Efficacy of Sr²⁺ and Cu²⁺ Ion Incorporation on the Mechanical, biological, and antibacterial properties of natural waste-derived borosilicate bioactive glasses for orthopaedic implants

Abstract

The inclusion of the metal based therapeutic ions into bioactive glasses received potential towards tissue regeneration applications due to their excellent biocompatible, mechanical and wear properties. In the current work, one such configuration is chosen, strontium (Sr) and Copper (Cu) ions based bioactive borosilicate glass system i.e., with varying composition of Sr and Cu for 0, 1 and 2 wt.%. The glasses were prepared through melt quench process and the as prepared specimens are characterized for their microstructural, mechanical, tribological, biocompatible and biological investigations. The results were found to appealing with microstructure and crystallographic peaks displaying the presence of extracted natural Silica (Si) and Calcium (Ca) powders. Further the same specimens are characterized for their mechanical strength and it was found to be 55.21MPa compressive strength and lowest specific wear rate overall for CuSr-1 wt.% composition. Being CuSr-1% displaying better mechanical properties, the same samples i.e., Cu, Sr for 1wt.%, were investigated for their biocompatible studies through Simulated Body Fluid immersion protocol. The SBF studies indicate the formation of hydroxyapatite that can be inferred from crystallographic peaks and Fourier Transform Infrared Spectroscopy. The samples were also exposed to antibacterial studies against Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus) and are found to exhibit excellent antibacterial activity. The observed selective antibacterial activity highlights the potential of these bioactive glasses as advanced materials for load-bearing orthopaedic implants and bone tissue engineering scaffolds, where the simultaneous requirements of mechanical robustness and antimicrobial efficacy are critical for clinical success.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-50704-x.

Introduction

Biomaterials are found to be predominant in the field of the tissue engineering and regeneration over decades^1,2. Amongst these, bioactive glass has been one of the keen areas of research for the scientists in the pursuit towards bone tissue engineering and its regeneration. Ever since the development of the first artificial material with an ability to form chemical bond with the bone by Larry Hench in 1969 in the University of Florida which was later termed as 45S5 Bioglass, henceforth earmarked to a new research field of bioactive glasses³. With decades of researches and advent of new technologies, various fabrication techniques, new and modified matrix in lieu of silica-based matrix have led to the development of various subgroups and classification of bioactive glasses. They have been further classified as borate-based (B₂O₃), silica-based (SiO₂) and Phosphate-based (P₂O₅) on the basis of major network former in the glass matrix⁴. Fabrication techniques such as solgel method from wet chemistry, melt quench, flame synthesis, microwave irradiation, powder sintering have also been in existence and new advancements have also been devised⁵. With the advent of technologies, further, the bioactive glass have provision to incorporate specific dopants such as Cu, Na, Ag, Zn, B, F, Mg, Ce, Ti, Co, Fe, Li, Sr, etc.⁶ as trace element in the glass matrix envelope the potential to tailor the properties such as osteoblast activity, osteoclast activity, bone metabolism, structure stabilization, angiogenesis, mechanical properties, and biocompatibility, anti-microbial activity^7,8. Though, in vivo studies of bioactive glass have indicated comparatively faster bond formation rate than bio-ceramics, and invitro studies have predicted their osteogenic properties via osteoprogenitor cells stimulation at the genetic level, in terms of commercial success, they lag behind bio ceramics⁹. Consequently, substantial amount of research has been carried out to enhance the potential of the bioactive glass in terms of biocompatibility, bone-tissue interfacing, degradation rate, fabrication process, manufacturing and production cost reduction¹⁰.

This work differs from earlier studies on Cu–Sr co-doped phosphate or silicate bioactive glasses primarily through the use of a borosilicate network, which modifies glass structure, dissolution behaviour, and therapeutic ion release. Unlike silicate glasses, where Cu and Sr mainly act as network modifiers influencing antibacterial and osteogenic responses, the incorporation of B₂O₃ alters network connectivity and accelerates yet controllable degradation, enabling a more balanced and sustained release of Cu²⁺ and Sr²⁺ ions^11,12. Compared with phosphate glasses, which often show rapid dissolution and limited long-term stability, borosilicate systems provide improved structural integrity while still promoting bioactivity¹³. Additionally, the presence of boron introduces distinct biological effects, including enhanced angiogenic and osteogenic signalling, which are not typically reported in Cu–Sr co-doped silicate or phosphate glasses alone^14,15.

Amongst all the bioactive glasses explored, borosilicate bioactive glass offer certain unique potentials such as compositional flexibility of dopants, controlled degradation, mechanical stability, tailorable bioactivity, thermal stability, lower risk of crystallization, multifunctional application and has bioactive boron benefits^16,17. The in-vivo & in-vitro studies on the borosilicate glass as a tool for bone tissue regrowth and damage repair have indicated excellent bioactivity in terms of simulating bone tissue regeneration and formation of extensive bond with bone at the molecular level with optimized rate offering healing and degradation in synchronization¹⁸. The composition of the borosilicate bioactive glass is primarily composed of silica oxide (SiO₂), boron oxide(B₂O₃), Sodium oxide (Na₂O), and Calcium Oxide (CaO)¹⁹.

Studies from Shaowen Cheng et.al.²⁰reported that boron could enhance osteogenic effect by stimulating osteogenic differentiation-related marker gene synthesis during the proliferation and differentiation phase in human bone marrow stromal cells (BMSCs) and could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering. In addition, the in-vitro studies evident that, Si alone can promote osteogenic differentiation, cell proliferation, and provide desirable gene expression for bone regeneration^21–23. Incorporating substantial amounts of transition metal oxides (TMOs) into bioactive glasses enables the tuning of their properties for targeted applications, improving mechanical performance while also providing antimicrobial activity. Among various metal oxides, the incorporation of strontium (Sr) and copper (Cu) into bioactive glasses can improve their functionality. Specifically, Sr enhances osteoblast activity and suppresses bone resorption by osteoclasts, whereas Cu provides pro-angiogenic and antibacterial effects. For instance, Yann C. Fredholm et.al²⁴ studied the effects of the Sr²⁺ substituted bioactive glass on osteoblasts and osteoclasts and fabricated the series of BGs (SiO₂–P₂O₅–Na₂O–CaO) in which calcium was substituted by strontium due to their charge and ionic radius similarity. Their in-vitro studies evident that strontium-substituted BG promotes anabolic effects on osteoblasts and an anti-catabolic effect on osteoclasts, promoting apatite formation and inhibiting bone resorption. In addition, Sadegh Firoozi et.al²⁵ reported about the antibacterial effect of Sr substituted 58S BG against methicillin-resistant Staphylococcus aureus (MRSA) bacteria. Their study was based on elucidating the effects on bioactivity, alkaline phosphatase, biocompatibility & antibacterial activity of the Sr substituted, sol–gel derived 58S-BG and to suggest the optimal content of SrO in BG-58S composition to achieve the highest cell proliferation and activity as well as antibacterial efficiency against MRSA.

On the other hand, studies investigating Cu-doped silica-based glasses by Marta Miola et.al²⁶ reported improved antibacterial efficacy against common pathogens like S.aureus and E.coli highlighting the antimicrobial effect against fungi, gram negative and gram positive bacteria over a broad spectrum. Peiman B. Milan et.al²⁷ in their studies of vascular grafts containing modified BGs indicated the better chemical stability and degradation rate of borate-based BGs for endothelialization and angiogenesis. And also highlighted that the melt-derived, Cu modified BGs enhance the vasculogenic and angiogenesis factors accelerating tissue generation patient-specific vascular prostheses. Alongside from bioactivity and physicochemical stability, tribological performance is also a critical consideration for bioactive glasses to be used in biomedical applications. Glass-based composite or coatings in implants may endure sliding, abrasion, and contact stresses at implantation and service and lead to wear and the formation of debris²⁸. Therefore, tribological performance testing of doped and co-doped glasses assists in determining their reliability and biological safety over an extended period.

