Physico-mechanical and antimicrobial performance of green nanocomposite Ca/Bentonite/Chitosan

doi:10.21203/rs.3.rs-7014132/v1

The need to balance biomaterials' mechanical and biological properties is driving the development of scaffolds for tissue engineering applications. A scaffold composed of Eggshell calcium (Ca), exfoliated bentonite (EXF-BE), and Chitosan (CS) was synthesized and studied as a bone tissue scaffold. The highest value of the surface area was 134.6 m²/g for the Ca/EXF-BE/CS with an average pore width of 44.5 Å. The mechanical properties of EXF-BE were enhanced with Ca and CS addition, which is clear in the values of ultimate tensile strength (σ_uts) 15.38 MPa, 16.19 MPa, and 17.84 MPa; Toughness 1.435± 0.23 MJ/m³, 1.713  MJ/m³, 2.067 MJ/m³, and strain at breakdown of about 28.5, 28.69 ± 0.3%, and 30.1 ± 0.33% for EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS, respectively. Antimicrobial properties, assessed by the disc diffusion method, showed inhibition zones of 27.13, 21.25, 19.25, and 20.88 mm against E. coli and S. aureus, demonstrating promising multifunctional properties.

Bentonite

mechanical

Anti-bacterial

toughness

Scaffold

In the last several years, bone tissue engineering has become a vital means of restoring bone functions that have been compromised by accident, illness, or ageing [1]. A scaffold's optimal design for bone replacement must have many characteristics to promote cell development, differentiation, and proliferation. A material must have the following properties: biocompatibility, osteoconductivity, an acceptable porosity ratio, a suitable specific surface area, and an ideal rate of biodegradation [2]. Tissue engineering and regenerative medicine treatments are becoming increasingly successful due to the advancements in cell selection, cell culture, and novel material formulations[3]. Numerous materials, including both manufactured and naturally occurring compounds, have been investigated for use in regenerative applications. In general, organically generated materials like collagens have appealing inherent qualities; yet their applicability is severely limited by the intricacies of purification, immunogenicity, mechanical characteristics, and pathogen transmission [4]. Because of this, increased focus has been placed on using designable contemporary material science to regulate material characteristics and tissue reactions [5].

The various clinical difficulties are being solved with the use of biomaterials, which are having a significant influence on medical therapies [6]. Every year, thousands of lives are saved in the medical profession by drug-eluting stents covered with polymers and controlled drug release systems made of biomaterials [7]. Biomaterials are essential components of dental implants, bio-adhesives, and surgical sutures, and they play a major role in the medical device industry [8]. In order to give novel approaches for tissue engineering regeneration, clays and clay minerals are developing materials for biomaterial design. Inorganic layered nanomaterials have been used for medicinal purposes since ancient times, including wound healing and hemorrhage inhibition [9]. Clays and clay minerals are used nowadays in cosmetics as creams, powders, and emulsions as well as in medications as active components or excipients[10]. Furthermore, the addition of clay to polymers improves the mechanical properties due to the formation of nanocomposites. The interactions between clay nanoparticles and drugs as well as other biological molecules have been thoroughly studied and are therefore utilized for controlled delivery[11].

Bentonite is a word used commonly in geology to describe a kind of sedimentary rock that is mostly composed of various types of clay minerals and smectite clay minerals, particularly montmorillonite. Its mineralogical makeup may also include other nonclay impurities. It’s a type of clay that is mined and processed in many different locations in the globe. It has several industrial and consumer uses [12]. Due to its numerous advantageous applications, there is a chance that consumers and workers may be exposed to it extensively. According to the toxicity and epidemiological studies that are now available, respirable dust inhalation by cohorts of people who are exposed at work is the main exposure route of concern. Although no regulatory or advisory authority has identified bentonite as a carcinogen, it is likely no more harmful than any other particle that isn’t controlled[13]. However, some bentonite may include varying quantities of respirable crystalline silica, which is known to be carcinogenic to humans. As a result, it is important to practice judicious management and adherence to occupational exposure limits[14].

