Zn-containing Wollastonite with Well-defined Microstructural and Good Antifungal Activity

Antimicrobial and antifungal materials we prepared from Zn-containing wollastonite set by wet precipitation method. Wollastonite, hardystonite, willemite and very little quartz were developed after sintering at 1100 °C/2 h, however, the Raman spectroscopy approved the later phases by their characteristic Raman shift bands. The microstructure exhibited accumulated rounded to irregular clusters containing nano-size particles (< 500 nm) developed in all sintered samples. Zeta potential; exposed negative values for all powdered samples from -2.64 to -17.6 mV (i.e., for Zn-free to highest Zn-containing samples). It can be easily noticed that the lowest ZnO-content exhibits a varied range of antibacterial activities in contrast to Gram-negative (E. coli) and Gram-positive (S. aureus & B. subtilis). Correspondingly, the CZS5 exhibits good inhibitory effect against the filamentous pathogenic fungus (A. niger). Wollastonite powder containing ZnO was prepared by wet method. Wollastonite, hardystonite, willemite and little quartz were developed in sintered at 1100 °C/2 h. The powder has wide effect on the A.niger fungi in the high ZnO-containing powder. The powder can also inhabit the growth of gram-positive and gram-negative bacteria.


Introduction
Several inorganic cements have been utilized for progress of hard tissues. The best regularly used cements as bone substitutes are designed from calcium silicates, sulfates and phosphate cements [1][2][3]. Reliant on the upright mechanical strength and biocompatibility of the calcium silicate (CS); it is used as a filler in dental and orthopedically surgery. Ceramics of calcium-silicate, validate antibacterial action owing to their alkaline possessions. A unique important reasons of infection in the body after grafting of scaffold are bacteria [4]. The integration of metallic antibacterial agents for instance (Cu 2+ , Ag + , Ce 4+ and Zn 2+ ) into the bioceramic matrix was endorsed [5,6]. Conferring to earlier reports, initiation of trace elements keen on biomaterials positively attained many extra bio-functions, for example antibacterial property, osteogenesis and angiogenesis [7]. Upon assimilation of Zn 2+ into the silicate structure of the calcium silicate; replacement of silicate joining by tetrahydra of zinc at the chain of terminal silicate will instruct constancy to the crystal structure. Zn 2+ has superior antibacterial activity, owing to the antagonistic effect of Ca 2+ and Zn 2+ release. Zinc ions certainly disturbs the antibacterial activity and cellular reaction, thus providing an attractive bone filler alternate [8,9]. By means of raising the concentrations of zinc, antibacterial action extended in the inhibition zones as mentioned by El-Bassyouni et al., upon investigating the doping of the hydroxyapatite (HA) with diverse percentages of Zn [10]. Wang et al., (2011) [11] decided that an exact amount of ZnO has no poisonous side effects on the human body and can correspondingly improve the cell proliferation.
Zinc ion demonstrates a favorable potential to be used as an antibacterial agent, it is convoluted in the rule of multiple cellular purposes and accomplishes some key functions. For antimicrobial actions, the characteristic immune system utilizes zinc. It is commonly informed that zinc oxide has antimicrobial activity devoid of toxicity and it has environmentally friendly effects compared with other biocide agents such as copper or silver [12]. Zn oxides show wide anti-bacterial spectrum, virus deactivation possessions and anti-fungal action. Hereafter, Zn-discharging biomaterials embrace immense healing value in several clinical uses [13]. Li et al., (2015), designated that the adding of ZnO to the wollastonite declines the total pore volume, thus demonstrating that the ZnO increase the density of wollastonite [14]. Likewise, Sirelkhatim et al., indicated the deliberation of zinc ions; antibacterial and antifungal properties in the form of pure zinc and zinc oxide nanoparticles [15]. Moreover, it was figured out that the impact of ZnO on the bioactivity and antimicrobial properties of the nano-sized hydroxyapatite improved the resistance of samples against bacterial activity [16]. One of the mechanisms liable for antimicrobial activity generally described in the texts is the production of reactive oxygen species (ROS) by metal oxide nanoparticles. ROS take account of hydroxyl radicals (HO − 2 ), superoxide anions (O 2 − ) and hydrogen peroxide (H 2 O 2 ), that may ground the demolition of the cellular constituents for instance protein, DNA and lipids [17]. Otherwise, the nanocomposite bioactivity could be anticipated by the zeta potential or ζ potential of the nano-additives. Therefore, the most inspiring finding was a negative zeta potential to allow the bone cell activity in a future in-vivo test [18]. Additionally; Raman Spectroscopy as a non-destructive chemical analysis, was used to provide comprehensive evidence about the chemical structure, crystallinity and molecular interactions, phase and polymorphy. It is established on the inelastic scattering of photons, known as Raman scattering with the chemical bonds within the material.
In the present work, wollastonite with/without ZnO was prepared through wet precipitation method [19]. Characterization of the sintered samples was considered by X-ray diffraction analysis (XRD), Raman spectroscopy (a non-destructive chemical analysis method), scanning electron microscopy (SEM) and zeta potential. The influence of powdered sintered samples as antibacterial and antifungal were evaluated.

