Zn containing mesoporous bioglasses with enhanced textural and antibacterial properties produced by three modifications of the sol-gel method

Journal of Materials Science: Materials in Medicine (2025) 36:105 https://doi.org/10.1007/s10856-025-06960-w BIOMATERIALS SYNTHESIS AND CHARACTERIZATION Original Research Zn containing mesoporous bioglasses with enhanced textural and antibacterial properties produced by three modifications of the solgel method Anastasia Beketova 1,2 ●Georgia K. Pouroutzidou3●Eleana Kontonasaki3●Veronica Giourieva4,5 ● Krisjanis Smits1,2 ●Valentina Stepanova1,2 ●Ioannis Tsamesidis3●Rajan Choudhary6●Kristaps Rubenis1,2 ● Toms Valdemars Eiduks7●Maria Bousnaki3●Dagnija Loca1,2 ●Rigini Papi4●Athanasia Pylostomou1,2 ● Janis Locs 1,2 Received: 18 May 2025 / Accepted: 21 October 2025 © The Author(s) 2025 Abstract Mesoporous bioactive glasses (MBGs) have potential applications in bone tissue regeneration around tooth implant and local drug delivery. Small amounts of zinc added to their composition could additionally provide antibacterial and ossteoinductive and anti-inflammatory properties. In this study, zinc-containing mesoporous bioactive glasses (5ZnO–25CaO–70SiO₂) were synthesised using three modified surfactant-assisted sol-gel methods: dilute water (MZ1), Stöber (MZ2), and microemulsionassisted (MZ3). X-ray diffraction (XRD) analysis confirmed that MZ1 and MZ3 were amorphous, while MZ2 exhibited a ZnO crystalline phase. The synthesised particles showed uniform morphology with sizes ranging from 10 to 500 nm. Brunauer–Emmett–Teller (BET) analysis revealed that MZ1 had the highest specific surface area (726 m²/g), approximately 4.1 times higher than MZ3 (176 m²/g). Haemolysis testing showed that MZ1 and MZ2 were non-haemolytic, whereas MZ3 caused lysis of erythrocytes. All samples were biocompatible with periodontal ligament fibroblasts, maintaining cell viability above 80% after three days of incubation. Antibacterial assays indicated that MZ2 exhibited over 60% inhibition of P. intermedia in a dose-dependent manner, but only ~20% inhibition of P. gingivalis. MZ2 demonstrated a bacteriostatic effect and was most effective in reducing anaerobic bacterial populations among all tested groups. These results highlight the potential of Zn-containing mesoporous bioactive glasses as multifunctional biomaterials for periodontal tissue engineering, suitable for such applications as scaffolds, bone cements, bone-filling granules, and antibacterial implant coatings. Furthermore, MZ2 material due to its antimicrobial properties, can potentially be a material of choice in periodontitis/periimplantitis therapy applications. *Anastasia Beketova [email protected] *Janis Locs [email protected] 1Institute of Biomaterials and Bioengineering, Faculty of Natural Sciences and Technology, Riga Technical University, Paula Valdena Street 3 k-1, LV-1048 Riga, Latvia 2Baltic Biomaterials Centre of Excellence, Headquarters at Riga Technical University, Riga, Latvia 3Department of Prosthodontics, Faculty of Health Sciences, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece 4Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 5Department of Botany, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 6School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland 7Institute of Materials and Surface Technologies, Riga Technical University, P. Valdena str. 7, LV-1048 Riga, Latvia 1234567890();,: 1234567890();,: Graphical Abstract 1 Introduction Antibiotic resistance has become a global challenge, according to the World Health Organisation. Every year, at least 700,000 people worldwide pass away from infections caused by resistant bacteria [1]. A reason for that is the prolonged use and overuse of antibiotics and implantation of medical devices, which might form a bacterial biofilm on their surface, becoming a source of chronic infection [2]. Recently, an increase of antibiotic resistance in patients with infection around tooth implants (peri-implantitis) was reported [3]. Peri-implantitis is a biofilm-induced infection caused by anaerobic oral microflora, such as P. gingivalis and P. intermedia. These bacteria are also responsible for such symptoms as gum bleeding, implant