In addition, some studies have demonstrated that co-doped bioactive glasses with combining the angiogenic and antimicrobial benefits of copper with the osteogenic effects of strontium²⁸. This co-doping strategy has been shown to improve the material’s performance in bone tissue engineering applications. Hanna Kankova et.al²⁹ developed Cu and Sr co-doped mesoporous silicate based MBGs and highlighted the influence of Copper-Strontium co-doping on bioactivity, cytotoxicity and antibacterial activity and found that, the overall properties are enhanced. However, only a few studies have focused on multifunctional Cu–Sr co-doped bioactive glasses prepared by either melt quenching or the sol–gel route. In the previous studies, doping Cu and Sr improved bioactivity and hydroxyapatite formation. Wherein Sr act as network modifier that enhances the glass dissolution thus aids to enhanced hydroxyapatite layer formation. Whereas, Cu can further influence surface reactions and protein adsorption. Together, they improve bioactive surface layer formation, promoting better cell attachment and bonding with native bone. Cu provides strong antibacterial activity, reducing infection risk at the implantation site. Sr helps stabilize the glass network and improves mechanical integrity.

In the current study, Sr and Cu co-doped borosilicate glass system with the composition was developed to evaluate the effects of co-doping on mechanical, tribological, bioactive, and antibacterial properties for tissue engineering applications.

Materials and methodology

Materials

Glass samples were fabricated with the compositions mentioned in the Table 1. Calcium oxide and silicon dioxide were extracted from natural resources called egg shells and rice husk. Remaining all chemical reagents such as boric acid powder (FINAR), strontium carbonate (SRL), sodium bicarbonate (FINAR), copper oxide (SRL) with 99.9% purity were procured. The base glass composition, previously reported in³⁰, was used as a reference.

Table 1.

Borosilicate bioactive glass composition.

Glass code composition (mol%)
B2O3	SiO2	Na2O	CaO	CuO	SrO
BSG-2% Cu	31	18	24.5	24.5	2
BSG-2% Sr	31	18	24.5	24.5	-
BSG-1% CuSr	31	18	24.5	24.5	1
BSG-2% CuSr	31	18	24.5	24.5	2

Methodology

Extraction of silica and calcium oxide from natural waste

Waste-derived silica and CaO differ from conventional high-purity chemical precursors primarily in terms of sustainability, resource efficiency, and functional bioactivity, while maintaining comparable or even enhanced biological performance. From a sustainability perspective, waste-derived sources (e.g., agricultural residues, industrial by-products) significantly reduce reliance on energy-intensive mining and chemical purification processes. Their use lowers carbon footprint, diverts waste from landfills, and supports circular-economy principles, aligning well with the growing emphasis on environmentally responsible biomaterials development. In terms of bioactivity, waste-derived silica and CaO often contain trace biologically relevant elements (e.g., Mg, Na, K, P) that are absent in ultra-pure commercial precursors. These trace ions can synergistically enhance glass dissolution behaviour, promote hydroxyapatite formation, and stimulate osteogenic cell responses. For bone tissue regeneration, such multi-ionic release profiles are advantageous, as they more closely mimic the ionic complexity of natural bone mineral environments.

Figure 1. represents the extraction process of and calcium oxide powders from natural waste materials. The raw rice hull was collected from the rice mills and washed using distilled water. This washing process removes physical impurities like dust, soil, and organic residues to have a cleaner raw material for silica extraction. After the cleaning process, the rice husk is treated with hydrochloric acid (HCl). The husk is stirred and soaked in HCl for 6 h, which extracts metallic impurities such as iron, calcium, and magnesium to get the quality of the final product. The mixture is filtered in order to separate the acid solution from the treated rice husk. This process eliminates the acid and dissolved impurities, leaving only the purified husk. The filtered substance is then deposited into a furnace and calcined at 700 °C for 12 h. This calcination process burns the remaining organic materials, and a white si-rich ash forms as a result.

Fig. 1.

Schematic representation of the extraction process: (a) silica from rice husk biomass and (b) production of calcium oxide from eggshell waste.

The process of extracting calcium oxide (CaO) from eggshells comprises a series of thermal and mechanical treatment processes to transform the calcium carbonate content in the eggshells to calcium oxide. Initially, waste eggshells were collected and well cleaned with hot water to eliminate organic residues and membranes. The shells were dried in an oven after cleaning to remove moisture. Once dried those shells were grinded to get fine powder, which results in calcium carbonate (CaCO₃). The eggshell powder is then calcinated at 900 °C for 6 h in a muffle furnace. The calcium carbonate decomposes at high temperature to give calcium oxide (CaO). The importance of such materials to use in the fabrication of BGs is material uniqueness and bioactivity, similarly due to their sustainability and cost effectiveness.

Bioactive glass synthesis

The borosilicate glass samples fabrication through melt-quenching is described in the Fig. 2a in the initial step the precursor powders were precisely weighed and properly grinded with pestle mortar to get homogeneity. The powder mixture is then poured into a crucible and melted in a high-temperature muffle furnace at 1100 °C to obtain a uniform molten glass. At the same time, a metal mould is preheated at 400 °C to avoid thermal shock. After the molten glass achieves the desired viscosity and homogeneity, it is slowly poured into the preheated mould. immediately moved to another furnace that was maintained at 300 °C for 3 h and allowed to cool slowly to room temperature to form a dense, cylindrical borosilicate glass (BSG) sample. This fast-quenching process contributes towards the preservation of the amorphous structure and efficient enhancement of the material’s bioactive and mechanical properties for biomedical use. Photographic view of synthesized borosilicate bioactive glass samples as shown in Fig. 2b.

Fig. 2.

Schematic depiction of (a) the BSG fabrication process and (b) the visual appearance of the fabricated BSG samples.

On the other hand, Porosity and trapped gas bubbles were minimized through careful control of raw material preparation and melting parameters. The precursors were thoroughly dried and finely ground to ensure compositional homogeneity and to reduce gas evolution during heating. A stepwise melting schedule with an initial decarbonation stage was employed to allow complete removal of CO₂ and other volatile species before reaching the final melting temperature. The melts were held at high temperature for sufficient time to promote bubble coalescence and escape, followed by casting onto a preheated mold and controlled annealing to prevent thermal shock and residual porosity. This combination of precursor conditioning, optimized thermal treatment, and controlled cooling effectively reduced trapped gas bubbles and bulk porosity in the final glasses.

Characterization- physical, mechanical and tribological studies

The experimental density, theoretical density, void fraction, hardness, compression and wear were studied by using following principles and corresponding equations. The experimental density(ρ_e) of all glass samples was determined using Archimedes’ principle, with water serving as the buoyant medium.

where, w_a is the weight in air, and w_b is the weight in water.

The theoretical density () and Void fractions are measured by following equations

where, x_i = mole fraction & ρ_i = density of oxides in glass samples.

wherein, is theoretical density, is actual desnity.

The samples hardness was determined Using a Vickers microhardness tester (VH1-MDX Economet Chennai Metco, India). In order to perform test, cylindrical glass samples with dimensions of 15 mm in length and 11 mm in diameter were used. For each sample, the test was conducted five times with a 1 kg force applied for 15 s each time, and the average value of hardness were noted. The samples were polished in prior to testing to provide a flat surface and better visibility of the indentation. A Universal Testing Machine (UTM) from (Advanced Equipment—AE 30kN), with a maximum load capacity of 3000 kg and a feed rate of 2 mm/min, was used to assess compressive strength. Further, the abrasion wear of the cylindrical glass samples was evaluated using a pin-on-disc tribometer (Magnum engineers, TE-165-LE). In this, the materials were set up in a pin-and-disk arrangement, wherein the pin was a small cylindrical glass sample and the disk was a larger flat one. The test was carried out following the ASTM-G99 standard and under room temperature. During the test, the disk rotated at a speed of 300 rpm, while the glass pin slid over a 200 m distance against SiC abrasive paper with a grit size of 150. Each cylindrical glass sample was tested at three different track radii (30, 45 & 60 mm). Scanning electron microscopy (SEM) was carried out to examine the alterations in surface morphology and to identify the prevailing wear mechanisms. Specific wear rate of specimens was calculated by measuring weight loss and using the following expression.

where ΔW, ρ_e, P and L represent weight loss (gm), experimental density (g/cc), applied load (N) and sliding distance (m) respectively.