Characterization
In the present work, wollastonite alone or containing ZnO was pre-prepared through wet precipitation method [19]. The design of the composition based on the CaSiO 3 : ZnO ratios 90:10, 70:30 and 50:50 were given the following codes [CS0, CZS1, CZS3 and CZS5] ( Table 1). The starting materials were CaCO 3 (98 wt %, El-Gomhouria Company Cairo, Egypt) as the source of CaO, amorphous silica (high grade purity, SiO 2 gel, Fluka Chemie GmbH, Switzerland) as the source of SiO 2 and hydrated zinc acetate [98.5 wt%, Zn(CH 3 CO 2 ) 2 ⋅2H 2 O, Qualikems Fine Chemicals Pvt. India] as the source of ZnO. Nitric acid was used to change CaCO 3 into the water soluble calcium nitrate (CaNO 3 ). Mixture of water soluble silica gel with CaNO 3 formed the base sample, however, the addition of soluble zinc acetate with different ratios to the mixture will form other samples. The gotten gel was being placed in an incubator to dry (at 100 °C/24 h) then heat-treated at 1100 °C/2 h.
As mentioned in the previous research paper, the identification of the developed phases after sintering at 1100 °C/2 h was considered using: XRD, PANalytical Empyrean diffractometer system Holand, with Cu-Kα radiation. XRD functioned at 40 kV and 40 mA; verified patterns in the range of 2θ = 10-70°. Differentiating of the crystalline phases were possible by matching the diffraction patterns of the prepared batches with ICDD (JCPDS) standards. As well the morphology and microstructure were investigated by field emission scanning electron microscopy (FE-SEM, Quanta 250 FEG, FEI, Netherlands). Moreover, to decide the electrical surface charges on the sample powder, Zetasizer (Zeta potential Analyzer, Malvern Instrument Ltd, UK) stimulated by means of a 633 nm laser was used. Fine powder of samples was well-distributed in distilled water by temperature 25℃ then used for the estimation of the zeta potential (each measurement being the average value of 12 runs). Raman Spectroscopy (i-Raman Plus 532S portable laser Raman spectrometer, USA) which gives evidence about the phase and polymorphy, crystallinity and molecular relations and chemical configuration, can help in conforming the major crystalline phase The spectral resolution was 3.5 cm −1 and the spectral range was in-between 300 and 1600 cm −1 .

Antibacterial and Antifungal Activities
Antimicrobial and antifungal activity of the sintered samples were tested in-vitro screened against different pathogenic strains of Gram positive (Staphylococcus aureus ATCC29213 and Bacillus subtilis ATCC6633), Gram negative (Escherichia coli ATCC25922 and Salmonella enterica ATCC25566) bacteria, yeast (Candida albicans ATCC10321) and filamentous fungi (Aspergillus niger NRC53) using the method of agar diffusion technique [20]. Spores suspension of all strains were primed and attuned to be approximately (1 × 10 6 spores −ml of fungi and 1 × 10 8 of bacteria). Into each plate holding 50 ml of sterile potato dextrose agar (PDA) a 1 ml of fungal and bacterial spore suspensions was inoculated to encourage the luxuriant fungal growth and nutrient agar medium (NA) for bacteria, respectively. 50 and 100 mg of each sample were placed individually on the inoculated agar plates and left for two hours at 4℃ to permit the diffusion of the compound. The plates were incubated for 24 h at 37℃ for bacteria and 72 h at 28℃ for fungi [21]. For all the verified organisms; the inhibition zones (IZ) were recorded in millimeters at three different points and the average values were reported as Mean ± SD using MS Excel.