mobility and oral malodour. In 56% of cases, this infection leads to implant failure [4]. Considering that the dissemination of oral pathogenic bacteria might implicate cardiovascular diseases [5], there is a strong need to develop alternative antibacterial therapeutic approaches to eliminate antibiotic-resistant oral pathogens. One of the novel strategies to prevent colonisation of the resistant Gram-negative anaerobic bacteria on the implant surface is the application of antibacterial bioactive glasses in situ to achieve a bacteria-free environment for reosseointegration [2]. Bioactive glasses are widely used as bone grafting materials in dentistry (e.g. Perioglas®, Novabone®) due to their osteoconductive and osteoinductive properties [6,7]. In the last two decades, nanoscale bioactive glasses with mesoporous ordered structure evolved as the latest generation of smart multifunctional materials. Mesoporous bioactive glasses (MBGs) have been proposed as bioactive fillers and drug carriers either alone or combined with other materials, taking advantage of their unique porous structure to load drugs, biomolecules and antibacterial ions [8] MBGs are produced by addition of structure-directing agents into sol-gel chemistry, so that they acquire a large pore volume, a highly organised structure of channel-like pores in the 2–50 nm range, and a large specific surface area (600–1500 m2)[9,10]. MBGs in the binary system SiO2-CaO exhibit better apatite-forming capacity, and superior biological properties, as compared to non-mesoporous sol-gel or melt-derived bioactive glasses (BGs) [11]. Their enhanced texture facilitates quick ion exchange between the glass surface and the surrounding biological media, leading to fast supersaturation and precipitation of surface hydroxyapatite. Furthermore, nanodimensional features of mesoporous glasses favour their direct interactions with cells and components of extracellular matrix. MBGs can incorporate different active metal ions like Zn, Ag, Cu, Ce, Ga to boost their antibacterial properties [6,11–13]. These antibacterial MBGs can prevent biofilm formation and induce bacteria-free environment for rapid healing. The mechanism behind their antibacterial action could be explained by rapid therapeutic ion release, and local changes of pH. The unique surface characteristics of MBGs could additionally provide non-specific action against bacterial membrane. In contrast to conventional metal oxide nanoparticles, such antibacterial glass formulations are non-toxic and biocompatible. Therefore, antibacterial MBGs seem promising materials for treatment of antibiotic-resistant peri-implant infection and promotion of de novo bone formation around tooth implant. Among various antibacterial ions, zinc is known for its relative low toxicity and excellent antibacterial and antiinflammatory properties. Zinc is a necessary trace element in the human body that comes from food sources. The total amount of zinc in the body is approximately 1.5 g in women and 2.5 g in men [14]. Zinc participates in cellular metabolism, including DNA and protein synthesis, cell signalling and division. 30% of Zn is accumulated in human 105 Page 2 of 16 Journal of Materials Science: Materials in Medicine (2025) 36:105 bones and it is actively involved in the synthesis of the collagen matrix, mineralisation, and bone remodelling [15]. Zinc also contributes to wound healing processes by promoting fibroblast and epithelial cell migration and proliferation [16]. Due to its antibacterial properties Zn is also formulated into many oral care products for control over plaque and calculus, as the main causes of periodontal disease [17]. In dentistry, zinc oxide eugenol cements are indicated for temporary tooth filling and indirect pulp capping in case of pulp inflammation. They relieve pain and elicit bacteriostatic and antiseptic action. Zinc has a major role in immune function regulation, bacterial infection control, inflammatory response balancing, and oxidative stress reduction—all of which are linked to the pathophysiology of periodontal disease [18]. Furthermore, the potential to inhibit biofilm formation of zinc-containing glasses has been reported recently [19]. Mesoporous