Ca derived from CaCO₃ will be precipitated as Ca (OH)₂ and may be as CaO due to the medium of formation. Both of them plays an important role in enhancing the bioactivity, mechanical properties, and antimicrobial effect especially when used as polymer composite[15]. Furthermore, several studies have shown that the creation of scaffolds by the insertion of montmorillonite into natural biomaterials, including as gelatin, collagen, silk, and chitosan promoted cell differentiation, proliferation, and cell-scaffold interactions[16].

Chitosan (CS), a linear polysaccharide, involves the deacetylation of chitin, the second most abundant chemical in nature behind cellulose. It is a linear copolymer of 2-amino-2-deoxy-β-d-glucopyranose and 2-acetamido-2-deoxy-β-d-glucopyranose connected to β-(1 → 4). Its highly valued characteristics include excellent bioavailability, nontoxicity, hydrophilicity, biocompatibility, and ease of modification[17]. This work composites are made of an organic phase (Chitosan) and an inorganic phase (Bentonite, and Ca). They get over the issues with auto- and allografts that were previously discussed and may lead to improved mechanical qualities by fusing the flexibility of polymers with the strength of ceramics[18]. The capacity to stimulate cells, improving adhesion and differentiation, and biocompatibility are two further benefits of creating composites with natural polymers. The primary degraders of natural polymers like chitosan and gelatin are enzymes [19]. The links between the glucosamine and N-acetyl-glucosamine units that make up the chitosan structure are progressively hydrolyzed by enzymes[20]. The sustainable developments goals (SDG) tackled in this piece of work started from the innovation of novel nanocomposite (SDG-9) using a green, simple, low-cost (SDG-8 & SDG-12) for multi-functional biomedical applications (SDG-3) such as bone scaffold together with antimicrobial activity.

The raw bentonite sample (BE) from the bentonite quarry in the Western Desert, Egypt was used in the preparation process. The sample composed chemically as follows: 54.82% SiO₂, 2.5% MgO, 9.5% Fe₂O₃, 17.56% Al₂O₃, 2.4% CaO, 2.6% Na₂O, 1.45% TiO₂, and 9.2% LOI. Cetyltrimethyl ammonium bromide (CTAB), and analytical-grade dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich, Egypt. Sodium hydroxide pellets (Thermo Scientific, 98%) and glacial Acetic acid (Thermo Scientific, 99.7%) were used.

2.1. Sample preparation

2.1.1. EGG-shell CaCO₃

Egg shells were collected from markets in the Beni-Suef governorate, Egypt. The collected samples of membranes were removed and then washed several times with distilled water (250 ml) to remove residues of both organic shells and any mechanical contaminants. The washed samples were dried 2h at 70 ^oC. The dried samples were crushed by mortar and grinded in ball mill for 2hours, sieved for getting homogenous size distribution of the samples. The grinded samples were etched by a solution of diluted HNO₃ (55 %), dried, and washed with 100 ml DI water. Overall, the final collected powder was calcinated at 600^oC to remove organic contaminants and increase the oxygen and defects in the samples.

2.1.2. Bentonite preparation.

All the steps used in section 2.1.1. was followed in section 2.1.2. except for the final calcination at 600 ^oC. 25 g of Bentonite (BE) was crushed in mixer, etched using diluted HNO₃ (55 %), and stirred for 12 h to remove any metal oxide contaminants from the collected field samples. The samples were dried at 70 ^oC for 12 h and sieved to get homogenous-size samples.

