In vitro cell responses of MG-63 osteoblast cells on bioactive diopside and wollastonite nano-bioceramics for biomedical applications

The present study aimed to synthesize and characterize diopside (CaMgSi 2 O 6 ) and wollastonite (CaSiO 3 ) nano-bioceramics via a combination of mechano-chemical and calcination processes. In vitro biomineralization and cell responses of wollastonite and diopside were performed on simulated body fluid (SBF) and MG-63 osteoblast cells. Results revealed proper tissue biomineralization of wollastonite and diopside through the generation of an apatite-like layer on the surface of nano-bioceramics. Cell responses of wollastonite and diopside eventuated non-cytotoxicity by MG-63 osteoblast cells, and their viability and cell proliferation were confirmed. Alizarin red staining of diopside and wollastonite evidenced great bioactivity and tissue biomineralization, and the ALP enzyme of diopside and wollastonite was enhanced in contact with the MG-63 osteoblast cells. Regarding the existence of Mg 2+ in the calcium-silicate network and the stability network, diopside illustrated high biological and cell responses in comparison to wollastonite, and both of them were suggested as bioactive and biocompatible nano-bioceramics for biomedical applications.


Introduction
Restoration of bone defects is a significant challenge in medical and biomedical sciences, and finding alternative materials for bone tissues is vital. Calcium silicates (CaSiO 3 and Ca 2 SiO 4 ) and calcium silicate-based ceramics have become the principal focus in biomaterials [1,2]. These materials have biological properties such as bioactivity and enhancement of cell interactions compared to traditional calcium phosphates like hydroxyapatite (HAp) or tricalcium phosphate [3]. Therefore, they are promising candidates as bone graft materials.
Moreover, incorporating metal atoms (Mg, Zn and Zr) into the crystalline structure of calcium silicates has improved their biological revenue [4][5][6][7]. In fact, silicate biomaterials including wollastonite and Ca -Si -M ternary ceramics (M = Ti, Mg, Zn and Zr) are the topic of many research projects for bone tissue restoration usages. A significant feature of silicate biomaterials is their ability to release the silicon (Si) ion which persuades the growth and distinction of osteoblast cells to some extent [8][9][10]. Additionally, a precise study of CaO -SiO 2 ceramics showed their direct connection with bone. This study showed that ceramics including CaO and SiO 2 have suitable bioactivity and connection power to bone [11].
wollastonite. Moreover, in the chemical composition of diopside, calcium atom is replaced by magnesium atom and the Mg -O bond is developed.
Since the Mg -O bonding energy is higher than that of the Ca -O bonding, the stability of the crystalline structure is increased [14]. On the other hand, the CaO -SiO 2 -MgO ceramic systems are appropriate for bone regeneration applications [15].
Duchin et al. found that the mechanism of bioactivity of CaO -SiO 2 -MgO system ceramics is similar to silicate glass, which is related to the direct release of the Si ions [16]. One of the significant bioceramic compounds is wollastonite (CaSiO 3 ), which has unique characteristics such as thermal stability and high hardness. Hence, it has acquired abundant applications in ceramic industry [17]. In addition, wollastonite has high bioactivity so that apatite layers appear on it and grow fast only after 3 days. Besides, the growth rate of the hydroxyapatite in nanostructured wollastonite was more than its growth rate in micro-structured wollastonite [18].
One of the most interesting synthesis processes of nano-structured bio-ceramics is mechano-chemical process. The particle sizes of nano-structured materials are less than 100 nm [19]. Due to their ideal physical and chemical properties, they attract much attention of scientific and research communities [20].
In this study, in vitro cell responses of MG-63 osteoblast cells and biomineralization on synthesized diopside (CaMgSi 2 O 6 ) and wollastonite (CaSiO 3 ) nano-bioceramics via a combination of mechano-chemical and calcination processes were studied for biomedical applications.

Synthesis of diopside and wollastonite
To synthesize diopside, 15.38%wt of MgO, 46.15%wt of SiO 2 , and 38.46%wt of CaCO 3 were chosen, and then mechano-chemical process (ball milling) was carried out. In this process, balltopowder weight ratio and milling speed were 10:1 and 350 rpm, respectively, and the milling times were 5 minutes, 10 and 20 h.
Similarly, the process was conducted to synthesize wollastonite, and the milling times were 5 min, 10 and 20 h. The raw materials included 37.5%wt of SiO 2, 62.5%wt of CaCO 3 , and the molar ratio was 1.125. The milling speed and ballto powder weight ratio were 350 rpm and 10:1, respectively. After this process, the milled powders were calcinated at 1200 ℃ for 2 h [21].

Characterization of diopside and wollastonite
To evaluate the phase structure of the milled and calcinated powders, XRD patterns of these samples were prepared using a PW3040 Philips X-ray diffractometer. The XRD patterns of the samples were obtained via Cu-k radiation with a wavelength = 1.54018 ° in the ranges of 15 -70, 10 -60 and 10 -50 degrees. To determine the crystallites size of the developed compounds, the width of the present peaks was used in the XRD patterns and Scherrer method.
The Scherrer equation is [22]: Where D is the crystallite size, λ is the wavelength, K is the shape factor (about 1), β is the full width at half maximum (FWHM), and is half the diffraction angle.
To investigate the morphology and micro-structure of the synthesized diopside and wollastonite, SEM images were prepared using scanning electron microscopy (FEI, Quanta, USA). To examine the chemical composition and distribution of elements of the synthesized diopside and wollastonite compounds, EDS and X-ray map analyses were prepared. To examine the micro-structure and nano-structure of the synthesized diopside and wollastonite, TEM images of the samples were prepared using transmission electron microscopy (EM 208, Philips, the Netherlands).

