Enhanced bioactivity, antimicrobial efficacy and biocompatibility of silver-doped larnite for orthopaedic applications

2.1 Materials Utilized

For the synthesis of ceramic materials, calcium nitrate tetrahydrate (98 %, SD fine), silver nitrate (99 %, Merck), citric acid (99.5 %, SD fine AR), tetraethyl orthosilicate (Sigma Aldrich, 98 %), and nitric acid were employed for the synthesis of pure and silver doped larnite. For the preparation of simulated body fluid medium the following chemicals were utilized: Sodium hydroxide AR (99.0 % SDFCL), Conc. Hydrochloric Acid LR (35–38 %, SDFCL)Sodium Chloride AR (99 %, SDFCL), Sodium Bicarbonate AR (99 %, SDFCL), Potassium Chloride AR (99.5 %, SDFCL), Di-potassium Hydrogen Orthophosphate AR (99.0 %, SDFCL), Magnesium Chloride AR (99.0 %, SDFCL), Calcium Chloride AR (98 %, SDFCL), Sodium Sulphate Anhydrous AR (99.5 %, SDFCL), Tris(hydroxymethyl)aminomethane AR (99.8 %, SDFCL) and double distilled water. Cultural media such as Luria-Bertani Broth, Mueller Hinton Agar and Potato Dextrose Broth media used for the antimicrobial and antifungal analysis were purchased from Hi-Media Pvt. Ltd., Mumbai. Ethylenediaminetetraacetic acid (EDTA) (HiMedia Pvt. Ltd.), NaCl AR (99.0 %, SDFCL), Sodium phosphate monobasic anhydrous Hi-AR (99.2 % HiMedia Pvt. Ltd.) and Sodium phosphate dibasic anhydrous, for molecular biology grade (99.8 %, HiMedia Pvt. Ltd.) were used for hemolysis activity.

2.2 Synthesis of Ag-doped larnite

Synthesis of Ag-doped larnite (Ca_2−xAg_xSiO₄, where x = 0, 0.02, 0.04, 0.06, 0.08, 0.1) was carried out via sol–gel combustion method by incorporating citric acid as the fuel. Initially, a homogenised solution of calcium nitrate (Ca(NO₃)₂·4H₂O) in a stoichiometric quantity was infused with fuel to synthesise larnite. A stoichiometric amount of tetraethyl orthosilicate (TEOS) was then introduced while being constantly stirred, which results in the formation of a heterogeneous system as found to be similar to that of our earlier reports (Venkatraman et al., 2021; Vijayakumar et al., 2022). To catalyse the hydrolysis of TEOS, concentrated nitric acid was added; which homogenised the system by converting TEOS into silanol and ethanol. The same method was carried out to synthesise larnite doped with silver using a stoichiometric quantity of silver nitrate. The gel obtained was aged and dried and then decomposed in a preheated muffle furnace at 400 °C. Following manual grinding, the resultant precursors were calcined at 900 °C for 6 h. The precursor after calcination was characterized by different analytical techniques.

2.3 Antimicrobial activity using broth dilution method

A wide range of target strains were used to study the antimicrobial spectrum of Ag doped larnite (Ca_2−xAg_xSiO₄, where x = 0, 0.02, 0.04, 0.06, 0.08, and 0.1). The Microbial Biotechnology Laboratory of Vellore Institute of Technology provided all clinical strains. Gram positive test strains like Escherichia coli and Pseudomonas aeruginosa and Gram-negative target strains like Staphylococcus aureus and Staphylococcus epidermidis were utilised in the course of the study and the protocols were followed from our published articles (Vijayakumar et al., 2023; Choudhary et al., 2018). Percentage of inhibition by silver doped larnite compounds was calculated as follows (Vijayakumar et al., 2023): $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{C}\mathrm{F}-\mathrm{B}\mathrm{C}\mathrm{F}}{\mathrm{C}\mathrm{F}}\times 100$$ where CF – Control broth containing test strain and BCF – Final concentration (2000 µL) of biomaterial containing target clinical strains. Interaction between clinically relevant strains and Ag doped larnite biomaterials were studied using SEM analysis.