2.1.3. EG-shell CaO & Bentonite (Ca/BE Nanocomposite)

The calcinated powder (5 g) was dissolved in HNO₃ (55%) (12 ml of HNO₃, where 10 ml caused the complete dissociation visually and an extra 1 ml to make sure the complete dissociation) were added drop by drop till complete dissociation. 88 ml H₂O were added (pH=2) and stirred for 15 min (Beaker 1). 10 g of BE were added into 100 ml DI Water, stirred for 15 min (Beaker 2). Beaker 2 was completely added into beaker 1 and stirred for 10 min. 15 g of green tea leaves were boiled in 100 ml DI water for 5 min, filtrated to get the extract (reducing agent). 100 ml of the extract were added to beaker 1. NaOH solution (1 M) was added drop wise into beaker 1 to adjust pH 10 of the final solution. A stirring step continued for 24 h. Finally, filtration, washing, and followed by drying step at 70 ^oC. The collected powder is Ca/BE nanocomposite.

2.1.4. EG-shell CaO/BE/Chitosan preparation (Ca/BE/CS Nanocomposite)

In beaker A, 2 g of high molecular weight of chitosan dissolved into 2% glacial acetic acid (W/V) in 400 ml DI water stirred for 2 h (1000 rpm). In beaker B, 2 g of Ca/BE nanocomposite were sonicated in 50 ml H₂O for 60 min, then added slowly into chitosan during the reaction (500 rpm) for 1 h. In beaker C, 0.66 g sodium tri-poly phosphates (TPP) was dissolved in 100 ml DI water and stirred for 30 min, then added drop wise into beaker A for ionic gelation (breaking larger chains into smaller chains). Then an alkaline solution of NaOH 0.1 M was prepared and added to the previously mentioned mixture. Where the pH was adjusted at 4.2 and left under magnetic stirring (500 rpm) for 12 h. Finally, the solution was filtrated and dried overnight in the room temperature to get the final powder (Ca/BE/CS).

2.2. Antimicrobial Test

Antimicrobial susceptibility testing using the disc diffusion method is a widely applied procedure to evaluate the efficiency of antimicrobial agents [21]. In the current test, the prepared compounds were investigated against Gram-negative (E. coli), Gram-positive (Staphylococcus aureus), and unicellular fungi (Candida albicans). Whereas a standardized bacterial suspension is prepared, ensuring the turbidity matches an optical density (OD) of 0.5 at wavelength 600 nm. The bacterial cultures are inoculated with (1.6%) according to a Nutrient Agar (NA) medium containing Peptone (5 g/L), beef extract (1 g/L), yeast extract (2 g/L), NaCl (5 g/L), and Agar (15 g/L) at 45°C via the pour plate method. Once the agar solidifies in a Petri dish, wells of 0.7 cm diameter are created using a sterile cork borer. Each well is loaded with 100 µL of the test agents (EXF-BE, Ca/EXF-BE, Ca/EXF-BE/CS) at concentrations of 1000 and 500 µg/mL, alongside Damson (negative control) and Cefotaxime (1000 µg/mL, positive control). The plates are incubated at 37°C for 18 h. Following incubation, the diameters of the inhibition zones around each well are measured, indicating the antimicrobial efficacy.

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of antimicrobial agents were determined using 10 mL sterilized test tubes filled with 2 mL of Nutrient Broth (NB), with concentrations of the compound that gave positive results from previous test which were (EXF-BE, Ca/EXF-BE, Ca/EXF-BE/CS) set at 50, 100, 200, 400, 600, 800, and 1000 µg/mL. A standardized bacterial suspension of freshly cultivated E. coli was inoculated into each test tube, achieving a final inoculum concentration of approximately 5 × 10⁵ CFU/mL. The test tubes were incubated at 37°C for 24 h, after which the tubes were inspected for turbidity to assess bacterial growth. For the MBC determination, 100 µL samples from all the tubes were streaked and spot-inoculated on sterile NA plates and incubated at 37°C for an additional 24 h. Positive controls (NB medium with bacterial suspensions and without antimicrobial agents) and negative controls (NB medium without bacterial inoculum and antimicrobial agents) were included to ensure accurate results.