Co-incubation Test for Antifungal Activity
Sample CZS5 was placed in 9 ml sterile potato dextrose broth (PDB). Culture medium was inoculated with 1 ml of A. niger spore suspension (1 × 10 6 spores −ml ) and then maintained in a shaker-incubator at 150 rpm, 28 °C for 12 and 24 h. After incubation time about 100 μl of the suspension was plated on the surface of the PDA medium and further incubated for 72 h for fungal colony forming to be easily counted. The reduction ratio was evaluated by the following equation: where, R is the percentage reduction ratio, A is the number of colonies from the untreated culture medium (devoid of testing materials) and B is the amount of colonies from the treated medium. The control sample was not treated by tested material [22].

Microscopic Investigation
The agar plate of A. niger was tested using the Olympus CX40 RF100 light microscope bonded a Canon A620 digital camera to approve the effect of the CZS5 on the hyphal growth and sporulation.

X ray Diffraction, Raman Spectroscopy and SEM Analysis
Identification of the crystalline phases designed in the sintered samples at 1100 °C/2 h were revealed in the XRD pattern as accessible in Fig. 1. It shows the crystallization of wollastonite (CaSiO 3 ) alone in case of the parent sample. While upon incorporation of little Zn content as in CZS1 will catalyze the crystallization of hardystonite (Ca 2 ZnSi 2 O 7 ) which becomes the major phase with higher content of ZnO in CZS3 and CZS5 samples. Willemite (Zn 2 SiO 4 ) was developed as secondary phases in the later samples (i.e. CZS3 and CZS5). The crystallization will concisely follow the next equations: The Raman spectroscopy of CS0 and CZS5 samples which gather the crystallization of wollastonite, hardystonite, willemite and inadequate low quartz are shown in Fig. 2. Wollastonite, which crystallized alone in the CS0 sample, displays clear Raman shift bands 402, 632, 856, 965, 1011 and 1084 cm −1 which are more or less similar to the reference data by Buzatu and Buzgar, 2010 [23]. They decided that the diverse types of vibrations detected in the Raman spectra are existing at different wavenumbers, reliant on the chemical composition and structure. On the other hand, the low Raman shift value at 402 is referred to the Ca-O bending whereas bands appear at 856, 965, 1011, 1084 cm −1 are attributed to stretching Si-O non bridging band (Si-O nbr ). A characteristic Raman shift was detected at 906, 663 and 614 cm −1 which refer to hardystonite [24], while the bands noticed at 876 cm −1 denote the willemite [25]. Furthermore, a weak Raman shift band was detected at 463 cm −1 which pointed out to the low quartz content [26].
The microstructures of the sintered samples at 1100 °C/2 h show that the incorporation of the ZnO into the nominal wollastonite which stimulate the crystallization of nanostructures (Fig. 3). The Zn-free wollastonite CS0 sample shows scattered accumulate rounded clusters in submicro-scale containing connected net-like particles in the nanometer size. Integration of the ZnO change the rounded clusters into irregular ones in the CSZ3 and CSZ5 samples containing submicron particles. However, sample CSZ1 shows nanoscale size but with little flake like crystals in between.