In addition, fungal strains including Aspergillus niger and Fusarium oxysporum were used for antifungal assay. Similar to the antibacterial activity, broth dilution experiment was conducted to evaluate the antifungal effectiveness of silver-doped larnite (Ca_2−xAg_xSiO₄, where x = 0, 0.02, 0.04, 0.06, 0.08, and 0.1) following the protocols from the author’s previous reports on the analysis of antifungal efficacy of silicate ceramics for orthopaedic applications. Percentage of antifungal activity were determined using the formula (Vijayakumar et al., 2023): $$Percentageofinhibition\left(\%\right)=\frac{\mathit{Fungaldryweightofcontrol}-Fungaldryweightoftests}{\mathit{Fungaldryweightofcontrol}}\times 100$$

2.4 Haemolytic activity

Haemolysis testing was performed to evaluate the compatibility to human blood of Ag doped larnite biomaterial. The experimental procedure involved the use of blood samples, which were sourced from healthy human volunteers. To prevent coagulation during the testing process, the blood was treated with Ethylenediaminetetraacetic acid (EDTA). The haemolysis assay was conducted using freshly obtained human red blood cells (RBCs). Fresh human RBCs were washed thrice with 150 mmol of NaCl (2500 rpm for 10 min) and the separated serum was suspended in 100 mmol of sodium phosphate buffer. Different concentrations of Ag doped larnite composites (0.5 mg/ml, 1.5 mg/ml and 2 mg/ml) were mixed with 200 µL of RBC solution. The reaction volumes were made up to 1 mL with sodium phosphate buffer. The reaction mixture was incubated for 1 hr at 37 °C before being centrifuged for 20 mins at 2500 rpm. Using sodium phosphate buffer as a blank, the optical density of the supernatant was measured at 541 nm. As a positive control, deionized water was employed (Henkelman et al., 2009; Palanivelu and Kumar, 2014). The experiment was repeated and concordant readings were recorded as shown in Table 4. The percentage of haemolysis were calculated using the formula (Vijayakumar et al., 2023): $$Hemolysis\left(\%\right)=\frac{\mathit{Absorbanceofbioceramics}-Absorbanceofblank}{\mathit{Absorbanceofpositivecontrol}}\times 100$$

2.5 In-vitro cell culture analyses

The method of cell isolation and culture was adopted from our published protocols (Venkatraman et al., 2022). The protocol was conducted with ethical approval from the Medical Research Ethics Committee of the University of Malaya Medical Centre (approval number: 20164-2398) to obtain patient infrapatellar fat pad. Human adipose tissue derived mesenchymal stromal cells (hAMSC’s) were collected from the infrapatellar fat pad of patients aged 50 to 70 years undergoing total knee replacement surgery. Isolation and culture of the collected cells was carried out according to the protocols mentioned in our previous reports. After culture process the cells were seeded onto the surface of the silver doped larnite scaffolds and incubated at 37 °C in 5 % CO₂ with 95 % humidity and the surface attachment of hAMSC’s were observed using micrographic analysis. To assess the impact of various silver-doped calcium silicate scaffolds on cell proliferation, a colorimetric assay utilizing Alamar Blue (AB) was employed. The proliferation assay was conducted at three time points: days 7, 14, and 27. Absorbance measurements were taken using a microplate reader at wavelengths of 570 nm and 600 nm. All experiments were performed in triplicate, with results presented as mean values accompanied by their standard deviations. The procedure involved in the cell culture analysis including isolation, attachment and proliferation of hAMSC’s were provided elaborately in the supplementary file.