2. Samples characterization

After the preparation process, the powder samples were examined with various devices as follows:

2.1. X-ray diffractometer: to detect the samples crystallinity, X-ray diffraction (XRD) using (PANalytical Empyrean, Netherlands), 40 kV (accelerating voltage), 35 mA (current), CuKα (radiation source) with (λ = 0.15406 nm).

2.2. Micrographs of the surface morphological characteristics were taken using Quanta FEG 250, Switzerland's field-emission scanning electron microscopy (FESEM).

2.3. The integration process on the essential functional groups was studied using the Bruker spectrometer FT-IR (Vertex 70).

2.4. Brunauer-Emmett-Teller (BET) Micromeritics Tri-Star II employs N₂gas as an adsorbent, 77 K for surface area measurements.

2.5. A Material Testing Machine (H5KS, TINIUSOLSEN) was used to assess the mechanical properties of the scaffold. All scaffolds were subjected to a load of 10 KN while in a room environment at 30 °C. The approach speed was set at 0.01 mm/s. The sample measured 8.0 mm in diameter and 2 mm in thickness. The compression strength, stress, and strain at break were assessed. Every data point represented the mean of five examined samples.

3.1. Structural Characterization

Fig (1) presented the XRD of the prepared samples. Structure A refers to EXF-BE with deformation, hesitation, and deviation in all the peaks, confirming the exfoliation success process (if compared to Bentonite in literature [22]. The present peaks mean the exfoliation process despite the presence of CTAB and Ultrasonication, the exfoliation process is better described as partially exfoliated bentonite. Structure B contains deviated peaks from (5.78°, 6.95°, 19.85°, 21.54°, 26.68°, and 28.56°) that are used to identify montmorillonite mineral with clay and non-clay impurities (minerals), which is the basal component in bentonite according to ICDD card No: 00-003-0010 [22][23]. The actual deviated peaks are (5.78°, and 6.95°) with noticeable destruction and shifting of the montmorillonite peak's position. The spacing value of the pure bentonite is reported as 15.3 Å based on the used card, despite the calculated value shows 18.65 Å which is a clear increase in the d-spacing value which is a proof of exfoliation process.

The precipitation of Ca particles on the surface of EXF-BE acquires the Ca/EXF-BE some crystallinity as shown in the Fig.2B. Noticeable shifting and broadening in the main peaks ensure the successful integration of the targeted matrix. The nature of polymer intercalations as amorphous material is the deformation of and clear broadening in all peaks. As well as remarkable background Fig.1. C. especially the peaks of monomontrollite and quartz at 21.46° and 24.56°, respectively[23].

3.2. Morphology and compositional analysis

Based on the FE-SEM micrographs, the surface characteristics of exfoliated bentonite, Ca/EXF-BE nanocomposite, and Ca/EXF-BE/CS nanocomposites were assessed. Normal bentonite without any further exfoliations exhibited layered structure creating a compressed and agglomerated cluster. Tiny pieces that might be connected to mineral impurities seem to be anchoring the surface of the bentonite clusters in Fig.2 A. The partially exfoliated bentonite shows a notable ordered layered structure of the clay minerals with the disordered pieces referring to the exfoliated parts (Fig. 3D & G). The precipitated Ca particles over the exfoliated bentonite Ca/EXF-BE composites appeared as rough surface Fig.2.B, E, and H. The chitosan encapsulation of the Ca/EXF-BE as a smooth surface (low roughness), indicating the composite's effective synthesis Fig.2. C, F, and I. The various scale SEM micrographs are shown in Fig.2.