Zeta Potential (ξ)
The average values of the twelve runs of ζ and their standard deviation values are accessible in Table 2 and Fig. 4. Zeta potential was used to designate the constancy of surface charges, particle sizes and suspensions for the prepared batches. It was observed that the zeta potential of all samples were of negative values. Such remark may be a beneficial property for the samples upon implantation in bone inclosing viable cells. The attained zeta potential values were -2.64, -9.38, -17.6 and -17.6 mV. As a result, it may be decided that the admission of the ZnO has initiated a decline in the zeta potential value [10]. The value of area % was 100% for CS0, CZS1 and CZS3; whereas for the sample with the higher percentage of zinc oxide (i.e., CZS5) it displayed two peaks of 96.6% for the first peak and 3.4% for the second peak. It could be decided that the gradual doping of the zinc oxide into the wollastonite had enlarged the negative value of their zeta potential particularly with increase in doping percentage (i.e., for CZS3 and CZS5). In contrast, the conductivity value of the CS0 scored the higher value in comparison with others; doped with different percentage of zinc oxide. Correlated to a greater electrostatic repulsion among particles, materials are showing tiny or no repulsive force for a larger zeta potential value [27].

Antibacterial and Antifungal
Antibacterial and antifungal effects of the prepared samples were tested in-vitro against pathogenic Gram-positive, Gram-negative bacteria, yeast and fungi using the agar diffusion assays. The inhibition zone (IZD) in millimeter was measured predicted on visual interpretations.

3
The results are presented in Table 3 and Fig. 5. It was designated that, CZS1 at high concentration of 100 mg weight exhibits a wide range of antibacterial activities against Gram-negative (E. coli) and Gram-positive (S. aureus & B. subtilis). Gram-positive (S. aureus) have the maximum sensitivity (15 mm IZD) to the CZS1. It also exhibited good inhibitory effect against the filamentous pathogenic fungus (A. niger), with zones of inhibition of 17 ± 0.61 mm. The outcomes also showed that all prepared samples at low (50 mg) and high (100 mg) concentrations have antifungal activities against the filamentous fungus (A. niger) with diverse effects in the range from 13-25 mm IZD higher than the base CS0 of 12 mm IZD as revealed in Fig. 6. The CZS5 showed the highest antifungal effects at 50 and 100 mg weights with IZD of 20 and 25 mm respectively that represent 17% and 21% inhibition compared to the effect of other samples. The CZS5 exhibited the best antifungal effect was further evaluated by co-incubation method to determine the inhibition rate of A. niger growth. The reduction in the fungal growth was evaluated after 12 and 24 h incubation in culture medium in comparison with control (untreated culture medium without adding testing material). The potential antimicrobial activity of calcium silicate-based material was previously attributed to their alkalinity and high release of the calcium ions [28]. Likewise, results indicated that, the fungal growth was inhibited by 40.74% after 12 h and 55.12% after 24 h (Fig. 7). Obtained results showed the effect of time, where the growth rate decreased directly with increasing the incubation time.
To approve the consequence of the CZS5 on the fungal growth, the fungal mycelium was moved into glass slides and tested via an ordinary light microscope. Figure 8, exhibited weak immature hyphae and low sporulation in colonies grown near to the zone of inhibition. In contrast, the fungal colonies grow at different places away from the effect of tested sample exhibited mature hyphae with large conidia carrying spores. Specimens exposed to the stated microorganisms verified different antifungal properties as revealed in Table 3. It may be determined that the best antifungal activity was attributed to CZS5 (the higher concentrations of zinc oxide content) in agreement with early research of [29]. The clarifying antibacterial activity of the calcium silicate matrix doped with ZnO is a promising reason for demonstrating their ability to transfer through the bacterial membrane along with  [30]. The impact of the Zn 2+ ions on the micro-organisms is assembled from their feedbacks with the negatively charged fungi cell walls and before the creation of complex compounds within the fungal membrane. Parallel studies were achieved but in the existence of nanoparticles, they established that the fungal activity of the composites is attributable to the strong oxidizing power of the Zn 2+ , which facilitated the creation of the reactive oxygen species (ROS) that impede the microbial growth [31].

Conclusions
The partial exchange of ZnO for CaO in the wollastonite system was implemented using the wet precipitation course. The current study aimed at the exploration of the effect of Zn-containing wollastonite on the, microstructure and anti-fungal characteristics in order to produce a viable ecofriendly physico-chemical material. Wollastonite with/  (20-25 mm) over all strains which may approve the prospective for Zn as a biologically active material. On that account, the intention of the current research work was to appraise the effect of the ZnO on the microstructure and antifungal activity of wollastonite to find a ceramic with enriched properties for the bioengineering uses.