bioactive glasses are typically produced by the sol-gel method with addition of pore-forming agents. Sol-gel technique has numerous advantages such as low processing temperatures, simplicity and possibility to tailor the materials for a specific application. In the most commonly used Stöber modification, a cationic surfactant cetyl trimethylammonium bromide, or CTAB, can be added to the synthesis solution to guide the formation of mesopores [20]. A silicate precursor tetraethyl orthosilicate (TEOS) and metal ion precursors are usually added into the water/ ethanol solution under basic conditions and carefully stirred. Then, the resulting nanoparticles are filtered, dried and calcined to form mesoporous nanoparticles. The Stöber technique may produce uniform MBG spheres, which typically have diameters between 100 and 400 nm [21]. By adjusting molar ratios, pH and added precursors it is possible to create a variety of nanoparticles with diverse morphologies and sizes. In the microemulsion assisted solgel process, organic molecules, such as ethyl acetate can also be added during the synthesis to achieve a more uniform shape of the particles or to increase their dispersity. Unfortunately, using traditional sol-gel based methods to synthesise highly dispersed MBGs with multiple metallic ions remains difficult because metallic precursors can weaken the stability of nanoparticles during synthesis, which leads to aggregation and irregularity in the final MBGs. To date, only a few studies exist regarding Zn-doped MBGs (Zn-MBGs). Neščáková et al. synthesised highly dispersed Zn-containing MBGs in the CaO-SiO2system using an emulsion assisted sol-gel method [22]. However, they had a relatively low specific surface area, and their antibacterial properties were not investigated. Another study reported that the extract of Zn-doped binary SiO2-CaO MBGs received after incubation in culture medium at a concentration of 0.1 mg/mL exhibited anti-inflammatory properties and promoted the osteogenic capacity of rat bone marrow stromal cells (BMSCs) [23]. Naruphontjirakul et al. used strontium (Sr)- and zinc (Zn) to partially substitute calcium and synthesised MBG nanoparticles (MBGNs) in the binary system SiO2-CaO using an emulsion assisted sol gel method and tested the antibacterial activity using the disc diffusion method against E. coli and S. aureus [24]. They found statistically significant increase in antibacterial effect against Gram-negative bacteria (E. coli). Pourshahrestani et al. applied the same method for doping MBG with Zn with molar contents of 1, 2 and 4% as substitutes of either calcium and silicon and observed a slight decrease of bacterial viability of S. aureus and E. coli at the highest concentrations of their extracts [25]. In another attempt, zinc-doped bioactive glass porous nanoparticles in the system 60SiO230CaO5ZnO-5P2O5 (mol%) were, developed and incorporated into alginate/ chitosan composite hydrogel as a wound dressing [26]. The authors observed efficient antibacterial effect against E. coli and S. aureus and wound healing in an in vivo rat model. Summarising the aforementioned reports, all the ZnMBGs were synthesised by the emulsion assisted sol-gel method, and exhibited promising antibacterial, osteogenic, and anti-inflammatory properties, although issues regarding the optimisation of their composition and improvement of antibacterial effectiveness are still open for further research, to fully explore their clinical potential. There are almost no reports on the use of the modified Stöber synthesis method to produce such materials, while there are no available data on the antibacterial properties Zn-doped MBGs with oral pathogens, that would exploit their potential as therapeutic agents in the treatment of peri-implantitis and periodontitis. The aim of this study was to synthesise Zn containing MBGs (5ZnO-25CaO-70SiO2%mol) using three different modifications of the surfactant-assisted sol-gel method: (a) modified Stöber method under dilute water conditions [27,28] (b) modified Stöber method with addition of ethanol as a solvent [29] and (c) the microemulsion-assisted sol-gel approach [30], and to compare their hemocompatibility, biocompatibility and antibacterial properties against anaerobic bacteria related to periodontitis and periimplantitis. 