3.3. FTIR

FTIR shows various bands of absorption related to the various component’s functional groups. The bands at wavenumbers 3400 cm⁻¹, 1640 cm⁻¹, 1000.2 cm⁻¹ and 918.3 cm⁻¹, respectively[24]. Additionally, the distinctive bands linked to Si−O−Al and Si−O−Mg in addition to Mg−Fe−OH may be responsible for the noticeable attenuated bands across the spectral range spanning around 400 to 1000 cm⁻¹ (Fig. 3A). Those previous bands are the same for bentonite without any further modifications. In the exfoliated samples, there is a clear shifting to the actual position, reduced intensities, and the absence of minor bands (Fig. 3B). This demonstrates both the effective dispersion of the functional alumina octahedron and silica tetrahedron units that started bentonite into discrete or unique sheets, as well as the potential annihilation of these units.

Ca/EXF-BE spectrum shows a clear shifting in the 1000.2 and 918.3 cm⁻¹ bands. In addition to the increased intensities of 400-500 cm⁻¹ bands which supports the Ca-O stretching vibrations [25]. The changes in the EXF-BE bands around 1500 and rising of bands 2850 cm⁻¹ enhances the possibility of carbon residues on the surface may be from the acidic medium or the eggshell organic parts that precipitated during the precipitation process [26].

Due to CS polymerization several bands as C–O at 1059.2 cm⁻¹, N–H at 1582 cm⁻¹, and C–H at 1416.7 cm⁻¹ as shown in (Fig. 3C)[27]. In addition to the variation in the main band position and intensities reduction. This confirms the hybrid structure successful integration. The various bands reduction refers to the contribution of these groups in the composite formation hydrogen binding or chemical complexation.

3.4. Surface area measurements

The samples textual properties were significantly impacted by the integration of the Ca, EXF-BE, and CS, as well as by the new structure's shape. The identity of BE is still present in the surface area plot, with higher values showing an increase in the surface area due to exfoliation process. The presence of Ca loaded over the surface of Bentonite exfoliated layers, increased the roughness and surface area. The higher obtained values due to CS encapsulation. The graph is close to type III and H₃ wedged shaped pores. The obtained values of surface area for EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS are 109.8 m²/g, 111.1 m²/g, and 134.6 m²/g. In addition to average pore width 44.5 Å, 46.8 Å and 43.2 Å for EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS, respectively.

3.5. Mechanical Properties

Fig. (5) Represents stress-strain curves of the various prepared samples. Similar behavior of stress-strain curves with Ca addition over BE matrix. Young’s modulus 3.73 N.mm^-2, 6.09 N.mm^-2, and 13.86 N.mm^-2 for EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS (calculated from the slope of the linear part (elastic limit)). The ultimate tensile strength (maximum stress can be afforded by the material before breakdown (σ_uts)) to be afforded by EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS are 15.38 MPa, 16.19 MPa, and 17.84 MPa, respectively. Toughness were 1.435± 0.23 MJ/m³, 1.713 ± 0.25 MJ/m³, 2.067 ± 0.31 MJ/m³for EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS, respectively.

Considering all the mechanical properties analyzed together, it was found that Ca/EXF-BE/CS, exhibited the highest toughness and strain at break and fairly high tensile strength as well as superior young’s modulus if compared to the other prepared samples. Because toughness represents the resistance of the composition to fracture, it is a crucial factor to consider in the design of a scaffold for bone replacement[28]. Here comes the key advantage of the polymeric matrices, given that they can absorb high energies before fracture and are typified by a comparatively high fracture toughness, which is opposite to ceramic behavior [29]. Using CS increased toughness by 40% than EXF-BE. Young modulus exhibited a similar trend, being the highest for the Ca/EXF-BE/CS composite and lowest for the EXF-BE sample. As far as the strain at break is concerned, the enhancement of the samples by Ca then and CS is also pronounced. The lowest value was detected in EXF-BE without any further composites, reaching about 28.5± 0.27%, while the highest value of 28.69± 0.3% in Ca/BE, and 30.1± 0.33% in Ca/BE/CS was detected.

Overall, Ca/EXF-BE/CS emerged from the mechanical testing analysis as the most favorable one and a great enhancement in the main BE structure. Interestingly, these results show that a quite small difference is enough to trigger considerable differences in material properties. The strain at break was rising despite the widespread tendency that as strength increases, the stiffer the materials and break at lower strain.

