Modification of Bioactive Glass-Ceramic by ZrO2/Ag2O Micro/Nanocomposite for Biomedical Applications

INTRODUCTION 
Hench and his colleagues pioneered the development of bioactive glasses, most notably the 45S5 Bioglass[1]. Both glasses and glass-ceramics are biocompatible and bioactive materials capable of bonding directly to bone tissue through the formation of biologically active hydroxyapatite (HA) layers. These HA layers form an interfacial bond and are structurally and chemically similar to the mineral phases found in bone [2]. However, the low hardness of bioactive glass has restricted its use to non-load-bearing applications [3]. It has been demonstrated that the mechanical properties of bioactive glasses can be enhanced by incorporating oxides such as alumina, magnesia, titania, or zirconia. Additionally, crystallizing bioactive glasses to form glass-ceramics can also improve their mechanical characteristics [4]. Glass ceramics in the CaO-SiO2-P2O5-Na2O system, when processed through sintering and subsequent crystallization, exhibit excellent mechanical properties and can form a chemical bond with living bones. These bioactive glass ceramics, which fall under the category of osteoconductive biomaterials, are ideally suited for medical applications. They are particularly effective in orthopedic and dental implants due to their strong bioactivity and established biocompatibilit.[5]. Generally, these bioactive glass ceramics form hydroxyapatite layers when exposed to simulated body fluids (SBF), which are buffered salt solutions that mimic the ionic concentrations of bodily fluids. This hydroxyapatite layer enhances their ability to bond with bone and supports tissue regeneration [6]. From a clinical perspective, one of the primary challenges surgeons face is the risk of post-implantation infections. Silver (Ag) has been shown to have significant antibacterial properties, which can help mitigate the risk of infections and combat bacterial growth around implants [7,8]. Over the past few years, biologists have extensively studied the biological properties of silver (Ag). These investigations have focused on its antibacterial effects, mechanisms of action, and potential benefits in medical applications, such as infection control in implants [9]. Therefore, incorporating silver oxide into biological implants is a reliable approach. Studies have shown that bacteria generally cannot develop resistance to silver (Ag) incorporated into biomaterials, making it an effective option for reducing the risk of infection [10,11]. Thus, numerous studies on silver-containing bioactive glasses (Ag-BGs) have been conducted to determine the optimal amount of silver needed in the glass structure for various applications. These investigations aim to balance the antimicrobial benefits with other desired properties of the bioactive glass. [12,13]. Zirconia was first used about 20 years ago to address the problem of ceramic brittleness and reduce the risk of implant failure. Its introduction significantly improved the mechanical strength and durability of ceramic implants [14], this is because zirconia provides an oxide ceramic with superior mechanical properties due to phase transformation toughening, which enhances its resistance to crack propagation and significantly improves its overall strength [15]. The utilization of composites made from zirconia (ZrO₂) and bioactive glass in the Na₂O–CaO–SiO₂–P₂O₅ system offers significant potential, particularly in applications like bone regeneration and implants [16], Combining the high strength provided by zirconia reinforcement with the excellent compatibility and bioactivity of bioactive glass presents a promising approach for creating durable, bioactive materials [17]. The high bending strength of the zirconia-toughened glass-ceramic composite was validated, with no signs of degradation noted after 12 weeks of in vivo implantation. The optimal zirconia content for achieving both high strength and bioactivity was found to be 30 vol% [18]. A composite coating layer of bioactive glass-ceramic with varying zirconia concentrations was utilized, and hardness tests indicated that increasing the zirconia content resulted in enhanced coating hardness. The incorporation of zirconia was aimed at mitigating ceramic brittleness and minimizing the risk of implant failure[14]. The objective of this study is to synthesize bioactive glass-ceramic with enhanced mechanical properties and antibacterial activity using the melting technique, aimed at applications in biomedical fields.

MATERIALS AND METHODS
The materials used in this study include Sodium oxide (Na₂O, 98%, India), Silica foam (SiO₂, 99.9%, India), Phosphorus pentoxide (P₂O₅, 98%, China), Calcium oxide (CaO, 98%, India), Zirconia (ZrO₂, 98%, China), and Silver oxide (Ag₂O, 98%, China).