3.1 Characterisation of Ag-doped larnite

Fourier Transform Infrared (FT-IR) spectroscopy was employed to analyze the presence of functional groups of finely ground silver-doped larnite samples, as shown in Fig. 1a. The existence of Si-O functional groups was evidenced by absorption bands at 843 and 887 cm⁻¹. The spectrum also showed a peak at 427 cm⁻¹, which can be attributed to O—Ca—O bending vibrations. Notably, the spectra exhibited peaks around 1400 cm⁻¹, characteristic of carbonate group stretching vibrations which was due to the adsorption of carbon dioxide (CO₂) from the atmosphere when exposed to air. This atmospheric carbonation can be explained by the adsorption of atmospheric carbon dioxide onto the ceramic material's surface and within its pores upon exposure to air (Tilekar et al., 2011; Choudhary et al., 2015). The identified vibrational bands for both pure and silver-doped larnite samples align with previous research findings (Palakurthy, 2019; Venkatraman et al., 2022; El-Khooly et al., 2023). Importantly, the spectral analysis suggests that the incorporation of silver into the larnite structure was not significantly altered the material's essential functional groups or overall structural integrity which was confirmed and supported with the XRD analysis.

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Fig. 1

(a) FT-IR spectra, (b) XRD spectrum of Ag-doped larnite calcined at 900 °C and (c) Magnified XRD patterns of Ag-doped larnite.

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X-ray Diffraction (XRD) analysis was conducted to determine the phase composition of the synthesized powders (Fig. 1b). The diffraction patterns obtained from samples calcined at 900 °C showed excellent agreement with the standard JCPDS data card (96-901-2790), confirming the successful synthesis of the target material. Previous findings divulged that inducing a metal ion into the Ca²⁺ site of the bioactive ceramics including calcium phosphates and other similar silicates of the same class leads to slight alterations in the crystal lattice of the parent material (Palakurthy, 2019; Shobana and Swamiappan, 2023; Dubnika and Zalite, 2013). This phenomenon can be attributed to the difference in ionic radii between Ag⁺ (1.15 Å) and Ca²⁺ (1.0 Å). The larger silver ions, when substituting for calcium, cause a slight expansion of the crystal lattice (Dubnika and Zalite, 2013; Mansour et al., 2017). At 2θ = 33.28°, a distortion brought on by Ag⁺ doping can be noted where the intense peak has been shifted due to lattice expansion (Fig. 1c). The deviation in the high intense peaks of larnite clearly indicated the successful incorporation of Ag⁺ into the crystal lattice of larnite bioceramics. Considering the previous reports on silver doped biomaterials, Palakurthy et al., (2019) observed the decrease in crystalline nature of wollastonite upon increased concentration of silver into wollastonite’s matrix without the presence of secondary phases. Similar research on silver doped wollastonite resulted in the formation of single phasic wollastonite with flake like morphology (Reddy and Pathak, 2018). While Erdem et al., (2021) investigation on silver incorporated HAp resulted in the formation of HAp along with the traces of β-TCP due to the higher concentration of silver in the crystal system of HAp. Despite the silver doping, the XRD patterns reveal the formation of a single-phase material, maintaining the monoclinic system characteristic of larnite. Furthermore, the successful incorporation of silver without significant structural changes indicates that the material's fundamental properties, such as bioactivity and mechanical strength, may be preserved while gaining additional functionalities from the silver doping. This balance between maintaining the base material's beneficial properties and introducing new characteristics through doping is a key consideration in the development of advanced biomaterials. Crystallite size and the lattice parameter values of larnite and Ag doped larnite were calculated and the values have been provided in Table 1.