3.6 Antimicrobial activity

The antimicrobial susceptibility testing results, shown in Figure 6, indicate that EXF-BE at concentrations of 1000 µg/mL and 500 µg/mL displayed no measurable zones of inhibition, signifying an absence of antimicrobial activity against the tested organisms. This result indicates that the active components of EXF-BE may be either absent or ineffective in targeting microbial cell walls or processes within this assay. The combination of Ca/EXF-BE at a concentration of 1000 µg/mL exhibited significant inhibitory activity, demonstrated by zone diameters of 27.13 mm for E. coli, 21.25 mm for Staphylococcus aureus, and 23 mm for Candida albicans. The inhibition observed at 500 µg/mL, with zone sizes measuring 22.75 mm, 20 mm, and 19.38 mm for E. coli, Staphylococcus aureus, and Candida albicans, respectively, indicates a dose-dependent response. The observed moderate inhibition may be linked to the introduction of calcium, which could enhance cell permeability or interfere with critical cellular processes. The nanocomposite Ca/EXF-BE/CS at a concentration of 1000 µg/mL resulted in inhibition zones measuring 19.25 mm for E. coli, 20.88 mm for Staphylococcus aureus, and 19.38 mm for Candida albicans. At a concentration of 500 µg/mL, the inhibitory effects reduced to 19.13 mm, 17.75 mm, and 17.25 mm, respectively. The results indicate that incorporating CS did not substantially improve the antimicrobial effectiveness of the formulation, suggesting an absence of synergistic interaction between CS and the other components. According to (Table 1), our results surpassed those of AgNPs/zeolite, which presented inhibition zones of 12.52 mm and 12.08 mm, with E. coli and S. aureus respectively[30], as well as AgNPs-halloysite nanocomposites[31]. However, the incorporation of chitosan did not improve antimicrobial efficacy, as evidenced by Co₃O₄/chitosan/bentonite, which exhibited inhibition zones of 17 mm for S. aureus and 20 mm for Vibrio sp. [32]. Although chitosan exhibits recognized antibacterial properties, its combination with calcium and bentonite may not yield synergistic effects, as indicated by our findings. In comparison to Chitosan/AgNPs-bentonite, which exhibited an inhibition zone of 35 mm against S. aureus.[33].

Conversely, (Figure 7) displays the outcomes of MIC and MBC experiments performed on E. coli utilizing two treatments: Ca/EXF-BE and Ca/EXF-BE/Cs. The findings demonstrate that both formulations exhibited a favorable antibacterial activity, with the minimum inhibitory concentration (MIC) determined at 50 µg/mL, where bacterial proliferation was markedly suppressed but not entirely eradicated. At this concentration, bacterial colonies were visibly diminished, validating the effectiveness of the therapies in inhibiting E. coli multiplication. Moreover, the minimum bactericidal concentration (MBC) was established at 100 µg/mL, signifying the value at which there was total elimination of bacterial colonies. The results indicate that both Ca/EXF-BE and Ca/EXF-BE/CS have considerable bactericidal capabilities, demonstrated by the lack of viable bacterial growth at this concentration. The comparative investigation of Ca/EXF-BE and Ca/EXF-BE/CS indicates that both formulations are suitable candidates for antibacterial applications. This observation underscores the necessity of assessing innovative formulations capable of efficiently addressing bacterial infections, particularly in an age of increasing antibiotic resistance. The findings highlight the potential of Ca/EXF-BE and Ca/EXF-BE/CS as economical substitutes for conventional antibiotics in treating bacterial infections. Expanding the investigation of these formulations across a wider range of microbial species could improve our comprehension of their antibacterial properties and guide their use in detergents, clinical, or agricultural contexts.