Statistical analysis

Experiments were performed at a fixed sliding distance of 200 m and a sliding velocity of 1 m/s, but systematically applied load (5, 10, and 15 N), track radius (30, 45, and 60 mm), and material composition varied. Every test was carried out twice for reproducibility purposes and the mean of the two readings was utilized for analysis. The repeated measurement variation was kept within ± 3% to provide experimental consistency. The signal-to-noise (S/N) ratio was calculated to analyse the performance characteristics with a view to minimizing SWR), according to the "smaller-the-better" criterion. Experimental data were analyzed through Analysis of Variance (ANOVA) to check the statistical significance and effect of each factor and their interactions on SWR.

Biocompatibility studies

To assess the in-vitro bioactivity of the synthesized bioactive glasses, the simulated body fluid (SBF) was prepared according to the standard method established by Kokubo³¹, which closely simulates the ionic composition and concentration of human blood plasma. The glass powders were submerged in the prepared SBF solution with a concentration of 1.5 mg/mL, where there was adequate contact between the glass particles and the physiological medium³². This arrangement was intended to allow ion exchange and the subsequent growth and nucleation of hydroxyapatite on the glass surfaces so that their bioactivity could be evaluated under physiological-like conditions. In the present work, Fourier Transform Infrared Spectroscopy (FTIR) was done using a Bruker Alpha II spectrometer with the KBr pellet technique to obtain spectra between 500–4000 cm⁻¹. Powder X-ray Diffraction (XRD) was performed using a PANalytical X’Pert Powder diffractometer with CuKα radiation, scanned from 10° to 60° with a step size of 0.02° and a counting time of 50 s per step.

Biological studies

Cell viability

Cell viability was determined through MTT assay. MTT assay was performed as per protocol. After reaching 75% confluency, cells were seeded into 96 well plate with density of 10,000 cells per well and they were grown at 37 °C in CO₂ incubator, once cells get attached to the wells, they were treated with set of compositions containing Cu-2%, Sr-2%, Cu&Sr-1%, Cu&Sr-2% in dose dependent manner in serum free media. After 24 h, media was removed and fresh media along with 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well. After 4 h of incubation 100ul of DMSO (Dimethyl Sulfoxide) was added and incubated for 10 min. Varioskan™ LUX multimode microplate reade (Cat# VL0000D0, Thermofisher scientific, Massachusetts, United States of America) was used to take absorbance. Absorbance was taken at 570 nm and cell viability (%) was calculated.

2wt. % Cu, 2wt.% Sr, and 1wt.% CuSr were selected to evaluate the anti-bacterial studies due to their positive results in cell viability assay. Different anti-bacterial activity confirmation studies performed such as spread plate method, UV spectrophotometer measurements, and Fluorescence microscopy imaging.

Antibacterial activity assessment

Spread plate method the basic principle of the spread plate method is dilution and even distribution of the sample on the agar plate for colony counting, as a result, shown as colony-forming units per milliliter (CFU/mL). We used two pathogenic bacteria, Staphylococcus aureus (S.aureus) and Escherichia coli (E.Coli) K12 strain; the former is a gram-positive bacteria, and the other is a gram-negative bacteria in this antibacterial study. Both the bacteria were inoculated in LB media with 1% pre-inoculum and incubated aerobically at 37 °C overnight for pure bacterial culture. The regular antibiotic Ampicillin is a positive control. The anti-bacterial activity was measured for 3 compositions 2wt.% Cu, 2wt.% Sr, and 1wt.% CuSr with different concentrations of 6 mg (the concentration at which the compositions have shown positive results for cell viability assay), 25 mg & 50 mg of each composition had added to 2 ml of bacterial culture in 2 ml Eppendorf tubes, Mix the samples for 2–5 min so that ions from the compounds will dissolve into the bacterial suspension. Under the same working condition, the positive control antibiotic ampicillin of 50 mg was added to 2 ml bacteria suspension. Samples were incubated aerobically at 37 °C for 2 h. before spreading these samples onto the LB agar plates, bacterial culture suspension was serially diluted in distilled water to achieve a cell concentration of approx. 104 CFU/ml. 100 μl was taken from the diluted sample and spread to the LB agar plates, incubating at 37 °C for 24 h. The colonies formed counted as colony-forming units (CFU/ml), and the antibacterial activity of the samples was measured.

Optical density (OD) by UV spectrophotometer UV spectrophotometer is one of the most convenient methods to measure bacterial growth by measuring the optical density (OD) of bacterial cultures at 600 nm. All three compositions, 2wt.% Cu, 2wt.% Sr, and 1wt.% CuSr of 50 mg, were added to 5 ml of LB media and 0.5 ml of bacterial suspension mixture. After mixing for 2–5 min, the mixtures were kept for incubation for 24 h at 37 °C in aerobic conditions. The final optical density (OD) values were measured by a UV spectrometer at 600 nm. OD values calculated the bacterial inhibition % by using the below formula,

A_c = absorbance of bacterial culture untreated.

A_t = absorbance of the test sample (bacterial culture treated with compounds).

Fluorescence microscopy imaging the pathogenic bacteria, gram-negative Escherichia coli K12 strain, and gram-positive Staphylococcus aureus, were grown in LB media at 37 ⁰C overnight in aerobic conditions. 2 ml of bacterial suspension from the overnight culture was taken in 2 ml Eppendorf tubes to this, 2wt.% Cu, 2wt.% Sr, and 1wt.% CuSr of 50 mg of each sample added in separate tubes and incubated for 2 h at 37 °C. The control sample was prepared under the same condition without bio-glass. Fluorescent slide preparation- After 2 h of incubation, collect the tubes and centrifuge the bacterial cultures at 12,000 rpm for 15 min; keep the pellet, discard the supernatant, and wash the cell pellet with 1xPBS buffer. Again, centrifuge at 8000 rpm for 10 min without disturbing the cell pellet, and discard the supernatant, leaving a small volume for cell suspension. The cell pellet was re-suspended, a small fraction of suspension was placed at one end of the slide, and a clear smear was prepared. The slide was fixed with methanol and air-dried. Then, the fluorescent dye acridine orange stain of 50ug/ml was added to each slide and incubated in the dark for 2–3 min, the excess stain was removed by rinsing under tap water and air dried. A Nikon eclipse Ti₂ inverted fluorescent microscope captured fluorescence imaging of each slide.

Results and discussions

Crystallographic investigations of as prepared samples

Figure 3 illustrates the XRD and SEM analyses of natural silica and calcium oxide powders, highlighting their structural and morphological characteristics. Figure 3a The broad diffuse hump centred around 22–25⁰, confirms that the silica is amorphous followed the XRD pattern as same as results obtained by²⁸. Figure 3b shows the XRD pattern of calcium oxide powder with sharp and strong diffraction peaks corresponding to crystalline planes like (101), (211), and (222), which established the crystalline nature³². The SEM images also reinforce these results, Fig. 3c shows a flaky and porous morphology, which increases surface area and reactivity³³. Figure 3d indicates CaO particles of eggshells are in the form of dense, granular agglomerates, which agree with characteristic morphologies of heat-treated calcium oxide³⁴. The amorphous silica and crystalline CaO mixture is suitable for the production of bioglass since the CaO provides calcium ions that function as modifiers, breaking the network of Si to promote bioactivity and dissolution rate in bioactive glasses³⁵.

