In-vitro Reactivity and Antibacterial Activity of Agro-waste Derived Silicate and Phosphate Glasses

: Two different categories of bioactive glasses prepared from biomass using SiO 2 and P 2 O 5 as glass formers are reported in this study. These glasses are prepared by melt-quench technique. The glasses are evaluated in-vitro for their bioactivity assessment with the help of simulated body fluid (SBF). The formation of hydroxyapatite (HAp) above the glass surface is taken as an indicator for the glasses to be bioactive. Hence, various experimental techniques like X-Ray diffraction, Scanning electron microscopy, Energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and Microwave plasma atomic emission spectroscopy are employed to assess the formation of HAp layer above the glass surface. All these results confirmed the formation of HAp layer. Further, drug loading and antibacterial studies were carried out to investigate the application of glass samples as drug delivery vehicles and antibacterial agents, respectively. These investigations proved that the as-prepared glass samples have high drug loading efficiency and antibacterial activity.


Introduction
The conservation of the environment is a big issue with growing population. Food bio-wastes are a major source of environment pollution. It causes human illness and diseases. This requires higher attention to manage these wastes. The recycling of these waste materials leads to productivity and sustainability. Rice is one of the popular food all over the globe. According to a recent study rice is approximately 50% of total dietary caloric intake [1]. The global rice demand reached up to 700 million tons. The rice husk produced from paddy is generally used in the thermal power plants [2]. Rice husk (RH) is a major source of silica [3]. Therefore, it can be employed in the formation of value added product like silica which can be used in various applications like glass decorative items, display devices, industrial materials and energy storage devices [4][5][6]. Other common food wastes are eggshells, which are also available everywhere. Eggshells are a rich source of Calcium carbonate (CaCO3) which on calcination produce Calcium oxide (CaO) [7]. CaO is very important component in the preparation of glasses [8]. These products are considered non-toxic and environmentally favourable [9][10][11].
Recently Sanjeev et al. [12] have evaluated the bacterial compatibility of SiO2 extracted from rice husk and found them significant for biomedical, clinical, and biological applications.
Another article by Jafari et al. [13] reported the loading of Ag2S with rice husk based MCM-41 nanoparticles. Their study assessed the antimicrobial character of as-prepared samples against gram-positive and negative bacteria and found significant inhibition zones.
Prokopowicz et al. [14] studied the in-vitro drug loading properties of mesoporous silica microparticles combined with calcium oxide. Chen et al. [15] provided the drug loading properties of rice husk derived mesoporous glasses grafted with folic acid. However, all these studies are mainly focussed on the materials derived utilizing RH. The present report aims to utilize RH based SiO2 with CaO extracted from egg shells and other required constituents to make biocompatible glasses.
Bioactive glasses (BG's) are being used in clinical applications since more than a decade as dental filler materials, bone grafts and scaffold formation due to their ability of bonding to bone, osteoinductivity, biodegradation and biocompatibility [16][17][18]. Hench was the first scientist who discovered 45S5 labelled as Bioglass [19]. The main constituents of BG's are silica (Si), phosphorus (P), calcium (Ca), magnesium (Mg), and sodium (Na) which are naturally present in the body. BG's are capable to connect the bone by simply forming hydroxyapatite layer on their surface. This layer works as a channel which interact with collagen fibrils of damaged bone that increases osteoinductivity and forms a new bone [20].
The surface reactivity of BG's improves osteoblast properties and gives strength to bond the bone with BG implants [21]. Apart from this, BG's have exhibited antibacterial effects [22,23] and enhanced angiogenesis and osteogenesis [20].
After Hench's discovery of bioglass, a variety of compositions have been studied with some modifications. However, all these studies lack in comparison studies of agro-waste derived silicate and phosphate glasses in terms of in-vitro reactivity and antibacterial studies.
To the best of our knowledge, agro-waste derived bioactive glasses containing more than 20% of MgO on molar percentage basis are not reported yet. Therefore, in order to fulfil this gap, in the present report two glasses with SiO2 and P2O5 as network formers have been taken under present investigation along with other constituents CaO and MgO. The variation in CaO and MgO content was done to see their influence on in-vitro bioactivity, drug loading properties, physical and thermal parameters.