2 Materials and methods 2.1 Synthesis of Zn-containing mesoporous bioactive glasses The reactants that were used for the synthesis: sodium hydroxide (NaOH), ammonium hydroxide 25%, hexadecyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), ethyl acetate, Ca(NO3)2.4H2O, Journal of Materials Science: Materials in Medicine (2025) 36:105 Page 3 of 16 105 Zn(NO3)2.6H2O were purchased from Sigma-Aldrich (now Merck KGaA, Darmstadt, Germany). Three types of Zn-containing mesoporous bioactive glasses (Zn-MBGNs) with the nominal composition 70SiO2–25CaO–5ZnO (mol%) were synthesised using three modifications of the surfactant-assisted sol-gel method described in the literature. MZ1 powder was synthesised using modified sol-gel method using water as a solvent. Initially, CTAB, a soft template for the mesoporous structure, was dissolved in the basic aqueous medium (pH=12), followed by the addition of TEOS as a source of Si and nitrate salts as a source of Ca and Zn metal ions. In detail, 0.315 g NaOH was dissolved in 785 ml of ultrapure water to achieve an alkaline medium (pH=12), where 1 gram of CTAB was dissolved. Then, 7.5 ml of TEOS were added to the mixture dropwise. After 30 min, the appropriate quantity of calcium nitrate tetrahydrate was added and mixed for 8 h at the T =80oC, followed by the addition of zinc nitrate hexahydrate and mixing for additional 1.5 h at the same temperature. The precipitate after washing was dried at 60 °C for 24 h and calcined at 550 °C for 5 h with a heating rate of 1 °C/min to remove the templating agent and nitrates. MZ2 material was produced by a modification of the sol-gel method with addition of ethanol as a solvent at room temperature [29]. The first step involved dissolving 1 g of the surfactant CTAB in the mixture of 150 ml of ultrapure water, 30 ml of ethanol, and 2 ml of ammonium hydroxide, which served as a catalyst. Following CTAB dissolution, the appropriate amounts of Ca(NO3)2.4H2O and Zn(NO3)2.6H2O were added simultaneously to the mixture and continuously stirred for 30 min. After that, 7.5 ml of TEOS was added dropwise and the resulting solution was stirred for additional 4 h. Then, the white precipitates were filtered and washed twice with distilled water. Samples were then dried for 24 h at 60 °C. Lastly, they were calcined at 550 °C for 5 h with a heating rate of 1°C/min. For the synthesis of MZ3 material the micro-emulsion assisted sol-gel method of the protocol of Neščáková et al. was strictly followed [22]. In brief, 2.24 g of CTAB was dissolved in 104 mL of ultrapure water for 30 min at 30 °C. 32 ml of ethyl acetate was then gradually added to the mixture while it was continuously stirred for additional 30 min. To keep the pH at 10.5 after that, ammonium hydroxide (28%) was added. Following that, 23.04 mL of TEOS was added and agitated for 30 min. Further, 4.34 g of calcium nitrate tetrahydrate and 1.09 g zinc nitrate hexahydrate were added with interval of 30 min and continuously stirred for additional 4 h. The suspension was centrifuged and washed twice with ultrapure water. Finally, the precipitate was dried in an oven at 60 °C for 24 h, and calcinated at 700 °C for 2 h at a heating rate of 2 °C /min. 2.2 Physicochemical characterisation 2.2.1 Fourier Transform Infra-red Spectroscopy (FTIR-ATR) A Fourier transform infra-red spectrometer (Thermo Scientific NicoletTM iSTM50, Waltham, MA, USA) in Attenuated Total Reflectance (ATR) mode was used to evaluate the molecular structure of the produced powders. The FTIR absorption spectra were obtained in range of the wavelengths between 4000 and 400 cm−1, with 64 scans, and resolution of 4 cm−1. The OMNIC software was used for the data processing. 2.2.2 X-ray Diffraction (XRD) X-ray powder diffractometry (PANalytical, Aeris, Netherlands) was used for crystalline phase analysis of the specimens. The X-ray source for the XRD apparatus was a copper (Cu) tube, which ran at 40 kV and 15 mA. The diffraction data were obtained within the range of 10 −70° 2θwith a step size of 0.0435° 2θdegrees. Complementary analysis was performed with X’PertHighScore and the International Centre for Diffraction Data PDF-2 (ICDD) database, while ICDD card #01–083-6338 was used for ZnO identification. 