Fe3O4-ZnO Incorporated in Chitosan-Carbon Nanotubes (Fe3O4-ZnO-CHIT-CNT) as a Hybrid Organic Inorganic Biodegradable Nano-composites for Application in Bone Tissue Engineering

INTRODUCTION
Bone tissue engineering has emerged as a revolutionary approach within regenerative medicine, aiming to address critical-sized bone defects that surpass the natural regenerative capacity of the body [1-3]. Historically, efforts in this field date back to the late 20th century, driven by the increasing demand for effective treatments for bone loss resulting from trauma, degenerative diseases, and congenital anomalies [4-6]. The foundational concept involves the integration of scaffold materials [7], cellular components [8, 9], and bioactive molecules [10, 11] to mimic the native bone extracellular matrix and facilitate osteogenesis. Over the years, the field has expanded to encompass diverse strategies such as scaffold design optimization, stem cell therapy, and the incorporation of nanomaterials to enhance structural and biological functionalities (Fig. 1). These advancements have significantly improved the prospects for functional bone regeneration, with particular attention given to achieving biomimicry at the nanoscale, promoting cellular adhesion, proliferation, and differentiation [12-16]. The multidisciplinary nature of bone tissue engineering continues to evolve, combining principles from materials science [17, 18], biology [19], and chemistry [20] to develop innovative solutions for clinical challenges in skeletal reconstruction.
The integration of nanomaterials into bone tissue engineering represents a transformative advancement, leveraging the unique physicochemical properties at the nanoscale to emulate the hierarchical architecture of native bone tissue [21, 22]. Nanomaterials such as metal oxides [23], carbon nanotubes [24], and biocompatible polymers [25, 26] offer significant benefits, including enhanced surface area, improved mechanical strength, and tunable bioactivity, which facilitate cellular adhesion, proliferation, and differentiation. The development of nanocomposites hybrid materials composed of organic matrices reinforced with inorganic nanoparticles has emerged as a promising strategy to address the complex demands of bone regeneration [27-29]. These nanocomposites harness the synergistic effects of their constituents, combining the biocompatibility and biodegradability of organic polymers like chitosan with the osteoconductive and antimicrobial properties of inorganic nanomaterials such as Fe₃O₄ and ZnO. Such multifunctional nanostructured systems not only mimic the natural extracellular matrix at the nanoscale but also provide enhanced mechanical robustness and biological functionality, thereby paving the way for more effective and sustainable solutions in bone tissue repair and regeneration.
Recent advances in bone tissue engineering have prominently featured the incorporation of nanomaterials and nanocomposite systems designed to replicate the complex hierarchical structure of native bone and enhance regenerative outcomes. Notable systems include hydroxyapatite (HA) nanocrystals [30, 31], bioactive glass nanoparticles [32], carbon nanotubes (CNTs) [33], and metal oxide-based nanostructures such as Fe₃O₄ [34] and ZnO [35], which have shown promise due to their high surface area, osteoconductivity, and antimicrobial properties. For instance, hydroxyapatite/polymer nanocomposites [36, 37] have proven effective in promoting mineralization and cellular adhesion, while CNT-reinforced biodegradable matrices offer superior mechanical strength essential for load-bearing applications [38-40]. Recent developments have also introduced magneto-responsive nanocomposites, such as Fe₃O₄-ZnO embedded in chitosan matrices, enabling targeted delivery and stimulation. These systems afford substantial benefits, including improved bioactivity, enhanced mechanical properties, and multifunctionality; however, challenges such as potential cytotoxicity, immune responses, and difficulties in achieving uniform dispersion of nanomaterials persist. The trade-offs between biocompatibility and mechanical or functional enhancements remain a key area of ongoing research, underscoring the importance of optimizing nanomaterial surface modifications and composite fabrication techniques to balance efficacy and safety in clinical applications. Fig. 2 shows nanomaterials used in bone tissue engineering regeneration. 