Scanning Electron Microscopy (SEM) analysis was conducted to examine the surface morphology of both pure larnite and silver-doped larnite samples, as illustrated in Fig. 2. The micrographs revealed distinct differences in surface characteristics between the undoped and doped materials. Both pure and doped samples showed irregular, and scattered-like formations which might be due to the multistep calcination process carried out during the synthesis of larnite (Vijayakumar and Swamiappan, 2022). The observed surface irregularity is characterized by highly agglomerated particles interspersed with pores distributed across the material's surface. Energy Dispersive X-ray (EDX) spectroscopy was performed in conjunction with SEM imaging to analyze the elemental composition of the samples. For the pure larnite, the EDX spectra confirmed the presence of all essential elements expected in single-phase larnite, validating the successful synthesis of the intended material. In the case of silver-doped samples, the EDX analysis provided crucial evidence for the successful incorporation of silver into the larnite matrix. The spectra for these samples showed a distinct silver peak, in addition to the characteristic elements of larnite. This observation strongly supports the formation of silver-doped larnite as the final product, without detectable secondary phases or impurities.

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Fig. 2

Micrographic images and elemental analysis spectra of pure and Ag doped larnite.

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3.2 In-vitro biomineralization

To evaluate the biomineralization process, X-ray Diffraction (XRD) analysis was performed on the scaffolds after their immersion in Simulated Body Fluid (SBF) for a period of 9 days. Fig. 3 (a–f) presents the XRD patterns of both pure and silver-doped larnite scaffolds following the bioactivity tests. The XRD data revealed notable changes in the diffraction patterns after 3 days of immersion in the SBF medium. It was noticed that pure and Ag-doped larnite scaffolds showed the formation of HAp layer at an escalating rate upon increase in the concentration of the Ag⁺ present in the material. This observation suggests that the presence of silver may influence the rate or extent of apatite formation on the scaffold surfaces. From the figure it was clearly witnessed that the dicalcium silicate ceramics had the ability to induce the apatite formation on the early stage of immersion due to its calcium rich composition along with the formation of calcite on the surface of the scaffolds. The calcite phase observed on the surface of the scaffolds might be due to the interaction between calcium (Ca²⁺) and carbonate (CO₃^2–) ions present in the body fluid medium (Choudhary et al., 2015). The formation of calcite crystals on the scaffold surface provides heterogeneous nucleation sites for apatite deposition, acting as templates that reduce the activation energy required for nucleation and promote the rapid growth of apatite crystals. This mechanism accelerates the mineralization process and enhances the bioactivity of the scaffold for bone tissue engineering applications (Choudhary et al., 2015; Zhao et al., 2009). On increasing the immersion period upto 9 days, the increase in the intensity of HAp peak at 2θ = 32.8° was observed along with larnite peaks in Ca₂Ag₀SiO₄, Ca_1.98 Ag_0.02SiO₄, and Ca_1.96 Ag_0.04SiO₄ respectively. Whereas larnite with higher concentrations of Ag⁺ resulted in the complete coverage of the scaffold’s surface with apatite layer. Silver, due to its larger ionic radius than the Ca²⁺ which could cause strain in the crystal lattice resulting in the rapid formation of active sites required for apatite formation (Piccirillo et al., 2015). Silver ions present in the crystal structure of larnite leads to the surface modification and charge distribution of larnite, enhancing its affinity for calcium and phosphate ions present in the physiological medium resulting in the formation of HAp layer at higher rate compared to the undoped material (Bigi et al., 2016). Palakurthy (2019) reported that incorporation of silver in the Ca²⁺ site of wollastonite significantly improved the rate of apatite deposition than pure wollastonite. Alongside hydroxyapatite doped with silver revealed higher rate of apatite deposition after 14 days of immersion into the HBSS solution (Swe et al., 2019). Research conducted by Shobana et al., reported that the partial substitution of Ag⁺ resulted in the formation of more active sites resulted in the superior apatite nucleation over 9 days of immersion in SBF medium. When the scaffolds were analysed on 9th day, the XRD patterns of pure, 1 % and 2 % Ag doped larnite showed apatite covering major surface with minor larnite peaks leftover. Whereas the larnite containing 3 %, 4 % and 5 % of Ag⁺ doping resulted in the complete coverage of the surface with HAp layer. Previous reports revealed that larnite synthesised from various synthetic and biowaste source showed the nucleation of apatite after 3 and 7 days of immersion at a moderate rate (Venkatraman et al., 2022; Vijayakumar et al., 2022; Choudhary et al., 2015). Whereas increased HAp nucleation was found to be improved with the presence of Ag⁺ doping in larnite. This enhanced apatite deposition can be ascribed to changes in chemical composition, lattice defects, surface energy, and silver's bioactive nature (Graziani et al., 2018).