Fig. 3.

XRD patterns and SEM micrographs of silica and calcium oxide extracted from rice husk and eggshell, respectively.

Melt quenching at 1100 °C produces glasses with a highly homogeneous and dense network, as the elevated temperature ensures complete melting, dissolution of precursors, and removal of residual organics or carbonates. As a result, melt-quenched glasses generally exhibit fewer intrinsic defects, such as closed porosity, residual stresses, or unreacted phases, compared to sol–gel–derived glasses, which often retain nanoporosity, hydroxyl groups, and microstructural heterogeneities due to low-temperature processing. In sol–gel systems, these features can act as defect sites that promote cracking and reduce mechanical strength, although they may enhance bioactivity. Therefore, melt quenching at 1100 °C favors structural integrity and mechanical reliability, while sol–gel processing prioritizes high surface area and reactivity at the expense of increased defect density.

Mechanical properties

Based on the hardness values shown in Fig. 4c, the Cu- and/or Sr-doped borosilicate glasses exhibit hardness values in the range of 5.39–5.71 GPa. In comparison, benchmark silicate bioactive glasses such as 45S5 Bioglass® typically report Vickers hardness values of 4.5–5.0 GPa, while 58S sol–gel-derived glasses are generally lower, around 4.0–4.6 GPa, due to their higher porosity and lower network connectivity. Quantitatively, this corresponds to an approximate 8–25% enhancement in hardness relative to 45S5 and a 15–40% improvement compared to 58S, depending on composition. This mechanical strengthening can be attributed to the borosilicate network, which provides higher packing density and improved resistance to deformation while still maintaining bioactivity.

Fig. 4.

Variation in (a) density, (b) void fraction, (c) hardness, and (d) compressive strength of borosilicate bioactive glass (BSG) with different concentrations of Cu and Sr dopants.

The actual and theoretical density comparison of the glass samples is exhibited in the Fig. 4a. The more massive Cu and Sr ions (molar masses equal to 63.546 g/mol and 87.62 g/mol) of their atom configuration can be broken and local density fluctuations can be caused by various structural defects observed in glasses, including glass modifiers, hanging or fragmented bonds. These defects also contribute to the overall reduction in densities. The increased packing of atoms in the glass structure due to the higher coordination number of Cu and Sr ions increases the density in overall³⁶. It is remarkable that for every sample, the actual density of the glass is constantly less than the theoretical density. It’s possible that trapped gas bubbles or gaps were created during the glass fabrication process, resulting in a porous structure that lowers the density overall. The lowest actual density is noted for the BSG-2% Cu composition at 2.56 g/cm³, while for BSG-2% Cu & Sr, a slightly increased value of 2.65 g/cm³ is observed²⁹. This overall trend of the actual density being less than theoretical density can be explained by the existence of voids, porosity, and other melt-quenching-caused microstructural defects. Theoretical densities were estimated from constituent oxide molar volumes and molecular weights, assuming ideal packing and complete densification. Of all the samples, the BSG-2% Sr and BSG-2% CuSr compositions exhibited the highest theoretical and actual densities, reflecting improved atomic packing and lower free volume defects. This may be due to synergistic interactions between Sr²⁺ and Cu²⁺ ions in altering the borosilicate network, leading to more compact network cross-linking^30,35.

Fig. 4 (b) shows the calculated void fraction for different glass compositions. rise in void content is due to adding of strontium and copper percentages in borosilicate glass, this is because of the atomic arrangement. hence homogeneity of the glass structure got disturbed. Moreover, copper ions will have the tendency to affect the melting viscosity during the synthesis process. The lowest void fraction (1.51%) was for the BSG-1% CuSr sample, which suggests that this composition possessed the densest and most homogeneous structure among the examined samples. The reduced void fraction suggests a denser microstructure that could enhance mechanical strength and bioactivity performance. The relatively higher void fractions in the other compositions show slightly more open structures, which are possibly due to unoptimized processing conditions or compositional mismatches that inhibit densification.

Further, the Fig. 4c illustrates the change in hardness (in GPa) for four various BSG compositions, comprising single and co-doped samples. BSG-1% Cu & Sr exhibited the hardness values of 5.71 GPa. The lowest hardness value was that of the BSG-2% Cu sample, which was recorded as 5.39 GPa. The hardness value increase by Sr doping explained by the capability of Sr²⁺ ions to function as effective network modifiers by increasing cross-linking density in the borosilicate matrix. Structural toughening would likely lead to increased indentation resistance³⁹. The co-doped samples also showed enhanced hardness over the Cu doping system only, suggesting a synergistic effect between Cu²⁺ and Sr²⁺ ions. These findings are consistent with previously reported work in literature, in which Sr doping was found to increase the compactness and hardness of glass networks³⁰. While the values of hardness obtained in this study are lower than standard ceramic implants such as alumina or zirconia, they are much greater than the hardness of natural bone and thus show promise for use in biomedical applications where moderate mechanical strength is necessary in addition to bioactivity. Sr²⁺ ions may enhance structural stability and minimize brittleness, while Cu²⁺ ions are capable of enhancing cross linking within the glass structure and facilitating energy dissipation upon loading. Compressive failure in the glass is less abrupt due to crack growth being restrained under compressive stress⁴⁰ attributed to the improved network connectivity, reduced porosity, and enhanced energy dissipation mechanisms imparted by Sr²⁺ and Cu²⁺ ion doping³⁶. The result highlights the potential for load bearing biomedical applications. which indicates by adding more percentages of Cu²⁺ and Sr²⁺ elements in the borosilicate glass gives good mechanical strength also.

As it can be inferred from Table. 2 When compared with the native bone, a compressive strength of approximately 55 MPa falls well within the range of cancellous (trabecular) bone, which typically exhibits compressive strengths of 2–12 MPa, and therefore exceeds the minimum mechanical requirement for this bone type. However, it remains significantly lower than that of cortical bone, whose compressive strength generally ranges from 100–230 MPa. Consequently, the reported strength indicates that the material is mechanically suitable for non–load-bearing or moderately load-bearing applications, such as bone fillers or coatings in trabecular regions, but it would not be sufficient as a standalone material for replacing cortical bone without additional reinforcement or composite design. Though glasses are fundamentally brittle and their strength is predominantly controlled by defects, comparative assessments of density, hardness, and compressive strength are informative. In this work, Cu–Sr co-doping decreased void fraction and increased hardness and compressive strength, indicating superior densification. Tribological experiments also verified diminished wear and lower friction, reducing surface damage and debris production. Notably, these enhancements were accompanied by robust bioactivity through the development of hydroxyapatite, which highlights the promise of co-doped glasses as biomedical materials.

Table 2.

Mechanical properties of the bioactive glass.

S. No	Mechanical property	Orthopaedic requirement	Current study
1	Compressive strength (MPa)	Greater than 100	50.67–55.21
2	Hardness (GPa)	0.06–0.08	5.39–5.71
3	Specific wear rate(mm3/N·m)	10⁻5	0.013–0.027

Finally, the Fig. 4d demonstrates the compressive strength data of the identical BSG compositions. In this case, BSG-1% CuSr exhibited better mechanical performance with the highest compressive strength of 55.21 MPa. Which is potential as coatings or as part of composites/scaffolds, whereas BSG-2% Cu registered 50.67 MPa. The greater compressive strength in Co-doped systems can be attributed to the high ionic bond strength of Cu²⁺, Sr²⁺ with the glass network, which is expected to impart a denser, more networked glass structure and inhibit crack propagation under compressive stress³⁰. Although the compressive strength values are lower than cortical bone (which is usually 100–200 MPa), they remain in an acceptable level to be applied to non-load-bearing or low-load biomedical applications like scaffolds, coatings, or fillers in bone defects. Additionally, the mechanical performance is further supported by the biological benefits of Sr and Cu, known to promote osteogenesis and exhibit antibacterial properties, respectively.