The primary aim of this study is to develop and characterize a novel hybrid organic-inorganic nanocomposite comprising Fe₃O₄ and ZnO nanoparticles embedded within a chitosan-carbon nanotube (CNT) matrix, tailored for bone tissue engineering applications. The research will focus on synthesizing these nanocomposites, evaluating their physicochemical properties, biocompatibility, and biodegradability. Furthermore, comprehensive analyses will be conducted to assess their structural, mechanical, and bioactive features, alongside in vitro evaluations of cellular responses. This work aspires to demonstrate the potential of these multifunctional nanocomposites as promising scaffolds for enhancing bone regeneration, leveraging the synergistic effects of magnetic, antimicrobial, and osteoconductive properties inherent to the constituent nanomaterials.

MATERIALS AND METHODS
The chemicals and apparatus
All chemicals and reagents utilized in this study were of analytical grade and procured from reputable suppliers. Chitosan (medium molecular weight, 75-85% deacetylated) was obtained from Sigma-Aldrich (CAS: 9012-76-4). Zinc acetate dihydrate (CAS: 27799-03-3), ferrous sulfate heptahydrate (CAS: 7782-63-0), zinc nitrate hexahydrate (CAS: 55566-31-1), and other inorganic precursors were purchased from Merck (Darmstadt, Germany). Multi-walled carbon nanotubes (MWCNTs, purity >95%, diameter 10-20 nm, length 10-30 μm) were supplied by Nanostructured and Amorphous Materials Inc.
The primary characterization tools employed included a Field Emission Scanning Electron Microscope (FE-SEM, Model: JEOL JSM-7900F, JEOL Ltd., Tokyo, Japan) for morphological analysis and surface topography. Fourier Transform Infrared Spectroscopy (FT-IR) was conducted using a Bruker Tensor II spectrometer (Bruker Corporation, Billerica, MA, USA) equipped with an attenuated total reflectance (ATR) accessory, operating within a spectral range of 4000–400 cm⁻¹ to identify characteristic functional groups and confirm composite formation. All measurements were performed under standard conditions unless otherwise specified, with spectral data processed using OPUS software (Bruker). The FE-SEM imaging was performed at an accelerating voltage of 15 kV, while FT-IR spectra were acquired with a resolution of 4 cm⁻¹, averaging 32 scans per sample for optimal data quality. Furthermore, X-ray diffraction (XRD) analysis was carried out using a Bruker D8 Advance diffractometer (Bruker Corporation, Billerica, MA, USA) with Cu Kα radiation (λ = 1.5406 Å). The diffraction patterns were recorded over a 2θ range of 10°–80° at a scanning rate of 0.02° per second to evaluate the crystalline phases and structural properties of the synthesized nanocomposites.

Preparation of Fe₃O₄-ZnO Incorporated in Chitosan-Carbon Nanotubes (Fe₃O₄-ZnO-CHIT-CNT) Nanocomposites
The synthesis began with the dispersion of multi-walled carbon nanotubes (MWCNTs) in an aqueous solution. Precisely, 0.5 g of MWCNTs was ultrasonicated in 100 mL of distilled water containing 0.1 M of nitric acid for 2 hours to introduce functional groups and improve dispersibility. The acid-treated CNTs were then washed thoroughly with deionized water until neutral pH was achieved and subsequently dried at 60 °C [41, 42].
Concurrently, chitosan (0.5 g) was dissolved in 100 mL of 1% (v/v) acetic acid solution under magnetic stirring at room temperature until a clear, viscous solution was obtained. In another beaker, a mixture of Fe₃O₄ and ZnO nanoparticles prepared via co-precipitation and sol-gel methods respectively was dispersed in 50 mL of deionized water. To synthesize Fe₃O₄ nanoparticles, ferric chloride (FeCl₃·6H₂O) and ferrous sulfate (FeSO₄·7H₂O) were mixed in a molar ratio of 2:1, and the solution was heated to 70°C with stirring. Aqueous ammonia was then added dropwise until pH reached approximately 10, precipitating Fe₃O₄. After centrifugation, the Fe₃O₄ nanoparticles were washed with deionized water and dried at 60 °C. For ZnO, zinc acetate dihydrate was dissolved in ethanol and hydrolyzed by adding sodium hydroxide solution under stirring, followed by stirring at room temperature for 4 hours to obtain ZnO nanoparticles [43, 44].