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Fig. 3

(a–f) XRD spectra of pure and Ag doped larnite after biomineralization.

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Fig. 4 illustrates the Fourier Transform Infrared (FT-IR) spectroscopic analysis of larnite samples following a 9-day biomineralization period. A peak observed at approximately 1660 cm⁻¹ can be attributed to H₂O stretching vibrations, while the band around 1400 cm⁻¹ corresponds to CO₃^2– stretching modes. The presence of these carbonate-related peaks suggests the formation of carbonated hydroxyapatite (HAp) during the initial stages of immersion in the physiological medium. Two distinctive peaks around 1030 and 970 cm⁻¹ were seen in the FT-IR spectra which was attributed to the stretching vibrations of PO₄^3-. On the other hand, the absorption bands corresponding to PO₄^3- bending vibrations were observed in the range of 450–600 cm⁻¹. These spectroscopic observations provide evidence for the development of an apatite-like layer on the larnite surface during the biomineralization process. The identification of both carbonate and phosphate groups is indicative of the formation of a biologically relevant calcium phosphate phase, resembling the mineral component of natural bone. These findings were in line with those of our previous reported literatures (Vijayakumar et al., 2023; Bakr et al., 2022). Fig. 5 depicts the surface morphology and corresponding elemental spectra obtained via SEM/EDX analysis after immersing the pellets for a periodic interval of about 9 days. From the micrographs it was observed that the appearance of bubble-like morphology over the surface of the material varies accordingly with difference in dopant levels of silver affirming the deposition of hydroxyapatite on the surface. The presence of essential elements in HAp like Ca and P ions were also identified on the scaffold surface with elemental analysis showing an active HAp deposition. The combined SEM and EDX results indicate that the silver doping level may play a role in modulating the deposition of hydroxyapatite. This finding aligns well with the XRD results obtained after biomineralization, reinforcing the relationship between silver content and bioactivity. The ability to induce HAp formation is considered a crucial characteristic for effective biomaterials, particularly in bone tissue engineering applications. Therefore, these observations provide valuable insights into the potential of silver-doped larnite as a bioactive material, with the silver content potentially serving as a tunable parameter for controlling bioactivity.

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Fig. 4

FT-IR spectra of pure and Ag doped larnite after biomineralization.

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Fig. 5

SEM micrographs and EDX spectra of larnite and Ag-doped larnite after immersion in SBF medium.

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3.3 Antibacterial activity assay

The O.D. of the 200 µl of suspension containing LB broth with Ag doped larnite and clinical test strains were added onto 96 well ELISA plate were recorded using ELISA reader and was tabulated in Table 2 and the results were shown in the Fig. 6 respectively.

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Fig. 6

Graph representing percentage of inhibition of clinical pathogen by Ag doped Larnite.