Glass-ceramic preparation
The synthesis of 45S5 bioactive glass was carried out using the conventional melting technique, comprising 45 wt% SiO₂, 24.5 wt% Na₂O, 6 wt% P₂O₅, and CaO (24.5 - X) with X wt% of ZrO₂/Ag₂O (where X = 0, 1, 3, or 5). For each variant of bioactive glass, a 10 g batch was created by thoroughly blending analytical reagents in a planetary ball mill (SFM-1, QM-3SP2) at 300 rpm for 6 hours. The resulting mixture was then melted in an alumina crucible using an electric furnace set to 1200 ± 10 °C for 2 hours to ensure complete melting. After this process, the molten glass was gradually cooled in the oven. The produced glass-ceramic was crushed in a mortar to obtain a semi-finished powder, which was subsequently milled in a planetary ball mill (SFM-1, QM-3SP2) at 300 rpm for 12 hours. The composition of each batch is detailed in Table 1. Compacted samples, measuring 10 mm in diameter and 30 mm in length, were sintered at 1200 °C with a heating rate of 5 °C/min. X-ray diffraction (XRD) analysis of the powder samples was carried out at room temperature using a Shimadzu 6000 with CuKα radiation (λ = 1.5405 Å). The scan range was from 10° to 80° (2θ) at a speed of 5°/min, with an applied power of 40 kV/30 mA to analyze the structure and phases.
The XRD peaks were compared with standard JCPDS files: NO. 00-022-1455 for sodium calcium silicate (Na₂Ca₂Si₃O₉) and NO. 00-012-0671 for sodium calcium silicate (Na₂CaSi₃O₈( . This study examines the impact of ZrO₂/Ag₂O weight percentage on the structure, mechanical properties, and biological behavior of bioactive glass-ceramics within the SiO₂, Na₂O, CaO, P₂O₅, ZrO₂/Ag₂O system. It explores how different concentrations of ZrO₂ and Ag₂O affect the material’s structural integrity, mechanical performance, and bioactivity.
FTIR was employed to verify the presence of ZrO₂/Ag₂O within the glass-ceramic matrix. The apparent porosity of the samples was determined using the Archimedes method, in accordance with ASTM C373-88 (1988). Compressive strength testing was conducted following ASTM C773-88 (1999). Vickers microhardness was measured in line with ASTM C1327-99, applying a 9.8 N indentation load and using the designated equation for calculation.

Hv=1. 854(p/d2)

Hv: Vickers hardness (Mpa), p: Loads (N), D: Diagonal lengths of the indentations impressions (μm).
The bending test was performed in accordance with the ASTM C1161 standard, utilizing a computerized universal testing machine set to a testing speed of 0.5 mm/min. The three-point bending strength was determined using the equations outlined below.

Qb=3 pf L/2wt2

Where: Qb: Bending strengths (MPa), Pf: Fractures loads (N), w: Samples width (mm), t: Samples thickness (mm).
In order to investigate samples bioactivity, (SBF) the simulated body fluid was prepared according to[19]. The sample was immersed in Simulated Body Fluid (SBF) at 37 ºC for 21 days. At the end of the immersion period, scanning electron microscopy (SEM) was performed on the sample surfaces to examine the formation of hydroxyapatite (HA) on the surfaces.

RESULTS AND DISCUSSION
X-Rays Diffractions (XRD) Analysis
The phases of the prepared powders were obtained using XRD. Fig. 1 shows XRD patterns of glass-ceramic powders with different of ZrO2 and Ag2O that melted in 1200 °C, two types of sodium calcium silicat that obtained in each sample which is (Na2CaSi3O8) (Na2Ca2Si3O9) that scanned in a diffraction angle ranging from 10º to 80º and agree with (JCPDS, card NO.12-0671) and (JCPDS, card NO. 22-1455) respectively.
 The various added dopants did not produce significant changes in the types of crystalline phases; rather, they resulted in only minor variations in the percentages of the main crystalline phases.
 In general, XRD results shows that the intensity of crystal phases and its quantity changed with the addition of ZrO2 and Ag2O for all samples [20].
For compact bioglass-ceramic samples sintered at 950 ºC, Fig. 1 presents the XRD results. The samples exhibit two types of sodium calcium phosphate: Na₂Ca₂Si₃O₉, which corresponds to JCPDS card No. 22-1455, and Na₂CaSi₃O₈, which is commonly associated with combeite in certain peaks. These findings are consistent with results reported by various researchers [21-23].