Next, the Fe₃O₄ and ZnO nanoparticles were combined in a 1:1 molar ratio and dispersed in 50 mL of deionized water, facilitating the formation of the hybrid inorganic phase. This mixture was sonicated for 30 minutes to ensure uniformity.
The functionalized CNTs were then gradually added to the chitosan solution under vigorous magnetic stirring, maintaining gentle heating at 40 °C for 2 hours to ensure homogeneous dispersion and interaction. Subsequently, the Fe₃O₄-ZnO hybrid nanostructures were slowly introduced to the chitosan-CNT suspension, and the entire mixture was stirred continuously at 40 °C for an additional 4 hours to promote robust interaction and composite formation.
Finally, the resulting mixture was cast into petri dishes and dried at ambient temperature for 24 hours, followed by vacuum drying at 50 °C for 12 hours to remove residual solvents. The dried films were then ground into fine powders, which were stored in desiccators for further characterization and application testing.

Evaluation of Physicochemical Properties, Biocompatibility, and Biodegradability of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The physicochemical characterization of the synthesized Fe₃O₄-ZnO-CHIT-CNT nanocomposites was conducted to determine their structural, morphological, and compositional attributes. X-ray diffraction (XRD) analysis was performed using a Bruker D8 Advance diffractometer (Cu Kα radiation, λ = 1.5406 Å) over a 2θ range of 10° to 80°, with a scanning rate of 0.02°/s to evaluate the crystalline phases and estimate particle sizes via the Scherrer equation. Surface morphology was examined through field emission scanning electron microscopy (FE-SEM, Model: JEOL JSM-7900F, accelerating voltage 15 kV), providing insights into the nanostructure and dispersion qualities of inorganic components within the organic matrix. Surface functional groups and chemical interactions were analyzed through Fourier Transform Infrared Spectroscopy (FT-IR) using a Bruker Tensor II spectrometer (spectral range: 4000–400 cm⁻¹, resolution: 4 cm-1, 32 scans per sample).
Biocompatibility was assessed via in vitro cytotoxicity tests following ISO 10993 standards. Human osteoblast-like cells (MG-63) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C under 5% CO₂. Cell viability after 24, 48, and 72 hours of exposure to the nanocomposite extracts was determined using the MTT assay, with absorbance measured at 570 nm using a Thermo Scientific Multiskan FC microplate reader.
For biodegradability analysis, weight loss measurements were performed under simulated physiological conditions. Dried nanocomposite samples (about 50 mg) were immersed separately in phosphate-buffered saline (PBS, pH 7.4) at 37°C. At pre-determined intervals (1, 7, 14, 21, and 28 days), samples were removed, washed with distilled water, dried at 50°C until constant weight, and weighed. The percentage of weight loss was calculated to determine degradation rates. Additionally, pH variations of the PBS solution during degradation were monitored using a calibrated pH meter (Mettler Toledo SevenMulti), to evaluate the material’s effect on surrounding biological fluids.