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According to Table 3, test strain growth was inhibited by the pH parameter's rise from 6.8 to 7.5 during a 24-hour incubation period at 37 °C. The rise in pH is mostly caused by the presence of a carbon source and biomaterial components in the medium. The media's pH increased as a result of the dissociation of Ca²⁺ and Si²⁺ ions into the medium (Venkatraman et al., 2022). The release of positively charged ions contained in biomaterials caused by a pH shift and antimicrobial action together raise osmotic pressure (Nowotnick et al., 2024). Larnite doped with silver play a pivotal role in inhibiting the growth of clinical test strains. These have bactericidal properties even at very low concentrations. One key element is the presence of Si-OH groups, which contribute to an increase in the pH of the surrounding medium (Catauro et al., 2015). This pH elevation has cascading effects on microbial physiology and metabolism. The alkaline environment created by the material interferes with the catalytic functions of various enzymes essential for microbial survival and growth (Song and Ge, 2019). Specifically, when the pH rises to the range of 7.3–7.5, it induces alterations in the physiological properties of microbes. One notable effect is the reduction in microbial respiration rates, which further compromises their viability and proliferation (Malik et al., 2018). This multi-faceted antimicrobial mechanism, combining the direct effects of silver ions with the indirect impact of pH modulation, positions silver-doped larnite as a promising material for applications where microbial control is crucial, such as in medical implants or wound dressings. Inhibition of test strains by Ag doped larnite powders were examined using plate assay method. Post incubation period of 24 hrs at 37 °C on Mueller Hinton agar medium showed good antimicrobial activity against both Gram-positive and Gram-negative test strains as shown in Figs. 7 and 8.

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Fig. 7

Control plate of S. aureus, with 2 mg/L of ceramics with different x values (Ca_2−xAg_xSiO₄) showing good antibacterial activity (B) x = 0, (C) x = 0.02, (D) x = 0.04, (E) x = 0.06, (F) x = 0.08 and (G) x = 0.1.

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Fig. 8

(A) Control plate of Escherichia coli on Mueller Hinton Agar media, (B) Control plate of Pseudomonas aeruginosa on Mueller Hinton Agar media, with 2 mg/L of ceramics with different x values (Ca_2−xAg_xSiO₄) showing good antibacterial activity (C) E. coli with x = 0, (D) E. coli with x = 0.02, (E) P. aeruginosa with x = 0.04, (F) E.coli with x = 0.06, (G) E. coli with x = 0.08 and (H) P. aeruginosa with x = 0.1.

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Silicates present in the Ag doped larnite powders disrupts the cell functions This interference extends to protein damage, cellular differentiation disruption, lipid degradation, and inhibition of adhesion and growth processes in clinical target strains. Consequently, these effects contribute to the prevention of biofilm formation, a critical factor in microbial persistence and antibiotic resistance (Gomes and Herrera, 2018). The interaction between the biomaterial and microbial cells is facilitated by electrostatic forces. The negatively charged cell walls of microorganisms attract positively charged ions, such as calcium and silicate ions, present in the biomaterial. This interaction leads to alterations in cell membrane permeability, potentially compromising the cellular integrity of the microorganisms. Calcium-silicate ceramics induces high alkaline nature of the medium thereby affecting the cell membrane and enzymatic activity of the clinical target strains (El Reash et al., 2019). Previous research on calcium silicate-based compounds has reported antimicrobial effects at concentrations as high as 15 mg/L (Janini et al., 2021). On the other investigation on the antibacterial activity of the composite containing silver substituted bioglass incorporated with sodium alginate and graphene oxide containing 2 % of silver exhibited enhanced bactericidal activity against the clinical pathogens compared to other concentrations (El-Khooly et al., 2023). In contrast, our current study demonstrates that silver-doped larnite exhibits significant antimicrobial activity at a much lower concentration of 2 mg/L. This enhanced efficacy at lower concentrations highlights the potential of silver-doped larnite as an efficient antimicrobial biomaterial.

The size of the microbes is nearly about 1–3 µm in length. Ag doped Larnite biocomposites cluster around the clinical test strains thereby inhibiting the cell growth by rupturing the proteins, lipids and DNA in the cell wall. Apoptosis of cells has been observed due to leaching of genetic material from the cell wall of organisms (Li et al., 2011). Scanning electron microscopy (SEM) was employed to visualize the effects of the biomaterial on bacterial morphology, as illustrated in Fig. 8. The micrographs reveal distinct differences between the control and treated bacterial samples. Fig. 9A, 9B, and 9C depict the control samples of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa exhibited smooth surface compared to post experiment test strain with biomaterial (Fig. 9 D–O). The observed morphological changes in the treated bacterial samples suggest that the biomaterial effectively interacts with and disrupts the cellular structure of these pathogenic organisms. This interaction likely contributes to the material's antimicrobial efficacy. Given these promising results, the biomaterial shows potential for various biomedical applications. Particularly, it may be suitable for use in bone graft substitutes and as endodontic fillers, where antimicrobial properties are crucial for preventing infection and promoting healing. These findings underscore the potential of this biomaterial in developing advanced medical implants and therapies that combine structural support with inherent antimicrobial activity.

