O5, ICDD 73-0618), diopside (CaMgSiO2, ICDD 78-1390) and sodalite (Na4Al3Si3O12Cl, ICDD 37-0476). Major phases were identified as sodalite, monticellite and akermanite. There is no evidence of residual NaCl in fabricated scaffolds according to XRD patterns.

Figure 1. XRD patterns of monticellite based porous scaffolds compared to boron derivative waste.

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3.2. Open porosity The selection of heat-treatment temperature as 800°C was related with the melting point of NaCl (803°C) [10]. The formation of crystalline phases up to 800°C provided the mechanical stability of the scaffold and liquid porogen was partially removed during dwell time. Then, the salt leaching led to removal of residual NaCl. Finally, all water sites replaced by room atmosphere during the drying stage. Considering the porous structure as a composite material is beneficial to understand the mechanism which was built from matrix composed of mainly sodalite and monticellite and porosity as secondary phase formed by porogen removal. The mass density (bulk density) and open porosity (apparent porosity) values with respect to NaCl content are given in Fig. 2. The increasing of NaCl content from 40 to 60 wt.% enhanced the open porosity value (from 70.8 to 81.5 %) and decreased the mass density (from 0.86 to 0.54 g/cm³). The porogen content as 50 wt.% was thought to be the threshold value for pore percolation where the higher number of NaCl particles are isolated and closed-to-open pore transition begins theoretically.

Figure 2. Mass density and open porosity values with respect to NaCl content.

3.3. Microstructural analysis SEM images of all MBPSs soaked in LRS for 28 days are illustrated in Fig. 4. It can be seen from the images at low magnification (100x) that the increment of porogen content from 40 to 50 wt.% caused an enhancement in pore size (Fig. 3. (a, c)). Not only that went further but also scaffold started to be degraded in LRS by 28 days when the porogen content was raised up to 60 wt.% (Fig. 3. (e)). Porous structures consisted of a pore distribution with pore

diameters ranging from a few micrometers to 300 m. Furthermore, the formation of interconnected macropores were observed. The maximum pore size was 150 m for MBPS-40%NaCl whereas that was found approximately 300 m for MBPS-50%NaCl respectively owing to the agglomeration of NaCl particles. MBPS-60%NaCl possessed also macropores; however, it is not appropriate to specify maximum pore size because the degradation gave rise to large cavities. The formation of flake-like layer on the wafer surfaces was obvious in SEM images (Fig. 3. (b, d, f)). The whole surface of wafers was covered with dense crystallites less than 1 m in length. The formation of Ca and P -based layer was expected referring bone-flake like apatite layer. However, EDS analysis revealed that calcium carbonate (CaCO3) crystals precipitated on the monticellite and sodalite particles. It is reported that the precipitation of polymorphs of CaCO3

could carried out in phosphorus-free simulated body solutions used as biomineralisation media [9]. The researchers investigated the potential of CaCO3

microparticles as templates for drug delivery applications by in vitro and in vivo experiments. The hydrophilic drugs, bioactive proteins and DNA were adsorbed on the surface of CaCO3 microparticles with the aim of solving the problems related to chemotherapy and radiotherapy [11]. For this reason, depending on the scaffold structure controlled by fabrication techniques and the characteristics of precipitated CaCO3 particles tailored during the biological synthesis process, monticellite based bioactive porous scaffolds can have a potential for being use as bioactive bone graft substitutes and as drug delivery carriers in the field of tissue engineering and regenerative medicine. 4. Conclusion

In the present study, monticellite based bioactive porous scaffolds were fabricated from boron derivative waste by porogen leaching technique. The effect of porogen content on phase evolution, open porosity content and microstructure of porous scaffolds and also, the effect of Lactated Ringer's solution on their bioactive characteristics were investigated. The fabricated scaffolds can have a usage potential as bioactive bone graft substitutes and as drug delivery carriers; however, further comprehensive studies are necessary.

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Figure 3. SEM images (SE mode) of (a, b) MBPS-40%NaCl; (c, d) MBPS-50%NaCl and (e, f) MBPS 60%NaCl soaked in LRS for 28 days at equal magnifications (100x and 10.000x).

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[6] L. Koroglu, E. Butev, Z. Esen and E. Ayas, Materials Letters, 209 (2017) 315-318. [7] B. Barutça, L. Köro lu, E. Ayas, A.T. Koparal, Ceramics International, 44 (2018) 8094-8099. [8] Y. Zou and J. Malzbender, Ceramics International, 42 (2016) 2861-2870. [9] A.C. Tas, Acta Biomaterialia, 10 (2014) 1771-1792. [10] J.B. Ferguson, J. Phys. Chem., 26:7 (1922) 626-630. [11] L.M. Manzine Costa, G.M. de Olyveira and R. Salomao, Adv Tissue Eng Regen Med, 3:2 (2017) 336-340.