Synthesis of bioactive glasses
In the present work, bioactive glasses containing SiO2 and P2O5 as glass formers have been synthesized by melt-quench technique. SiO2 and CaO were extracted from agro-waste rice husk (RH) and chicken eggshells (CES), respectively. The details of their extraction process has been given in as earlier publications [24,25]. Agro-waste derived SiO2 and CaO, with P2O5 (purity > 99.5%), MgO (purity > 99.5%) and CoO (purity > 99%) which are purchased from HIMEDIA Co. India were used. These agro-waste extracted minerals were further utilized along with P2O5 and MgO in the preparation of bioactive glasses. The raw materials were weighed appropriately and mixed homogeneously with the help of mortar and pestle. These mixed batches were transferred to alumina crucibles and melted at 1400 ℃ in an electric furnace. Further, the bubble free melt was poured on a copper plate and quenched with another copper plate. The as-quenched glasses were immediately shifted to a furnace at 500 ℃ for annealing. This was done to remove thermal stresses developed in the glasses during melting process. The obtained glasses when cooled down to room temperature were ground to powder for further characterization. Attempts were made to synthesis the similar glass composition using commercial SiO2. Unfortunately, the glass could not melt even at 1550 ℃ for long holding time. Hence, the comparison study with commercial constituents is not given in the present work. The details of glass composition along with their label is given in table 1.

Physical properties of bioactive glasses
Physical parameters: The physical parameters like molar volume, molar mass and density have been calculated. The following equation is employed to calculate molar volume as a function of the mole fraction of each constituent: Molar volume (Vm) = (1) Where and represent mole fraction and molecular weight of the i th component, and ρ is the density of the bioactive glass.
Density: For determining the density of glass samples Archimedes' principle is employed, where benzene was taken as an immersion liquid with density 0.876 g/cm 3 . All the measurement was done at room temperature. The following equation is employed to measure the density of bioactive glass samples [26], Where Wa and Wb are the masses of sample in air and benzene, respectively and is the density of benzene.

Vickers hardness:
The micro hardness of the bioglass samples was measured by using a micro hardness tester (Model: Mitutoyo micro-hardness tester) at room temperature by applying load of 500g for a dwell time of 15s.

SBF preparation and In-vitro reactivity
In-vitro reactivity was assessed by immersion of the as-synthesized glass samples in the SBF solution. The SBF solution was prepared by following a protocol given by Kokubo [27]. In a typical procedure, the fixed amount of NaCl, NaHCO3, KCl, Na2HPO4, MgCl2.6H2O, CaCl2.2H2O, Na2SO4, Tris Buffer and HCl were taken. It is assumed that SBF has ion concentration equal to human blood plasma, therefore it is suitable for the determination of bioactivity of bioactive materials [27]. Further, for assessing the bioactivity, the appropriate amount of glass samples was soaked for two weeks with SBF solution. The pH of the solutions was measured every day for an interval of 14 days to observe the pH variations due to hydration of samples with pH meter Toledo pH meter (USA). After completion of time period, samples were dried and characterized with XRD, FTIR, SEM, EDS, MP-AES to investigate the development of apatite layer.

Degradation studies
Weight loss or degradation behaviour of as-prepared glasses was investigated by measuring the weight of glass samples before and after immersion in the SBF solution. For measurement of weight, samples were taken out from the SBF solution after two weeks and dried for one day. The following equation was used to calculate weight loss: The calculated values of weight loss after two weeks are summarized in Table 2.