It can be inferred from the Fig. 4c–d, the co-doped Cu–Sr samples consistently show higher mean hardness and compressive strength than the single-doped counterparts; however, the degree of statistical significance depends on the associated scatter. The hardness increases from 5.39–5.48 GPa (single-doped) to 5.71 GPa (1%CuSr) and 5.52 GPa (2%CuSr), while compressive strength rises from 50.7–52.4 MPa to 55.2 MPa and 52.7 MPa, respectively. Considering the overlap of error bars, the improvement observed for the 1%Cu&Sr co-doped glass is likely statistically significant (p < 0.05) compared to single-doped samples, whereas the difference between 2%CuSr and single-doped compositions appears marginal to moderate. Overall, co-doping introduces a clear positive trend in mechanical performance, with the most pronounced and statistically meaningful enhancement observed at the optimized (1%CuSr) composition.

Tribological studies

Figure 5. represents the tribological investigation at the 10N load indicated considerable variation in the wear performance of the borosilicate glass compositions doped with Cu, Sr, and combinations of both. Figure 5a presents a comparative visual comparison of wear behaviour for all compositions at different track radii 30, 45, and 60 mm. It is observed that, the BSG-2%Cu has highest specific wear rate with 0.027 mm³/N·m at track radius 30 mm, while the BSG-1% CuSr composition has the lowest wear value of 0.013mm³/N·m at 60 mm track radius. in addition, the CuSr-1% showed the lowest specific wear rate overall, indicating synergistic wear resistance due to co-dopants in the composition. Over-doping can weaken wear resistance by excessive alteration of the silicate network, whereby an elevated concentration of network modifiers (Cu²⁺ and Sr²⁺) breaks Si–O–Si bonds and favours the generation of non-bridging oxygens (NBOs), thus destabilizing the glass network. Moreover, an excess of network-modifying oxides beyond the optimal concentration resulted in diminished mechanical integrity and increased wear rates in the bioactive glasses³⁷. While bioactivity and ion release are of high concern with bioactive glass, tribological properties are also of relevance in applications which can encounter contact and abrasion. Wear changes surface topography and creates particulate debris, both of which can have effects on ion release kinetics and biological interactions. Therefore, conducted comparative sliding tests to determine if co-doping of Cu and Sr changes friction and wear behaviour and to comment on the potential implications for biomedical application.

Fig. 5.

(a) Variation in specific wear rate and (b–d) coefficient of friction as a function of sliding distance at different track radii (30, 45, and 60 mm) for various borosilicate bioactive glass compositions.

Additionally, Fig. 5b–d demonstrate the behaviour of the coefficient of friction (µ) for all compositions under a constant load and different sliding distances ranging from 0 to 200 m. It is found that, among all the tested compositions, the highest value of the coefficient of friction (µ ≈ 0.71) was consistently recorded by BSG-2%Cu, and the lowest value (µ ≈ 0.5) was seen in BSG-2%CuSr. During the initial stage of sliding, all the samples depicted a steep rise in friction coefficient because of initial asperity interaction.

The friction traces of co-doped glasses maintained a more stable and lower value along the sliding distance than single-doped ones. Although intermediate µ values (0.66 and 0.61, respectively) were represented by BSG-2%Sr and BSG-1%Cu&Sr, respectively, the higher Cu content dual-doped composition BSG-2%Cu&Sr exhibited optimal lubricity and surface stability. Such performance can be attributed to the development of protective tribolayers or solid lubrication effects due to microstructural interaction between Cu and Sr in the glass matrix³⁸.

Based on Fig. 5a and Table. 3, the Cu- and Sr-doped borosilicate glasses exhibit specific wear rates in the range of 0.013–0.027 mm³ N⁻¹ m⁻¹, depending on composition and track radius, with the lowest wear observed for the Cu–Sr co-doped glass. In comparison, reported wear rates for TiO₂-based implant coatings typically lie in the range of 10⁻⁴–10⁻³ mm³ N⁻¹ m⁻¹, reflecting their high hardness and excellent wear resistance, while hydroxyapatite (HA) coatings show higher wear rates, commonly 10⁻³–10⁻² mm³ N⁻¹ m⁻¹, due to their brittle nature and weak adhesion. Although the wear resistance of the present borosilicate glasses is lower than that of TiO₂ coatings, it is comparable to or better than conventional HA coatings, particularly when Cu and Sr are co-doped, indicating their suitability for bioactive and moderate-load tribological applications where biofunctionality is prioritized alongside acceptable wear performance.

Table 3.

Specific wear rate of the bioactive glass samples at various track radius.

S.No	Composition	Specific wear rate at 30 mm Track radius	Specific wear rate at 45 mm Track radius	Specific wear rate at 60 mm Track radius
1	BSG-2%Cu	0.027	0.023	0.019
2	BSG-2%Sr	0.022	0.021	0.017
3	BSG-1%CuSr	0.018	0.016	0.013
4	BSG-2%CuSr	0.021	0.018	0.014

Taguchi analysis of SWR

The Taguchi approach was used to optimize the tribological parameters by investigating the signal-to-noise (S/N) ratios, thus reducing the experimental trials, time, and expense. Experiments were planned employing an L₃₆ orthogonal array, and the results were statistically examined with MINITAB 17 software. S/N ratios for the specific wear rate (SWR) obtained using the "smaller-the-better" criterion. The mean S/N ratio at each level of the factors was compiled to find individual parameter influences, while corresponding main effect plots indicated trends in each response.

Analysis of variance

The results of the ANOVA for the specific wear rate of Sr²⁺ and Cu²⁺ ion-incorporated borate-based bioactive glass are summarized in Table 4. The analysis reveals that all three parameters material composition, track radius, and applied load have a statistically significant effect on the specific wear rate (P < 0.05). Among these, the applied load emerged as the most dominant factor, contributing 82.94% to the total variation in specific wear rate, with an F-value of 341.68. This indicates that the wear resistance of the bioactive glass is highly sensitive to changes in the applied load, as higher loads increase the contact stress at the sliding interface, leading to enhanced material removal.

Table 4.

ANOVA table for specific wear rate.

Source	DF	Seq SS	Contribution (%)	Adj SS	Adj MS	F-Value	P-Value
Material composition	3	0.000165	6.96	0.000165	0.000055	19.12	0.000
Track radius	2	0.000158	6.69	0.000158	0.000079	27.58	0.000
Load	2	0.001962	82.94	0.001962	0.000981	341.68	0.000
Error	28	0.000080	3.40	0.000080	0.000003	–	–
Total	35	0.002366	100.00	–	–	–	–

The material composition and track radius also showed significant effects, contributing 6.96% and 6.69%, respectively. The influence of material composition can be attributed to the incorporation of Sr²⁺ and Cu²⁺ ions, which modify the glass network structure and affect its mechanical strength and wear behavior. Meanwhile, variations in track radius alter the sliding distance and contact conditions, thereby influencing the wear mechanism. The low error contribution (3.40%) indicates minimal experimental variability and a good fit of the model to the experimental data. Overall, the results confirm that load is the primary governing factor in determining the specific wear rate, while composition and track radius play secondary yet significant roles in wear performance optimization.

Signal-to-Noise (S/N) ratio

In Fig. 6a, the main effects plot for means shows the variation of the average wear rate with respect to material composition, track radius, and applied load. It can be observed that the slope corresponding to the load is the steepest among all the parameters, indicating that load has the most significant influence on the wear rate³⁹. The rate of wear is significantly increased by an increase in load, while that of track radius and composition is comparatively smaller. In Figure b, the S/N ratios’ main effects plot (where smaller is better) also illustrates that load has the greatest influence on wear behaviour, as shown by the steep drop of the S/N ratio with increasing load. Material composition has moderate influence, and the least change is observed in track radius, indicating a decreased impact on wear performance. Generally, both plots verify that the applied load is the most influential in determining the wear rate of the borosilicate glass composites, followed by composition of the material and track radius.