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Fig. 9

SEM micrograph of test strains (A) control S. aureus, (B) control E. coli, (C) control P. aeruginosa, (D, E) Clustering of Ag doped larnite powder 1 around S. aureus and E. coli, (F, G) Clustering of biomaterial powder 2 thereby inhibiting growth of S. aureus and E. coli, (H, I) Growth inhibition of S. aureus and P. aeruginosa due to clustering of Ag doped Larnite powder 3, (J, K) Clustering of Ag doped larnite powder 4 around S. aureus and E. coli, (L, M) Growth inhibition of S. aureus and E. coli due to clustering of Ag doped Larnite powder 5, (N, O) Clustering of biomaterial powder 6 thereby inhibiting growth of S. aureus and P. aeruginosa.

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3.4 Antifungal activity

Dry weight of fungal strains with Ag doped larnite ceramics of varying concentration were recorded and percentage of inhibition were tabulated in Table 4 and Fig. 10.

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Fig. 10

Graph representing percentage of inhibition of fungal strains by Ag doped Larnite.

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The percentage of inhibition of fungal strains (Aspergillus niger and Fusarium oxysporum) showed increase at 2 mg/ml concentration in each ceramic’s compounds. Fungal osteomyelitis is one of the commonly found due to the prolonged antifungal drugs exposure or debridement of certain surgical or materials used for bone tissue or replacement product (Karr and Lauretta, 2015). According to a study conducted by Stacchi et al., (2019) calcium and magnesium acts as micro elements in growth promotion and proliferation of fungal strains. Porosity of biomaterial ceramics lead to the fungal colonisation thereby providing ideal substrate for adhesion and proliferation of fungal strains (Sohn et al., 2009). The obtained results indicated that a good percentage of inhibitory effect of fungal pathogens compared to the earlier studied reported with larnite compounds. SEM micrograph showing different variations in fungal mycelium post incubation with Ag doped larnite composites were depicted in Fig. 11. Smooth fungal spores (Aspergillus niger) on control were observed compared to the treated spores of the fungus. Zhao et al., studied about the Sr-N doped TiO₂ and n-HA fillers for dental resins (Hou et al., 2008). Composite concentration of 2 mg/ml showed good antibacterial activity against Streptococcus mutans, and mineralization properties were proposed. However, present study confirmed good antibacterial activity against fungus (Aspergillus niger and Fusarium sp.) even at low concentration of 0.02 mg/ml.

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Fig. 11

(a) Control Aspergillus niger spores, Ag doped larnite composite treated with fungal spores (b) with x = 0 concentration, (c) with x = 0.02, (d) with x = 0.04, (e) spores with x = 0.06 composite concentration, (f) with x = 0.08 and (g) with x = 0.1 showing the least fungal growth (Fusarium oxysporum).

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3.5 Haemolysis activity

Haemolytic activity showed value <5 % thereby proving Ag doped larnite compounds has mild activity against erythrocytes as shown in Table 5.

From the Table 5, it can be concluded that the percentage of haemolysis was higher as the concentration of Ag-doped composite increased. This attributes to slightly higher haemolysis activity of the biocomposites. The permissible limit for haemolysis using human RBCs was at 5 % and present study showed results in range of 0.73 %–2.07 % which is lower (Hou et al., 2008). The cations present in the compounds leach into the solution thereby deforming the RBCs and causing haemolysis (Hossain et al., 2020). According to study conducted by Wan et al., ceramics showed haemolytic as high as 2.4–3.2 % at high concentration of 5 mg/ml (Wan et al., 2021). Present study states that larnite compound has effectively prohibited the haemolysis activity at low concentration of Ag-doped larnite (2 mg/ml) as shown in Fig. 12.