The proposed antibacterial mechanism of the prepared composites involves significant electrostatic attraction between their charged surfaces and bacterial cell membranes. Both composites exhibit improved antimicrobial activity. The positively charged surfaces of Ca/EXF-BE and Ca/EXF-BE/CS interact with negatively charged bacterial membranes, resulting in enhanced membrane permeability, substantial rupture, and leakage of intracellular components [34]. The positively charged structures effectively adsorb negatively charged teichoic acid molecules on the bacterial surface, leading to membrane disruption. Cationic amphiphilic peptides and synthetic antimicrobials are potential agents for addressing bacterial infections, especially those caused by drug-resistant strains. These chemicals engage with negatively charged bacterial membranes via electrostatic interactions, resulting in membrane rupture and cell death[34]. The method of action entails pore creation, alteration of membrane thickness, and facilitation of nanoparticles, all of which undermine membrane integrity [35]. The creation of supramolecular pores increases membrane permeability and disturbs ion homeostasis, negatively impacting bacterial survival [36]. The existence of negatively charged lipids, like cardiolipin in Gram-positive bacteria or phosphatidylethanolamine in Gram-negative bacteria, is essential for the antibacterial efficiency of these substances. Further, divalent metal ions such as Ca²⁺ and Mg²⁺ may also facilitate the activation of synthetic antimicrobials in cardiolipin-rich.

The study presents a successful preparation and characterization of green nanocomposites made from eggshell, bentonite, and chitosan (Ca/EXF-BE/CS) for potential bone tissue engineering applications. The mechanical properties of the composites were enhanced, with increases in Young's modulus, ultimate tensile strength, and toughness (σ_uts 17.84 MPa; Toughness 2.067 MJ/m3, and strain at breakdown of about 30.1 ± 0.33). These parameters are critical for scaffolds intended for bone regeneration. The combination of inorganic (Ca, bentonite) and organic (chitosan) phases can effectively mimic the composite nature of natural bone, offering a balance between strength and ductility. The increase in surface area and maintenance of suitable pore sizes in the Ca/EXF-BE/CS composite are advantageous for cell attachment, nutrient diffusion, and vascularization, key factors for successful bone regeneration. The optimized composite preserved and enhanced the textural properties of the scaffold, which is essential for supporting osteoconductivity and facilitating tissue in growth. The antimicrobial assessment revealed that the Ca/EXF-BE and Ca/EXF-BE/CS composites possess considerable antibacterial and antifungal activities, with inhibition zones surpassing those of several previously reported (such as AgNPs-halloysite, and AgNPs-Zeolite) nanocomposites (inhibition zone for Ca/EXF-BE: 27.13 mm, and 21.25mm. Ca/EXF-BE/CS 19.25 mm, and 20.88 mm against E. coli and S. aureus respectively). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values confirm the efficiency of these composites against E. coli, indicating their potential to reduce infection risks in clinical applications. However, the addition of chitosan did not result in a synergistic improvement in antimicrobial activity, suggesting that the interaction between chitosan, calcium, and bentonite in the current formulation may limit the availability or activity of chitosan's functional groups responsible for antimicrobial action. Despite all these promising results testing against a wider array of pathogens, including antibiotic-resistant strains, would help establish the scaffold's clinical relevance. Incorporation of other bioactive metal oxides or polymer matrices could further enhance both mechanical and antimicrobial properties. Surface modification or functionalization of the composite may improve cell adhesion, proliferation, and antimicrobial efficiency.