Biomineralization and cell responses
The evaluation of biomineralization and formation of an apatite-like layer on the diopside and wollastonite nano-bioceramics surface was conducted via SBF solution provided according to the Kokubo method [23]. The nano-bioceramic samples were immersed in 25 ml of the SBF solution (pH=7.4) and put into an incubator at 37 °C for 28 days. After the immersion of the scaffolds for this period, they were washed with distilled water and dried in an oven at 50 °C for 48 h.
The cytotoxicity evaluation was conducted according to ISO 10993-5 standard.
The sterilization of the wollastonite and diopside particles was performed in an autoclave. Subsequently, the particles were immersed in Dulbecco's modified eagle medium (DMEM) culture medium using ultrasonic irradiation for 30 min. the samples, and the same process was repeated. After 1, 2 and 3 min, optical absorption was read over 405 nm, and then its difference was determined from the last minute. Finally, these three differences and also their mean were calculated, and the final number was multiplied by factor 3433.

Statistical analysis
The experiments were performed for n=3. All data were suggested as mean ± SD.
One-way analysis of variance (ANOVA) was used to compare the results. A pvalue of less than 0.05 was determined statistically significant. Finally, these changes increase the speed of the reactions between the milled particles. Transformation of the milled powder structure is so high that the primary lattice is perfectly altered. During the milling process, various phenomena like mixing and chemical reaction in contact with particle levels and so on might occur, causing chemical reactions to take place during the milling process and reactivity of the milled product to increase [24].

Results and discussion
The main peaks of SiO 2 phase are not identifiable in the ranges of diffracted angles of 20 -30 degrees in the XRD patterns (Figs. 2(a-c)). As a result of the milling process for 20 h (Fig. 2(c)), the pattern lacked the diopside phase (CaMgSi 2 O 6 ).
Subsequently, to develop the diopside phase, the milled powders were calcinated at 1200 ℃ for 2 h. phase (JCPDS 01 -077 -1149) with a tetragonal structure was observed in the XRD pattern of this sample (Fig. 2(d)).
In the calcinated sample of the milled powder at 10 h (Fig. 2(e)), the peaks related to the diopside phase increased, and those related to the akermanite phase decreased (Fig. 2(e)). In the calcinated sample of the milled powder at 20 h (Fig 2. (f)), the pattern lacked the akermanite phase, and only the diopside phase was clearly observed because of appropriate milling time (20 h) among the raw materials. Formation of the diopside phase is according to relations 2 and 3: To obtain the optimum milling time, the milling process was conducted among raw materials like CaCO 3 and SiO 2 for 5 min and 10 and 20 h. Figs. 3(a-c) present the XRD patterns related to the milled powders at 5 min, 10 and 20 h. In the XRD pattern in Fig. 3(a), only the phases related to the raw materials like CaCO 3 exist, and by increasing the milling time to 10 h, the peaks widen ( Fig. 3(b)) because of decreasing of the crystallite size and increasing of the lattice strain during the milling process [25]. In this pattern ( Fig. 3(b)), only the phase related to the raw materials compound was observed. By increasing the milling time to 20 h ( were observed because of extending the milling time to 20 hamong the raw materials. Formation of the wollastonite phase is according to relations 2 and 4.

CaO + SiO 2→ CaSiO 3 (4)
To calculate the crystallite size of the synthesized diopside and wollastonite, the Scherrer method was used. Tables 1 and 2   showed the morphology of the wollastonite structure continuously in a plate shape.
According to Fig. 4(c) and Table 3 According to Fig. 4(d) and Table 4, the results of the synthesized wollastonite showed that oxygen (O), silicon (Si) and calcium (Ca) were the constituent elements of the wollastonite, and its non-availability was proved by the EDX analysis. It reveals that Si 4+ and Ca 2+ ions exist, meaning an appropriate primary distribution among the raw materials after the milling process which made uniform wollastonite.   [26,27]. With respect to the discussions, it is concluded that wollastonite and diopside are bioactive nano-bioceramics which can possess bone tissue biomineralization for biomedical applications. Fig. 7 depicts the mechanism of apatite formation. MG-63 osteoblast cells and diopside nano-bioceramic after alizarin red staining for 7 and 14 days. After 7 days, red veins ( Fig. 9(a)) are observed. Fig. 9 showing that wollastonite is bioactive nano-bioceramic.  days of alizarin red staining is much better and more outstanding than increasing calcium activity after 7 days of alizarin red staining related to two compounds.
Moreover, by comparing the results of alizarin red staining between diopside and wollastonite, it is concluded that diopside has offered more acceptable and excellent performance compared to wollastonite. In fact, bioactivity of diopside was higher than that of wollastonite.