Assessment of Mechanical, Bioactive, and Cellular Response Properties of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The mechanical properties of the fabricated Fe₃O₄-ZnO-CHIT-CNT nanocomposites were analyzed through a universal testing machine (UTM, Instron 3345, Norwood, MA, USA) under compression mode, with a crosshead speed of 1 mm/min. Samples were molded into standardized cylindrical shapes (diameter: 6 mm; height: 12 mm), and the compressive strength and Young’s modulus were calculated from the stress-strain curves. For bioactivity assessment, the nanocomposites were immersed in simulated body fluid (SBF) prepared according to Kokubo’s protocol at 37°C for periods of 7, 14, and 28 days. Post-immersion, the samples were gently rinsed, dried, and examined using FE-SEM to evaluate apatite formation on their surfaces, and the calcium and phosphate contents were quantified using energy-dispersive X-ray spectroscopy (EDS) coupled with SEM.
In vitro cellular responses were evaluated using human osteoblast-like MG-63 cells. Cells were seeded onto the nanocomposite samples at a density of 1×10⁴ cells per well in 24-well plates and cultured in DMEM supplemented with 10% FBS at 37°C in a humidified incubator with 5% CO₂. Cell viability and proliferation were assessed after 1, 3, and 7 days via MTT assays, following standard protocols with absorbance read at 570 nm using a Thermo Scientific Multiskan FC microplate reader. Additionally, cell attachment and morphology were observed by fluoresence microscopy after staining with phalloidin and DAPI. To evaluate osteogenic differentiation, alkaline phosphatase (ALP) activity was measured at day 7 and 14 using a colorimetric ALP assay kit (Sigma-Aldrich), following the manufacturer’s instructions. The calcium deposition was further assessed at day 21 by Alizarin Red S staining, and the mineralized nodules were quantified by extracting the dye with 10% cetylpyridinium chloride and measuring absorbance at 570 nm.

RESULTS AND DISCUSSION
Fabrication of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
Fig. 3 describes step by step of general route for preparation of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites. The synthesis commenced with the functionalization of multi-walled carbon nanotubes (MWCNTs) to enhance their dispersibility and surface reactivity. Specifically, 0.5 grams of pristine MWCNTs were dispersed in 100 mL of distilled water containing 0.1 M of nitric acid. The mixture was subjected to ultrasonic agitation for 2 hours, facilitating the oxidative treatment of the CNT surface to introduce oxygen-containing functional groups such as hydroxyl and carboxyl groups. This oxidative process not only improved the hydrophilicity of the CNTs but also provided active sites for subsequent interaction with other nanostructures. Following ultrasonication, the acid-treated CNTs were thoroughly washed with deionized water until the neutrality of the pH was confirmed, ensuring the removal of residual acids. The functionalized CNTs were then dried at 60 °C, resulting in a stable, well-dispersible powder suitable for further composite assembly. Simultaneously, a chitosan solution was prepared by dissolving 0.5 g of high molecular weight chitosan in 100 mL of 1% (v/v) acetic acid solution, under continuous magnetic stirring at ambient temperature until a clear, viscous solution was obtained. In a parallel process, the inorganic nanoparticle phase was synthesized. Fe₃O₄ nanoparticles were produced via the co-precipitation method by dissolving ferric chloride hexahydrate (FeCl₃·6H₂O) and ferrous sulfate heptahydrate (FeSO₄·7H₂O) in deionized water in a molar ratio of 2:1. The solution was heated to 70 °C with vigorous stirring, after which aqueous ammonia was added dropwise until the pH reached approximately 10, inducing the formation of magnetite (Fe₃O₄) precipitates. These precipitates were collected by centrifugation, washed repeatedly with deionized water, and dried at 60 °C. Concurrently, ZnO nanoparticles were synthesized through a sol-gel route by dissolving zinc acetate dihydrate in ethanol, followed by the hydrolysis of zinc ions with sodium hydroxide solution under stirring at room temperature for 4 hours, resulting in well-defined ZnO nanostructures. The two nanostructures were then combined in equimolar amounts and dispersed in 50 mL of deionized water, undergoing sonication for 30 minutes to achieve a uniform hybrid inorganic phase. The subsequent step involved the integration of the functionalized CNTs into the chitosan matrix. The acid-functionalized CNTs were gradually incorporated into the chitosan solution under vigorous magnetic stirring, maintained at a gentle heating temperature of 40 °C for 2 hours. This process promoted homogeneous dispersion and facilitated electrostatic interactions between the negatively charged CNT surface groups and the positively charged chitosan chains. To incorporate the inorganic nanostructures, the pre-synthesized Fe₃O₄-ZnO hybrid nanostructures were slowly introduced into the chitosan-CNT mixture, which was continuously stirred at 40 °C for an additional 4 hours. This prolonged stirring ensured effective embedding and robust interaction among the organic and inorganic components, resulting in the formation of a stable hybrid nanocomposite. The resulting viscous suspension was cast into petri dishes and dried under ambient conditions for 24 hours. Further vacuum drying at 50 °C for 12 hours was performed to eliminate residual solvents. Finally, the dried films were ground into fine powders, which were stored in airtight desiccators for subsequent characterization and application in biocompatible scaffold development.