The development of scaffolds for tissue engineering applications is driven by the need to balance the mechanical and biological properties of biomaterials. The scaffold prepared in this work is a successful composite Ca driven from eggshell loaded over EXF-BE and encapsulated using CS as a commercial low-cost bone tissue scaffold. Surface area increased with Ca addition and CS polymerization over the EXF-BE. The mechanical properties showed a stunning scaffold as Toughness values are 1.435 ± 0.23 MJ/m³, 1.713 ± 0.25 MJ/m³, 2.067 ± 0.31 MJ/m³ for EXF-BE, Ca/EXF-BE, and Ca/EXF-BE/CS (Ca/EXF-BE/CS exhibited the highest toughness and strain at break and fairly high tensile strength and elasticity modulus in comparison with other samples). The antimicrobial properties investigated by the disc diffusion method against Gram-negative and Gram-positive bacteria showed inhibition zones of 27.13 mm, 21.25 mm, and 19.25, and 20.88 mm, for both Ca/EXF-BE, and Ca/EXF-BE/CS respectively (Both Ca/EXF-BE and Ca/EXF-BE/CS have considerable bactericidal capabilities, demonstrated by the lack of viable bacterial growth at 100 µg/mL).

Ca	Calcium (Eggshell-derived)
BE	Bentonite
EXF-BE	Exfoliated Bentonite
CS	Chitosan
Ca/EXF-BE	Calcium/Exfoliated Bentonite Nanocomposite
Ca/EXF-BE/CS	Calcium/Exfoliated Bentonite/Chitosan Nanocomposite
TPP	Sodium Tri-poly Phosphate
CTAB	Cetyltrimethyl Ammonium Bromide
DMSO	Dimethyl Sulfoxide
FTIR	Fourier Transform Infrared Spectroscopy
XRD	X-ray Diffraction
FESEM	Field-Emission Scanning Electron Microscopy
BET	Brunauer-Emmett-Teller
MIC	Minimum Inhibitory Concentration
MBC	Minimum Bactericidal Concentration
OD	Optical Density
SDG	Sustainable Development Goals
σ_uts	Ultimate Tensile Strength
E. coli	Escherichia coli
S. aureus	Staphylococcus aureus
AgNPs	Silver Nanoparticles
LOI	Loss on Ignition

Ethics approval: Not applicable.

Funding: Not applicable.

Recommendation

Extend this work with other metal oxides and polymer matrices to enhance the mechanical properties and the anti-bacterial activity.

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Table 1. Comparison of antibacterial activity of various antimicrobial materials against different bacterial strains.

Antibacterial materials	Bacteria strain	Inhibition zones (mm)	References
Chitosan/AgNPs	Bacillus subtilis	8.5	[38]
Chitosan/AgNPs	E. coli	8.7	[38]
AgNPs/zeolite	Shigella dysenteriae	9.03	[30]
	S. aureus	12.08
	E. coli	12.52
AgNPs-halloysite nanotube nanocomposite	E. coli	12	[31]
AgNPs-halloysite nanotube nanocomposite	S. aureus	13	[31]
Chitosan/AgNPs-bentonite	S. aureus	35	[33]
Chitosan/AgNPs-bentonite	P. aeruginosa	16	[33]
Co₃O₄@chitosan/bentonite	S. aureus	17	[32]
Co₃O₄@chitosan/bentonite	Vibrio Sp.	20
Co₃O₄@Cetyltrimethylammonium bromide/bentonite	S. aureus	22
Co₃O₄@Cetyltrimethylammonium bromide/bentonite	Vibrio Sp.	35
Exfoliated Bentonite and Calcium (Ca/EXF-BE)	E. coli	27.13	This study
Exfoliated Bentonite and Calcium (Ca/EXF-BE)	S. aureus	21.25
Exfoliated Bentonite, Calcium and chitosan (Ca/EXF-BE/CS)	E. coli	19.25
Exfoliated Bentonite, Calcium and chitosan (Ca/EXF-BE/CS)	S. aureus	20.88

No competing interests reported.

SchematicflowchartforthesimpleCalcium.docx
Schematic flow chart for the simple Calcium/ Bentonite/ Chitosan preparation and Anti-microbial and mechanical properties.

Physico-mechanical and antimicrobial performance of green nanocomposite Ca/Bentonite/Chitosan

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Abstract

Figures

1. Introduction

2. Materials and Methods

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

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