Fig.6.

Effect of control factors on specific wear rate (a) mean (b) S/N ratio.

Regression equation

Interpretation of contour and surface plots of specific wear rate

The contour plot in Fig. 7 shows that the specific wear rate increases with increasing load for all compositions of materials. The darker areas represent higher wear rates, meaning that at higher loads, the material wears more. Of the compositions, samples with Cu–Sr combinations show comparatively lower wear rates than the individual Cu or Sr-doped samples, which indicates a synergistic effect of the double dopants in increasing wear resistance.

Fig.7.

Representation of the variation of specific wear rate (a) contour plot (b) surface plot.

The surface plot again supports this trend with a sharp increase in specific wear rate as load is increased. The surface gradient shows that load is the dominant parameter controlling wear behaviour, followed by material composition. The minimum specific wear rate is achieved for CuSr1% composition at lower load conditions. Generally, both plots show that the composites specific wear is greatly influenced by the load applied, and material composition optimized can significantly reduce wear under mechanical loads.

Morphology of worn surfaces of BSGs

The FE-SEM image of wear patterns on the surface of the borosilicate glass specimen is evident as shown in the Fig. 8. with indication of the sliding direction by marked arrows. Track lines, resulting from persistent sliding over alumina emery paper, are evident with recognizable indication of gradual roughening of the surface texture. The morphology of the worn surface of borosilicate glasses (Fig. 8a–d) shows grooves, cracks, pits, and debris particles, validating the presence of abrasive wear and brittle fracture simultaneously. Track lines in the direction of sliding signify ploughing action, while pits and rough surfaces reflect material pull-out and localized fracture. Third bodies in the form of embedded debris enhanced wear severity and surface roughness. As a whole, wear behavior is ruled by a synergy of abrasion, brittle fracture, and debris-supported interactions. Notably, CuSr-1 and CuSr-2 specimens with increased levels of copper oxide and strontium oxide have deeper track lines than Cu-2wt.% and Sr-2wt.%. Surface grooves and randomly distributed worn-out particles further damage surface integrity, due to sustained rotations during wear testing⁴⁰.

Fig. 8.

FE-SEM micrographs of worn surfaces of (a) Cu-2%, (b) Sr-2%, (c) CuSr-1%, and (d) CuSr-2% doped borosilicate bioactive glasses tested using P150 abrasive paper.

In case of individual copper and strontium glass systems, surfaces exhibit more extreme irregularities, deeper troughs, and pits as signs of higher abrasion and erosion, directly attributed to strontium presence in the mixture. 1%CuSr glass had some minimal wear characteristics, indicating improved mechanical integrity and better wear resistance. Therefore, the control of doping concentration is of primary importance for adjusting the wear resistance of glass systems. Excessive doping can cause defects or phase separation, compromising performance.

The SEM wear-track analyses clearly indicate the coexistence of abrasive, adhesive, and fatigue wear mechanisms, with their relative contribution depending on composition and sliding conditions. The presence of parallel grooves and well-defined track lines (Fig. a–d) is characteristic of abrasive wear, arising from hard asperities and entrapped debris during sliding. Adhesive wear is evidenced by material smearing, worn-out regions, and transfer layers, particularly visible as irregular surfaces and adhered debris particles (Fig. 8a–c). Additionally, the observation of microcracks, pits, and spallation features (Fig. b and d) suggests fatigue-driven damage, caused by repeated cyclic stresses leading to crack initiation and propagation along the wear track. Overall, the SEM features confirm a mixed wear mechanism, with abrasion being dominant and adhesion and fatigue contributing to material removal, especially in glasses with lower wear resistance.

Invitro studies

XRD analysis

X-ray diffraction spectra of borosilicate glass samples measured inprior and after immersion in SBF are shown in Fig. 9. The diffraction pattern at 0 days revealed a broad halo within the 20–35° range, which is typical for an amorphous glass structure, evidencing that no crystalline phases existed straight after synthesis. This amorphous character is critical in the case of bioactive glasses, as it supports ion exchange and the bonding potential of bioactive glasses with biological fluids. after 7 days of immersion in SBF, new crystalline reflections were found to appear at 2θ values of 25.82°, 32.01°, and 45.55° corresponding to the (002), (211), and (222) planes of hydroxyapatite, respectively, as identified using the JCPDS card no. 09–0432. The emergence of these peaks shows the initial stages of apatite nucleation on the glass surface in direct connection with the dissolution of Ca²⁺, Sr²⁺, and Cu²⁺ ions into the surrounding solution with subsequent re-precipitation in the form of calcium phosphate phases³⁶. With continuation of immersion time for 14 days, the peaks became sharper and more pronounced, reflecting continuous crystallization and growth of hydroxyapatite^41,42,43. The progressive evolution of clear-cut peaks indicates a high tendency of Cu and Sr dopant doped borosilicate glasses to produce a stable and crystalline apatite layer replicating the mineral content of natural bone. The combined impact of Cu and Sr dopants is seen to enhance nucleation and stabilization of hydroxyapatite at a faster rate, consistent with their role of enhanced bioactivity²⁴. The above results further support the FTIR findings, hence proving that the synthesized glasses have the capacity to cause the growth of a bone-like apatite layer upon exposure to physiological conditions.

Fig. 9.

XRD analysis of borosilicate bioactive glasses at different immersion times in SBF: (a) 0 days, (b) 7 days and (c) 14 days.

FTIR analysis

FTIR spectra of the borosilicate glass specimens before and after 7- and 14-days immersion in SBF are shown in Fig. 10. Before immersion, there were characteristic silicate network absorption bands at ~ 703–789 cm⁻¹ (bending by Si–O–Si) and ~ 965–1060 cm⁻¹ (Si–O stretching for Q²/Q³ units and non-bridging oxygens)⁴⁴. A wide band at ~ 3450 cm⁻¹ and a maximum at ~ 1642 cm⁻¹ are associated with O–H stretching and H–O–H bending vibrations, respectively, representing adsorbed water molecules on the glass surface⁴⁵. After immersion for 7 days, new bands at ~ 565–606 cm⁻¹ are found, which can be attributed to P–O bending vibrations of phosphate groups and confirm the onset of apatite layer development. The continuity of the silicate network in the 789–1038 cm⁻¹ region indicates continuing partial dissolution of the glass matrix⁴⁶.

Fig. 10.

FTIR spectra of borosilicate bioactive glasses: (a) before immersion, (b) after 7 days in SBF, and (c) after 14 days in SBF.

By 14 days, the bands associated with phosphates at ~ 560–604 cm⁻¹ intensified, together with a shift in the principal stretching band to ~ 1046 cm⁻¹, as expected for the development of a crystalline hydroxyapatite layer. Other bands relating to carbonate content at ~ 1409–1642 cm⁻¹ suggest the partial replacement of phosphate by carbonate groups in the apatite lattice structure⁴⁷.The wide O–H stretching band at ~ 3450 cm⁻¹ also increased and further confirms the development of a bone-like apatite layer that is hydrated. These findings validate that Cu- and Sr-substituted borosilicate glasses are bioactive on their surface and capable of nucleating hydroxyapatite when exposed to SBF.