2.2.3 Brunauer–Emmett–Teller (BET) and Brunauer–Joyner– Halenda (BJH) analysis The specific surface area (SSA), and the volume and size of pores in the synthesised powders were analysed using a nitrogen gas adsorption/desorption system (Quadrasorb evo, Quantachrome instruments, Boynton, FL, United States) by Brunauer-Emmett-Taylor (BET) according to ISO standard 9277:2010 (E) and Brunauer–Joyner–Halenda (BJH) methods. Before analysis, the specimens were vacuumdegassed at ambient temperature for 24 h. 2.2.4 Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray Analysis (EDX) and Scanning Transmission Electron Microscopy (STEM) The surface morphology of the mesoporous materials was visualised by scanning electron microscope (Verios 5UC (Thermo Scientific, USA)). For SEM measurements, samples were secured using an electrically conductive doublesided adhesive carbon tape on standard aluminium pin stubs. Images were captured in vector scanning mode using a TLD SE detector with an acceleration voltage of 2 kV. Prior to SEM-EDS measurement, samples were sputtercoated with carbon using a Leica EM ACE200 sputter coater (Leica). An X-MAX 150 EDX (Oxford) analysis was performed to qualitatively investigate the sample 105 Page 4 of 16 Journal of Materials Science: Materials in Medicine (2025) 36:105 composition. The pore morphology, configuration and particle size of the synthesised Zn-MBGs were evaluated by STEM. For sample preparation a small amount of powder was dispersed in isopropyl alcohol and sonicated in an ultrasonic bath for 5 min. Further, the specimens were placed on a carbon coated grid and dried before analysis. For STEM measurements, samples were transferred to a holey carbon film 400 Cu mesh (AGS147-4). For STEM imaging, an STM3+detector was used at 30 kV. 2.2.5 X-ray fluorescence spectroscopy (XRF) Qualitative and semiquantative bulk analysis of the specimens was performed on a Rigaku supermini, sr.no. 16019-3 XRF wavelength dispersive spectrometer equipped with an Pd tube, with three analysing crystals: LiF1 (for elements Ti-U), PET (for elements Al-Ti), RX25 (for elements F-Mg). The detectors were a SC scintillation counter, set up with LiF1 crystal for Ti-U and F-PC flow proportional counter, set up with PET and RX25 for F-Ti. Samples were analysed at 50 kV and 4 mA tube-operating conditions, F-U element range with step sizes: 0.02 deg (Ti-U), 0.05 (F-Ca) and scanning speed 15 deg/min. Sample spin was turned on, making the sample cup rotate during measurement. 2.3 Bioactivity evaluation For the evaluation of the in vitro bioactivity, the synthesised materials were immersed into Simulated Body Fluid (SBF) solution prepared according to Kokubo et al.‘s protocol for 21 days [31]. All specimens, equal in volume, were placed into sterile, clean bottles and incubated in SBF at a concentration of 75 mg/50 mL at 37 °C under static conditions. Filtration was used to extract the powdered samples from the SBF, which were subsequently cleaned with ultrapure water and dried for 24 h at 60 °C. XRD, FTIR and SEMEDS were employed to analyse the materials for calciumphosphate or apatite crystals formation. 2.4 In vitro biological properties evaluation 2.4.1 Haemolysis assay For the haemolysis assay, whole blood was taken from healthy adults after their written consent. Red blood cells (RBCs) were isolated from leucocytes and plasma by centrifugation for 5 min at a speed of 2000 rpm and washed three times using phosphate buffered saline (PBS). A final suspension of 2% RBCs volume was obtained by diluting RBCs in PBS (2% haematocrit). After that, red blood cells were mixed separately with various MBGs concentrations (0,06, 0,125, 0,5, and 1 mg/ml) derived from a stock solution (5 mg/mL) and incubated for 24 h at 37 oC. The untreated red blood cells’supernatant (Ctrl-) served as the negative control, while the red blood cells treated with lysis buffer served as the positive control. After the treated RBCs were centrifuged at 2000 rpm for one minute, the supernatants were collected and analysed using a microplate reader (Epock, Biotek Instruments, Inc., Winooski, Vermont, USA). The