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Fig. 12

In-vitro haemolytic activity of Ag-doped larnite compounds.

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3.6 In-vitro biocompatibility

3.6.1

3.6.1 hAMSC’s attachment

The hAMSC’s attachment on Ca₂Ag₀SiO₄, Ca_1.98 Ag_0.02SiO₄, Ca_1.96 Ag_0.04SiO₄, Ca_1.94Ag_0.06SiO₄, Ca_1.92Ag_0.08SiO₄ and Ca_1.90Ag_0.10SiO₄ scaffolds was shown in SEM micrographs (Fig. 13). The SEM micrographs demonstrated that cell attachment was evident in all groups. The morphology of attached cells in all groups was in spindle shape which indicates the typical morphology of mesenchymal stem cells in naïve condition. This characteristic infers the notion that Ca₂Ag0SiO₄, Ca_1.98 Ag_0.02SiO₄, Ca_1.96 Ag_0.04SiO₄, Ca_1.94Ag_0.06SiO₄, Ca_1.92Ag_0.08SiO₄ and Ca_1.90Ag_0.10SiO₄ scaffolds is biocompatible. In our previous findings, we have shown a better cell attachment on larnite and this phenomenon was observed again in all larnite groups although Ag particles are incorporated to improve its antimicrobial properties without limiting the cell attachment (Venkatraman et al., 2022).

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Fig. 13

Scanning electron microscopy micrographs showing attachment of hAMSC (A) Ca₂Ag₀SiO₄, (B) Ca_1.98 Ag_0.02SiO₄, (C) Ca_1.96 Ag_0.04SiO₄, (D) Ca_1.94Ag_0.06SiO₄, (E) Ca_1.92Ag_0.08SiO₄ and (F) Ca_1.90Ag_0.10SiO₄.

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3.6.2

3.6.2 hAMSC’s viability and proliferation

An excellent biomaterial for orthopaedic use should support cell viability and encourage long-term cell doubling (Dhivya et al., 2018). The proliferation of seeded hAMSC’s on Ca₂Ag₀SiO₄, Ca_1.98 Ag_0.02SiO₄, Ca_1.96 Ag_0.04SiO₄, Ca_1.94Ag_0.06SiO₄, Ca_1.92Ag_0.08SiO₄ and Ca_1.90Ag_0.10SiO₄ was steadily increased from day 7 to day 27 (Fig. 14). It was observed that the proliferation of hAMSC’s seeded on Ca_1.90Ag_0.10SiO₄ was significantly increased (p < 0.05) as compared with Ca₂Ag0SiO₄ on day 7, 14 and 27. However, the hAMSC’s seeded on Ca_1.92Ag_0.08SiO₄ was only significantly increased (p < 0.05) on day 7 and 14 when compared with Ca₂Ag₀SiO₄. This scenario supports the hypothesis that the maximum concentration of Ag used in this study has no negative effect in terms of cell viability and proliferation over the different time points.

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Fig. 14

Viability/proliferation of hAMSC’s seeded on Ca₂Ag₀SiO₄, Ca_1.98 Ag_0.02SiO₄, Ca_1.96 Ag_0.04SiO₄, Ca_1.94Ag_0.06SiO₄, Ca_1.92Ag_0.08SiO₄ and Ca_1.90Ag_0.10SiO₄ on day 7, 14 and 27. (One-Way ANOVA: cell seeded larnite scaffolds on day 7, 14 and 27 vs. day 1 culture). The assessments were reported to be statistically significant based on One-Way ANOVA if *p < 0.05, **p < 0.01 and ***p < 0.001.

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