.Elemental compositional analysis

The Ca/P ratio data shown in Fig. S1–S8 a clear compositional- and time-dependent evolution toward stoichiometric hydroxyapatite (Ca/P ≈ 1.67). As summarized in Table X, Cu- and Sr-only doped glasses exhibited relatively higher Ca/P ratios after 7 days (e.g., 2.48 for Cu-2% and 2.22 for Sr-2%), which gradually decreased after 14 days, indicating progressive calcium incorporation and apatite maturation. In contrast, the Cu–Sr co-doped glasses displayed consistently lower Ca/P ratios, reaching values as low as 1.75 after 14 days for CuSr-2%, suggesting faster and more effective HA formation kinetics. The accelerated reduction in Ca/P ratio for co-doped compositions reflects enhanced surface reactivity and more rapid transformation toward bone-like apatite.

Similarly, the crystallite size analysis provides quantitative evidence of HA nucleation and growth kinetics across compositions. All samples exhibited nanoscale crystallite sizes, confirming the formation of poorly crystalline, biologically relevant HA. The crystallite size showed only minor variation with immersion time (7 to 14 days), remaining within the range of ~ 0.34–0.39 nm for Cu-, Sr-, and CuSr-doped glasses. This indicates that while immersion time promotes chemical maturation (as reflected by Ca/P ratios), the HA layer remains nanocrystalline, which is desirable for bioactivity. The slightly more uniform crystallite sizes observed for Cu–Sr co-doped samples further suggest controlled HA growth kinetics.

Cell cultural study and antibacterial activity

Cytocompatibility

The human osteosarcoma cell line MG-63 was procured from the National Centre for Cell Science (NCCS), Pune, India. The line is part of the NCCS authenticated cell repository and was supplied with accompanying documentation detailing its provenance and quality-control status. MG-63 cells originate from a primary osteosarcoma derived from a 14-year-old male patient and are widely used as an osteoblast-like in vitro model in bone and cancer research.

Figure 11. cytotoxicity response of MG-63 osteoblast-like cells for different glass compositions, demonstrating their relative biocompatibility by invitro studies. Since the materials are being synthesized for biomedical applications, they must be noncytotoxic toward MG-63 cells ²⁷.

Fig. 11.

Cytocompatibility of osteoblast-like MG-63 cells treated to Cu and Sr doped borosilicate bioactive glasses.

The in vitro cytotoxicity profile for the set of compositions was assessed using a standard cell viability assay, which were mentioned in methodology was evaluated and found that Cu-2% showed enhanced cell growth with cell viability reaching approximately 140%, due to Cu²⁺ ions, which may stimulate cellular proliferation through mechanisms involving angiogenesis and upregulation of growth factors⁴⁸. Sr-2% contained bioactive glass composition also exhibited enhanced cell viability of nearly 122%, consistent with the known osteogenic and mitogenic roles of Sr²⁺ ions in promoting cell proliferation and differentiation, particularly osteoblast like cells⁴⁹. Co-doped 1%CuSr retained cell viability around 130%, suggesting a synergistic or additive action at a specific concentration, most likely balancing cytocompatibility and rate of ion release. Cu&Sr-2% had moderate decrease in cell viability compared to untreated controls. Proposing concentration-dependent cytotoxic threshold, most likely from excessive ionic releasing and causing oxidative stress or cellular homeostatic disruption. In total, all Cu- and Sr-doped samples were found to have good cell viability and proliferation, indicating the potential of the glasses developed for bone tissue engineering.

Antibacterial activity

Spread plate method

Figure 12. illustrates the antibacterial activity of bioactive glass samples doped with copper, strontium and co-doped CuSr against Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus). Control (A) exhibits heavy growth of bacteria, i.e., the normal growth of E.coli in the absence of any antibacterial substance. B and C exhibit moderate growth inhibition of bacteria, while D exhibits strong antibacterial activity with much clearer plate. Antibacterial activity is enhanced with increasing dosage of Cu-doped glass. Copper ions interfere with bacterial membranes and produce reactive oxygen species (ROS), causing bacterial death⁵⁰. E, F and G demonstrate low to moderate inhibition. F and G have visible bacterial colonies suggesting weak antibacterial activity. Sr doped glasses are obviously seen to have reduced antibacterial activity in comparison to Cu. Strontium boosts osteogenic activity mainly but has less intense antibacterial behavior⁴⁶. Plates (H-J) are significantly clearer compared to single Sr doped samples even Cu-doped at reduced doses. J (50 mg) exhibits strong inhibition, almost equaling the optimal Cu-only sample. It is evidently proved that co-doping synergistically improves antibacterial activity. Copper contributes bactericidal activity, and Sr can help cell compatibility, forming a balanced bioactive material.

Fig. 12.

Antibacterial activity of various concentrations of doped and co-doped BSGs against E. coli and S. aureus strains after 24 h of incubation at 37 °C.

Notably, even the CuSr composition at low dose (6 mg) remained highly antibacterial, highlighting the synergistic possibility of the pairing of bioactive ions towards dual action antibacterial from Cu and osteogenic promotion from Sr. The findings clearly show that copper-containing formulations are far more effective than Sr alone in fighting S. aureus. Control (A) represents the extensive growth of S.aureus colonies confirms a healthy bacterial population in the absence of antibacterial agents. Where B indicates moderate number of colonies at 6 mg concentration of bioactive glass, shown noticeable antibacterial effect. C indicates fewer colonies means shown better antibacterial activity with increased in dose. Similarly D exhibited, strong inhibition with a smaller number of colonies. Therefore, Cu²⁺ ions released from the glass show effective antibacterial activity against S.aureus, likely by disrupting cell membranes, producing reactive oxygen species and inhibiting enzyme activity. Sr-2% doped glass of 6 mg concentration shows high colony count which indicates weak inhibition. As the concentration increase to 25 mg, a smaller number of colonies, similarly colony count is high at 50 mg concentration when compared to Cu and CuSr samples. Hence Sr²⁺ ions provide minimal antibacterial effect against S.aureus. strontium is known for osteogenic properties than antimicrobial activity. H indicates 1%CuSr exhibited moderate inhibition bacterial colonies at 6 mg concentration of Bioglass. In case of (I) Considerable reduction and better than either single dopant at same dose. J(50 mg) represents very strong inhibition, i.e. almost complete bacterial suppression. CuSr combination exhibits a synergistic effect with significant antibacterial activity at lower doses (6 mg), showing that it could find application in biomedical fields such as wound dressings and antimicrobial coatings where metal ion concentrations are to be low. Interestingly, Sr alone was not as effective, consistent with literature implying that Sr’s main function is in tissue compatibility and bone regeneration, not direct antimicrobial action. But at higher concentrations (50 mg), it still exhibits moderate antibacterial activity, perhaps through enzyme interference or osmotic stress. CuSr composites offer a balanced approach, combining antibacterial efficacy with possible biocompatibility benefits. Strontium alone is not sufficient, but may enhance composite performance.

From Fig. 12. it was concluded that all the 3 compositions are showing more anti-bacterial activity on gram positive bacteria Staphylococcus aureus compared to gram negative bacteria Escherichia coli. Even the smallest concentration of 6 mg had shown its activity on S.aureus but the same concentration had no effect on E. coli. When the concentration of the 3 compositions increased to 50 mg they have shown almost equal anti-bacterial activity on both the pathogenic bacteria. Among tested compositions, 2%Cu doped glass showed the most potent antibacterial activity, followed by 1wt.% CuSr. The results suggest that Cu²⁺ has a major influence on antimicrobial activity, and co-doping with Sr²⁺ can introduce a tunable and synergistic effect on the basis of concentration. These results affirm the promise of doped Borosilicate BG systems as multifunctional biomaterials with both antibacterial and osteogenic functions, particularly well-suited for use in orthopedic and dental applications.

Figure 13. and Table. 5 lists the antibacterial activity of the copper (Cu), strontium (Sr) doped and codoped borosilicate bioactive glass compositions was tested against gram negative and gram positive strains using colony-forming unit (CFU) assay. The results were expressed as CFU/ml(104) for different concentrations of 6 mg, 25 mg and 50 mg of each composition. The 1%CuSr codoped BSG had a dose dependent antibacterial activity. At the lowest dose of 6 mg, CFUs of E.coli and S.aureus were decreased to 30 and 12 × 104 respectively. The dose was increased to 25 mg, which further lowered CFU counts to 20 (E.coli) and 10 (S.aureus), while 50 mg nearly completely prevented bacterial growth, and CFU counts were 0 and 4.