absorbance of the released haemoglobin was measured at 541 nm, with a reference wavelength of 700 nm. The percentage of haemolysis was calculated based on three independent trials using the following equation: Hemolysis%¼½sample absorbance negative control= ½positive control negative controlX100 2.4.2 In vitro cytocompatibility assay Primary cell culture of periodontal ligament fibroblasts (PDLCs) was established from periodontal ligament tissue received from a healthy young donor after extraction of his third molars and signing the informed consent form. The potential cytotoxicity of the synthesised MBGs was assessed in direct contact with PLDFs using the MTT (3-(4,5dimethylthiazol–2-yl)-2,5-diphenyltetrazolium bromide) assay. Initially, 96 well plates were seeded with 103cells each in DMEM culture medium (Dulbecco Modified Eagle’s mediumBiosera, Nuaille, France) supplemented with 10% FBS serum (Foetal bovine serum 10%, GibcoBRL, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and antibiotics/antimycotics (penicillin, amphotericin B, streptomycin (Gibco-BRL, Thermo Fisher Scientific Inc., Waltham, MA, USA)). The cells were left for 24 h to adhere in an incubator with 5% CO2atmosphere at 37 °C. Then, MBGs were precisely weighted and sterilised with UV light for 30 min. 1 mg/ml stock solutions of MBGs were prepared and precultured in a culturing medium for 24 h. Further, a sequence of dilutions with nanoparticles at two concentrations (0.25 and 0.5 mg/mL) were added to well-plates and cell viability was evaluated after 24 and 72 h of incubation. Cells that were not in contact with the materials were used as the positive control. To determine the mitochondrial dehydrogenase activity of living cells, Dimethyl sulfoxide (DMSO) was added (Sigma Aldrich, St. Louis, MO, USA), to dissolve purple formazan crystals. Using the microplate reader measurements were performed at the wavelength of 570 nm, with a 630 nm reference filter. Tests were conducted in quintuplicate. The outcomes were given as a percentage of the control average optical density. For all the in vitro biocompatibility a statistical t-test and one-way analysis of variance (ANOVA) were performed via the use of the GraphPad Prism 8.4.2 software programme, and the significance level was determined at p< 0.05. Journal of Materials Science: Materials in Medicine (2025) 36:105 Page 5 of 16 105 2.5 Evaluation of the antibacterial properties 2.5.1 Bacteria preparation The antibacterial activity of powders was investigated using the Prevotella intermedia (DSM 20706) and Porphyromonas gingivalis (DSM 20709). Strains were grown in modified peptone yeast glucose (PYG) medium for 3–4 days under anaerobic conditions (80% N2, 10% H2, 10% CO2) and agitation (130 rpm) at 37 oC. Bacterial stocks were kept as frozen glycerol aliquots in −80 °C. The GasPak EZ Gas Generating Pouch Systems (BD, Benex Limited, Dublin, Ireland) were used to generate the anaerobic environment for both antibacterial evaluation in liquid broth dilution assay and in agar plate dilution methods. 2.5.2 Liquid broth dilution assay For the liquid broth dilution assay, fully grown anaerobic bacteria were incubated in the presence of the materials for 3 days at 37 °C, at 130 rpm and under anaerobic conditions. At first, materials were sterilised under UV lamp for 20 min, sonicated for dispersion in PYG medium in water bath for 20 min at 37 °C and subjected to serial dilutions. Bacteria growth was evaluated by optical density (OD600nm) using the JENWAY 6305 spectrophotometer. Fully grown bacterial cultures were used to prepare the 10% (v/v) suspension (corresponding to 108cfu/ml) in PYG medium for each strain that was added to the materials suspensions at the concentrations 0.125, 0.25, 0.5, 1 and 2 mg/ml. Bacterial growth was determined by measuring the optical absorption OD600nm at 600 nm. Control growth (medium without inoculum) and blanks (medium with tested materials) were also evaluated. The % absorbance was determined as the absorbance ratio of material treated bacteria suspension to fully grown suspension (control). Means and standard deviations were calculated to evaluate tendency and variability for each condition. 