Drug loading
For assessing the loading efficiency of as-prepared glass samples, Vancomycin hydrochloride (VAN) was chosen as a model drug. Drug solution was prepared using distilled water as a solvent. A stock solution with a concentration of 100mg/mL was prepared which was used for drug loading studies. A standard curve was plotted for the assessment of loading efficiency.
UV-Visible spectrophotometer was used for the determination of absorbance of drug solution before and after loading at a wavelength 240 nm. were chosen to test the antibacterial activity. For this, the two bacteria were cultured overnight and spread on petri plates using pour plate technique. Then filter paper discs loaded with glass particles were placed on these petri dishes. Further, these plates were incubated in a Biochemical oxygen demand (BOD) incubator for 24 hours. After this, the plates were examined for antibacterial activity and inhibition zones were measured. For quantitative analysis of bacterial resistance, the glass samples were loaded with bacterial strains in ELISA plate reader in triplicate.

Bioactive Glasses Characterization
The as-prepared glass samples were characterized with X-ray diffractometer (model Xpert Pro  is a decrease observed with increase in the CoO concentration whereas for BGP1 and BGP2 density is increasing. As density and molar volume have an inverse relation, therefore molar volume increases in silicate system and decreases for phosphate system. The molar volume is calculated by the formula reported in and given in eq. 1 [28].  [31]. The bond length of Co-O bond is 2.13 Å which is larger than P=O, but the bond strength is greater which give a strong network. Therefore, the molar volume is reduced.

Density, Molar volume and weight loss of glass samples
The weight loss profiles after SBF treatment for both glass systems are given in Fig. 2. It can be observed that weight loss is less in silicate glasses as compared to phosphate glasses. In case of phosphate glasses, the mechanical strength is low. Hence, these glasses dissolve more as compared to silicate glass system. Also, weight loss increases with addition of CoO in both systems.

Network connectivity and hardness studies
Network connectivity (NC) is an important parameter while investigating bioactive behaviour of glasses. Also, it plays a significant role to predict the glass structure which is favourable for bioactivity properties. This parameter defines the physiological feasibility for the development of HAp layer when it lies close to 2.0 [32]. NC is calculated with the help of following equation: The obtained NC values are enlisted in Table 2. These values show an ascending trend in both glass systems with the inclusion of CoO. It can be observed from the

Thermal analysis
For evaluating different thermal parameters such as glass transition (Tg), onset crystallization (Tx), crystallization temperature (Tc) and thermal stability factor (ΔT) and the effect of CoO inclusion in both glass systems, DTA studies were performed. DTA thermograms are given in Fig. 3. All the values for thermal parameters are represented in Table 2. It can be clearly observed from the Table 2 that silicate glasses possess higher glass transition as compared to phosphate glasses. Furthermore, with increase in the concentration of cobalt, the Tg values increases but there is no significant increase in Tx and Tc. The increase in the Tg after CoO insertion is due to better network connectivity, which brings compactness and need more enthalpy to break the bonds in the glass network. This increase in enthalpy further increases Tg [35]. Hence the prepared silicate glasses are more stable than phosphate glasses. These glasses can be used in bone tissue engineering applications.  observation is significant as compared to previous studies discussed above [35,36]. on having a look on the sample surface after the SBF treatment, it is observed that all the samples surface is covered with a homogeneous cauliflower like HAp layer. In case of samples BGS1 and BGS2 the formed layer is more compact as shown in Fig. 5 (e and f) as compared to BGP1 and BGP2 which is given in Fig. 5 (g and h). The corresponding EDS spectra represent the composition of constituent particles. The article reported in literature by Vikas et al. [29] observed the HAp layer development with SEM and compared it with 45S5 glass. The present study shows more homogeneous HAp particle distribution as compared to their study. This might be possible due to use of agro-waste materials.