Fig. 13.

Antibacterial activity, as determined by CFU counts and inhibition rate, of 2% Cu, 2% Sr, and 1% CuSr-doped borosilicate glass samples against E. coli and S. aureus.

Table 5.

Inhibition Rate .

S.No	Composition	E.coli	S.aureus
1	Control	0	0
2	BSG-2%Cu	50	75
3	BSG-2%Sr	10	15
4	BSG-1%CuSr	55	95

These findings showed that the synergistic effect of Cu²⁺ and Sr²⁺ ions could increase membrane disruption and reactive oxygen species (ROS) production and play a role in broad spectrum antibacterial activity. 2wt.% Cu doped sample excellent bactericidal action against both strains. whereas CFU values were equal for E.coli from 6 to 25 mg, S.aureus reduced from 23 to 15. Bacterial inhibition was maximum at 50 mg, which completely inhibited E.coli and almost inhibited S.aureus 2 × 104 CFU/mL. Sr-2% doped glass also exhibited dose dependent antimicrobial action, though weaker than Cu-2% composition.

Optical density (OD) by UV spectrophotometer

Figure 13d. Illustrates the bacterial inhibition rate of 2wt.% Cu, 2wt.% Sr, and 1wt.% CuSr-doped borosilicate glass samples against E. coli and S. aureus and calculated using the equation mentioned in the previous section (materials and methods). Three samples were treated at 50 mg concentration with gram positive and gram-negative bacteria and noted the results. CuSr-1% glass system had more anti-bacterial activity on both pathogenic bacteria E. coli & S. aureus. The co-doped BSG with 1%CuSr exhibited the better antibacterial activity, achieving inhibition rates of 55% for E.coli and a remarkable 95% for S.aureus. this synergistic effect likely arises from the combined biochemical roles of Cu²⁺ and Sr²⁺, wherein Cu provides potent antimicrobial effects while Sr potentially modulates ion release and biocompatibility, enhancing Cu efficacy over time. Notably 1wt.% CuSr codoped outperformed individual dopants, especially against S.aureus, suggesting a promising strategy for developing bioactive glass systems with dual functionality enhanced antibacterial activity and favourable cytocompatibility.

Fluorescent imaging

After preparing fluorescent slides of the compositions treated with pathogenic bacteria, imaging was done using a Nikon eclipse Ti₂ inverted fluorescent microscope.

Bioglass compositions had clearly shown their effect in changing the structure, shape, and size of both the bacteria⁴⁵; these changes in both the bacteria prove that compositions 2wt.% Cu, 2wt.% Sr and 1wt.% CuSr had notable anti-bacterial activity against both the pathogenic bacteria. Compared with gram-negative Escherichia coli K12, the compositions had intense anti-bacterial activity against gram-positive bacteria Staphylococcus aureus. The difference in anti-bacterial activity of the compositions against both bacteria was distinctly shown in the Fig. 14.

Fig. 14.

Fluorescence microscopy images showing E. coli and S. aureus activity for (A) Control, (B) 2wt.% Cu, (C) 2wt.% Sr, and (D) 1wt.% CuSr-doped bioactive glasses, stained with acridine orange dye.

On the other hand, the antibacterial activity of the Cu–Sr–doped borosilicate glasses is primarily attributed to ion release–mediated mechanisms, with secondary contributions from membrane disruption, rather than direct evidence of reactive oxygen species (ROS) generation. Copper ions (Cu²⁺) released from the glass matrix are well known to exert antibacterial effects by interacting with bacterial cell membranes, leading to increased membrane permeability, protein dysfunction, and intracellular damage. Strontium ions (Sr²⁺), while not strongly bactericidal on their own, are reported to enhance antibacterial performance by modifying surface chemistry and ionic strength, thereby facilitating Cu²⁺ interaction with bacterial cells. The significantly enhanced inhibition observed for 1% CuSr co-doped glass (Fig. 13d) suggests a synergistic effect driven by combined ion release. The fluorescence microscopy images (Fig. 14) show a marked reduction in bacterial density and disrupted cellular morphology for both E. coli and S. aureus upon exposure to Cu-, Sr-, and CuSr-doped glasses, supporting a mechanism involving cell membrane damage and loss of viability. However, no direct assays for ROS generation (e.g., DCFH-DA or oxidative stress markers) were conducted in this study; therefore, ROS-mediated antibacterial action cannot be conclusively confirmed.

In summary, based on CFU reduction trends and fluorescence observations, the antibacterial behavior of Cu–Sr–doped borosilicate glasses is predominantly governed by Cu²⁺/Sr²⁺ ion release–induced membrane disruption, with potential but unverified contributions from oxidative stress. Further studies involving ion release profiling and ROS quantification are planned to fully elucidate the antibacterial mechanism.

Conclusions

Borosilicate glass samples were successfully synthesized via the conventional melt-quenching method using partially biogenic precursors such as rice husk for SiO₂ and eggshells for CaO. Mechanical and tribological assessments, including hardness, compressive strength, and wear resistance, were conducted. Co-doping with Cu and Sr increased the actual density from 2.61 to 2.65 g/cm³. The BSG-1wt.% CuSr sample exhibited the highest hardness (5.71 GPa) and compressive strength (55.21 MPa). However, at 2%CuSr, network disruption by Cu²⁺ and Sr²⁺ ions reduced the mechanical properties. With increasing track radius, specific wear rate decreased across all samples; yet, as CuSr content increased from 1 to 2wt.%, the wear rate increased slightly from 0.013 to 0.014 mm³/N·m at 60 mm track radius, due to greater material removal. SEM of worn surfaces showed smoother, denser morphology for 1%CuSr, indicating better micro-fracture resistance. In contrast, the 2% sample showed rougher surfaces, consistent with reduced stability. The synthesized borosilicate bioactive glasses showed strong in-vitro bioactivity, as indicated by the nucleation and development of hydroxyapatite layers on the surface of the glasses, as verified by FTIR (phosphate and hydroxyl vibrational signals) and XRD (sharp hydroxyapatite crystalline peaks) after immersing in SBF for 7 and 14 days thus confirming their suitability for bone-bonding purposes. Cu/Sr-doped glasses demonstrated excellent cytocompatibility in MG-63 cells and significant antibacterial activity, especially against E. coli and S. aureus, even at low concentrations. The 1wt.% CuSr and 2wt.% Cu samples showed the highest inhibition at 50 mg, confirming dose-dependent antibacterial efficacy via Cu-mediated membrane disruption and oxidative stress. Combining enhanced mechanical strength, cytocompatibility, and broad-spectrum antibacterial activity, BSG-1wt.% CuSr emerges as a promising material for orthopaedic implants and bone tissue engineering applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Locherla Daloji: Writing—Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Resources; Validation; Writing—original draft; Raghavendra Gujjala: Conceptualization, Formal analysis, Data curation, Supervision, Investigation, Writing—review & editing. Shakuntala Ojha: Data curation, Validation, Methodology, Formal analysis. G. Dheeraj Kumar: Methodology; Resources, Data curation. Prakash Saudagar: Investigation, Resources; Validation. Manuela Reben: Resources, Funding acquisition, Formal analysis. P. Syam Prasad: Investigation, Validation, Writing—review & editing. Aswani Kumar Bandaru: Investigation, Validation, Writing—review & editing.

Funding

The authors declare that no funds, grants, or other support.

Data availability

The data is made available through the domain [https://zenodo.org/records/17640952].

Declarations

Competing interests

The authors declare no competing interests.