2.5.3 Colony forming unit CFU assay The antibacterial activity of MZ1, MZ2 and MZ3 were evaluated by spread agar plate CFU (colony forming unit) counting. The treated and untreated bacterial culture was serially diluted and 25 μL spread in anaerobe CDC blood agar plates (Bioprepare) and grown at 37 °C under anaerobic conditions for 2 days. Colonies were counted and inhibitory activity was determined by the following equation: %inhibition ¼ðlog10CFUcontrol log10CFUtreatedÞ100=log10CFUcontrol where, CFUcontrol: number of colonies from non-treated specimens, CFUtreated: number of colonies in contact with materials. All experiments were performed under anaerobic conditions with two replicates. 3 Results 3.1 FTIR The FTIR spectra of all three Zn containing MBGs show the distinctive bands of amorphous silicate glasses [32]. (Fig. 1a) In particular, the band at 800 cm−1is ascribed as Si–O–Si bending vibration, while the band at 470 cm−1is assigned to the Si-O-Si rocking vibration. The IR spectra also revealed a broad band between 1000 and 1250 cm−1 that was made up of the transverse optical mode (TO1) of the Si–O–Si stretching vibration at 1090 cm−1and a shoulder at 1220 cm−1that was ascribed to the TO2 mode [33]. Additionally, there is a shoulder at about 960 cm−1in materials MZ1 and M2, which is associated with the Si–OH bonds’stretching vibration [33]. However, no discernible alterations, that might be attributed to the presence of ZnO phase were detected on FTIR spectra. The main absorption band for the Si-O-Zn bond (stretching vibration) is located around 457 cm−1[34] or 474 cm−1[35], which is probably overlapped with the Si-O-Si (rocking) vibration, located at 470 cm−1. 3.2 XRD The powders MZ1, MZ2 and MZ3 had a typical XRD pattern (Fig. 1b) of amorphous silicate materials, with a characteristic scattering pattern at around 2θ=18–28° which corresponded to amorphous silicates [36]. MZ2 material also contained ZnO crystalline phase with characteristic diffraction peaks located at 2θdegrees =∼31.7, 34.4,36.2, 47.3,56.6, 62.7, 62.9, 66.4, 67.7, 69.1 °2θthat can be assigned to hexagonal ZnO. (ICDD card #01–083-6338). 3.3 BET/BJH analysis The N2 adsorption-desorption isotherms of MZ1, MZ2 and MZ3 powders are presented in Fig. 2. MZ1, MZ2, and MZ3 are classified as mesoporous silica nanoparticles. These nanomaterials show isotherms resembling Type IV in their nitrogen adsorption-desorption isotherms. In addition, it appears that MZ1 has a definite hysteresis loop, most probably of the H1 type, which indicates that it has large cylindrical mesopores containing a significant volume of adsorbed substance and is highly porous. The MZ2 sample, on the other hand, not only contains considerable interparticle pore space, but also has a larger hysteresis loop that represents a blend of orderly and irregular mesopores with a wider range of pore sizes (H1/H2 type). Between MZ1 and 105 Page 6 of 16 Journal of Materials Science: Materials in Medicine (2025) 36:105 MZ2 on one side and MZ3 on the other, there appears to be a gradual increase in the pore structure’s homogeneity since the latter has a finer hysteresis loop relative to the two former samples. Especially in connection with their clear differences in pore structure and adsorption behaviour, the latter view explains the reason why these materials could be applicable for drug delivery systems, catalysis processes and adsorption techniques. Surface characteristics of the MZ1, MZ2 and MZ3 powders according to BET/BJH analysis are depicted in Table 1. 3.4 SEM/EDS and STEM analysis The morphology of the prepared particles was analysed by scanning electron microscopy (SEM) and STEM, that were depicted on Fig. 3. MZ1 material shows homogeneous, slightly agglomerated particles with average size 30–50 nm. In MZ1 material hexagonal ordered mesoporous channels running parallel to one another are visible in the STEM pictures, suggesting that the Fig. 2 N2 adsorption-desorption isotherms of MZ1, MZ2 and MZ3 powders Table 1 Surface characteristics of the MZ1, MZ2 and MZ3 powders according to BET/BJH analysis Specimen CTAB/ TEOS ratio Specific Surface area (SSA, m2/g) Pore volume (cm3/g) BJH pore diameter (nm) MZ1 1/7.5 726.9 1.54 2.97 MZ2 1/7.5 634.4 