pH analysis
The pH variation provides the information about the development of HAp layer above the glass surface. This pH variation is shown in Fig. 6. The initial rise in pH variations indicates the rapid release of alkaline ions (Ca 2+ and Mg 2+ ) from the glass samples into the SBF solution.
These ions get exchanged themselves easily with H + ions present in the SBF solution according to steps proposed by Hench [39]. Once all the ions are leached out from glass surface to SBF solution, a decline in the pH is observed. Therefore, pH either become constant with time or reduced. It can be observed from the graph that pH for BGS1 and BGS2 has shown more variation whereas BGP1 and BGP2 show a less but visible variation. Hence, pH studies for all samples exhibited the variation which supports the formation of HAp layer in these samples. These results further support the findings observed in XRD and SEM-EDS results. release is more in these two samples. Whereas, in case of BGP1 and BGP2 the quantity of Si is less, so release is also less. The release of Si ions is beneficial in the formation of osteoblast which makes bone [40]. The data shows that the release of Ca 2+ ions is more for all the glass samples as compared to other ions. The leaching of alkaline ions shows the replacement of these ions with H + ion which is an important part for bonding and helps in formation of bones.

Quantitative ion analysis
BGS1 and BGS2 release more Si ion as compared to BGP1 and BGP2 because higher content of the phosphate ions inhibit the release of other ions from sample surface to SBF which may lead to delay in the osteogenesis process [41]. However, the data shows that P ion is released in SBF solution in larger quantity in case of BGP1 and BGP2, which is directly related to its concentration in the parent glasses. Therefore, all the glasses show an exchange of ions that occur with variation in the constituent quantity.

Band gap studies
Energy band gap is calculated in order to observe the development of apatite layer on glass samples. The band gap variation in bioactive glasses before and after SBF treatment are shown in Fig. 8. The band gap of as-prepared BG's and after dipping in the SBF solution are calculated by Tauc's method using following equation [42].

αhν = A(hν-E) n (6)
Where E = energy bandgap, A = constant, hν = photon energy and n=1/2. The band gap is determined by extrapolating the linear portion of the graph to the x-axis where (αhν) 2 = 0.
Here, indirect band gap is calculated because the transitions of apatite like materials are supposed to be indirect [43]. The calculated values of band gap are given in Table 2. This table clearly shows that there is a change observed in the band gap values after the SBF treatment.
The change in band gap values can be explained in terms of entrance of CoO in the glass network. As CoO enters on the expense of MgO in both systems, due to structural units balance with modifier ions, there is no creation of non-bridging oxygens (NBO's) [8]. Also, compactness of structure increases and hence more energy is required for the movement of electrons leading to increased band gap values. Another reason for these changes might be due to formation process of HAp layer, during which breaking and making of bonds takes place which leads to structural changes. Thus, band gap values get modified after insertion of CoO.
It is reported earlier that the band gap values of HAp layer lies from 6 eV to down 3.95 eV [44,45]. The band gap values observed is approximately near to this range. There are various studies reported on bioactive properties of Co-doped glasses but these studies did not provide any band gap related information [35,46,47]. Hence, present study is providing a sight for HAp development and are consistent with previous studies done for HAp band gap.

FTIR analysis
The DLE of all samples at different drug concentrations is represented in Fig. 10

Antibacterial analysis
The The observed inhibition zones are shown in Fig. 12 wall and damage the cell membrane which lead to cell death [55]. Hence, the introduction of CoO inside the glass system improves it against bacterial growth.

Conclusion
Two glass series with two different glass formers SiO2 and P2O5 were prepared by melt-quench

Availability of Data and Material
All the data and material incorporated in the present manuscript will be made available whenever required.

Disclosure of potential conflict of interest
Authors do not have any conflict of interest including any financial, personal or other relationships with other people or organizations.

Compliance with ethical standards
Formal consent is not compulsory for the above type of work.

Funding Details
The authors did not receive any financial support from any funding agency Weight loss pro les of SBF treated samples  Different ion concentration (mg/mL) in SBF before and after 14 days immersion of glass samples Figure 8 Band gap variation in bioactive glasses before and after SBF treatment. FTIR spectra of glass samples before and after immersion in the SBF solution Figure 10 Drug loading e ciency for all glasses at different drug concentrations Figure 11 VANCO (Drug molecules) interaction with as-prepared glasses in the presence of cobalt oxide.

Figure 12
Inhibition zones for glasses against S. aureus and E. coli Cell growth inhibition (%) against S. aureus, E. coli and B. subtilis

Supplementary Files
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