Results of FTIR analysis
FTIR spectra of bioglass-ceramic doped with different amount of ZrO2 and Ag2O samples melted at 1200 °C are shown in Fig. 2. According to previous FTIR studies on bioglasses-ceramic [24], it’s clear that bands in the wavenumber 625, 1120, 1050 cm−1 corresponds to rocking vibrations of Si-O-Si bridges, Zr-O-Si and Si-O-Na bridges respectively.  As well as characteristic wavenumbers 932 cm−1 related to Si-Si bridge [25]. The improvement in mechanical properties of bioglass-ceramic samples is attributed to the formation of Zr-O-Si and Si-O-Ag bonds. Because zirconium ions was occupied interstitial sites within the bioglass network [26].

Density and Mechanical Properties Measurement
Table 2 presents the bulk density and apparent porosity of the bioglass-ceramic samples as a function of ZrO₂/Ag₂O weight percentage. It is evident that as the ZrO₂/Ag₂O wt.% increases in the glass-ceramic, the density rises from 1.98 to 3.32 g/cm³. This increase is attributed to the substitution of the lighter element Ca (density = 1.55 g/cm³) with the heavier elements Zr (density = 6.52 g/cm³) and Ag (density = 10.49 g/cm³). Additionally, structural changes occur due to the addition of modifying metal oxides (ZrO₂), which coordinate the NBOs (Zr-O-Si) and (Si-O-Ag), forming links between atoms. This leads to improved network connectivity and dimensionality, resulting in greater compactness and packing efficiency within the bioglass-ceramic structure. The increase in density is also linked to a reduction in average interatomic spacing during sintering, which in turn decreases the porosity from 32.4% to 11.43%.
Table 3 shows that increasing the ZrO₂/Ag₂O content from 1 to 5 wt% results in a substantial improvement in compressive strength, bending strength, and Vickers microhardness. This enhancement is attributed to the formation of new strong bonds, such as Zr-O-Si and Si-O-Ag, as confirmed by FTIR analysis. Additionally, the incorporation of Zr and Mg ions into interstitial sites within the bioglass-ceramic network contributes to increased density and improved mechanical properties, reinforcing the glass-ceramic structure.

In Vitro Bioactivity Tests
SEM Results of Bioglass-ceramic after Immersing in SBF Solutions
 Significant changes were observed in the bioglass-ceramic samples after 21 days of immersion in SBF, as shown in Fig.3. Agglomerations of HA were visible on the surfaces of both the (A0) and (A3) samples, consistent with the findings reported in [28,29].

CONCLUSION
A 45S5 bioactive glass-ceramic can be toughened by ZrO2/Ag2O particles as reinforcing phase. The experimental results showed the positive effect of ZrO2 and Ag2O introducing to bioactive glass-ceramic which was 1, 3 and 5 wt% of ZrO2/Ag2O together. The bending strength improved from 23.6 to 53.3 MPa while the compresive strength improved from 48.7 Mpa for the pure glass-ceramic to 108.8 Mpa for glass-ceramic sample involves 5wt% ZrO2 and Ag2O. The formation of a hydroxyapatite layer on the bioglass surface after immersion in simulated body fluid (SBF) was investigated using scanning electron microscopy (SEM). This in vitro study analyzed the behavior of the bioglass-ceramic sample in SBF. It can be seen increasing in the hydroxyapatite value with ZrO2/Ag2O addition. This indicates that with increasing the immersion period and ZrO2/Ag2O addition, the bioactivity of the samples had increased.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.