1.03 3.84 MZ3 1/10.28 176.1 0.57 3.88 Fig. 1 aFTIR spectra of the synthesised powders, initial spectra and after 21 days in SBF bXRD analysis of the Zn-MBGs before and after immersion to SBF Journal of Materials Science: Materials in Medicine (2025) 36:105 Page 7 of 16 105 distinctive mesoporous long period order has not been impacted by Zn ion doping. The powdered MZ2 exhibits apparent nano-porosity and dispersed, spherical particles with an average size of 450–550 nm. Alongside the mesoporous structure, STEM images also show spherical, fluffy particles with a rough surface and tiny void pores. According to SEM analysis MZ3 powder appears nonhomogenous with different forms of mesoporous agglomerates and ultra small particles of an average size of few nanometres. In STEM images, the material presented loose agglomerates consisting of 10–13 nm rounded particles. 3.5 XRF The chemical composition of all Zn-MBGs, observed by XRF is presented in the Table 2. By comparing the nominal and detected composition, a limited incorporation of calcium ions can be noted, especially in the specimen MZ1 (1.87%). MZ1 powder had the highest molar percent of ZnO (9.63%), while the lowest content of ZnO was in MZ3 powder (3.63%). 3.6 Bioactivity evaluation FTIR and XRD analysis revealed lack of alterations in specimens MZ1_21 d and MZ2_21 d in SBF, as compared to the initial materials (Fig. 1a, b). MZ3–21d material shows two additional crystalline diffraction peaks of low intensity at 31.5 and 45.2 2θdegrees, which we were not able to identify. SEM analysis showed no considerable surface changes in specimens MZ1 and MZ3 and obvious increase of surface roughness in material MZ2, attributed to dissolution-precipitation processes on its surface in SBF (Fig. 4). EDX analysis of the initial materials demonstrated Fig. 3 SEM/STEM images of the synthesised materials Table 2 Chemical compositions (mass %) of Zn-MBGs determined from the XRF results Nominal Composition MZ1 MZ2 MZ3 SiO270.0% 88.5% 89.44% 84.84% CaO 25.0% 1.87% 2.63% 11.53% ZnO 5.0% 9.63% 7.93% 3.63% Total 100.0% 100.0% 100.0% 100.0% 105 Page 8 of 16 Journal of Materials Science: Materials in Medicine (2025) 36:105 the presence of calcium and zinc peaks in the corresponding spectra, indicating that these ions were successfully incorporated into the materials’structure (Fig. 4). EDX spectra of the specimens that were maintained in SBF for 21 days showed only small quantities of Ca, P, Cl and Na. (Fig. 4). Therefore, all three Zn-MBGs did not exhibit pronounced ability to form hydroxyapatite surface layer. 3.7 Haemolysis evaluation Two of the tested materials MZ1 and MZ2 presented no haemolysis (<2%) at the tested concentrations, while MZ3 appeared to be haemolytic in a concentration-dependent manner (Fig. 5a). 3.8 In vitro biocompatibility assay Figure 5b presents relative cell viability (%) of PDLCs in contact with the tested materials. MZ1 specimens did not show any negative effect on the viability of PDLCs at both tested concentrations of 0.25 mg/ml and 0.5 mg/ml, as compared to the control. MZ2_C1 and MZ3_C1 specimens caused a reproducible, statistically significant (p< 0.05) reduction of cell viability/proliferation of 9.3% at 1st day and 15.2% at 3rd day, respectively, as compared to the control. Significant differences were observed between intergroups across both days and doses, with MZ2_C1 and MZ3_C1 showing consistently lower cell viability compared to other conditions, as indicated by distinct statistical groupings (p< 0.05). No significant differences were detected between Day 1 and Day 3 within most groups, suggesting that the observed lower cell viability is primarily dose-dependent rather than time-dependent. However, all tested materials are considered biocompatible, as their viability was above 80% after 3 days of incubation with cells. 3.9 Antibacterial studies Antibacterial activity of MZ1, MZ2 and MZ3 was evaluated using two methods: liquid broth dilution method Fig. 4 SEM-EDS analysis of the Zn-MBGs before and after immersion to SBF Journal of Materials Science: Materials in Medicine (2025) 36:105 Page 9 of 16 105 46. Liang Q, Hu Q, Miao G, Yuan B, Chen X. 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