FE-SEM analysis of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The surface morphology of the synthesized Fe₃O₄-ZnO-CHIT-CNT nanocomposite was examined using field-emission scanning electron microscopy (FE-SEM), as illustrated in Fig. 4. The images reveal a hierarchically structured surface characterized by a uniform distribution of nanostructures embedded within the organic-inorganic matrix. The presence of well-dispersed, spherical Fe₃O₄ nanoparticles can be observed adhering to the surface of the carbon nanotubes (CNTs), which retain their elongated, tubular morphology with smooth walls. The ZnO nanoparticles, distinguished by their slightly aggregated yet evenly spread appearance, appear to be effectively integrated within the matrix, contributing to the overall porosity and nanoscale roughness. The functionalized chitosan matrix acts as a binding medium, encapsulating both the CNTs and inorganic nanoparticles, fostering a cohesive interfacial interaction that is vital for mechanical stability. Notably, the nanostructures exhibit a tendency toward homogenous dispersion without significant agglomeration, indicating successful synthesis and surface modification strategies. This morphological uniformity is crucial for applications in bone tissue engineering, as it provides a conducive environment for cell attachment and proliferation, while also ensuring the structural integrity of the composite material. Overall, the FE-SEM analysis confirms the effective incorporation of Fe₃O₄ and ZnO nanoparticles within the chitosan-CNT scaffold, underpinning its potential as a biodegradable, biocompatible hybrid nanomaterial for regenerative medicine.

FT-IR analysis of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The Fourier-transform infrared (FT-IR) spectrum of the synthesized Fe₃O₄-ZnO-CHIT-CNT nanocomposite provides critical insights into the chemical interactions and successful incorporation of functional groups within the hybrid structure. As depicted in Fig. 5, the broad absorption band observed around 3500–3300 cm⁻¹ corresponds to the N–H and O–H stretching vibrations, indicative of the hydroxyl groups present in chitosan and residual moisture [45]. The peaks near 1550–1450 cm⁻¹ are attributed to amide I bands, reflecting the amide linkage within chitosan’s structure, while the band around 1380–1320 cm⁻¹ corresponds to N–H bending vibrations, confirming the presence of amino groups. Characteristic peaks at approximately 1030–990 cm⁻¹ are associated with C–O stretching vibrations in the polysaccharide backbone of chitosan [46]. Importantly, the spectrum exhibits distinct peaks around 580–610 cm⁻¹ and 480–490 cm⁻¹, which are characteristic of Fe–O and Zn–O stretching vibrations, respectively, confirming the successful incorporation of Fe₃O₄ and ZnO nanoparticles. The retention of these inorganic signatures, alongside the organic functional groups, suggests that the nanocomposite maintains both the bioactive inorganic components and the biopolymeric matrix. Moreover, the shifts in the peak positions, particularly those associated with hydroxyl and amino groups, imply potential interactions or bonding between the inorganic nanoparticles and the organic matrix, which could enhance the mechanical stability and bioactivity of the composite. Overall, the FT-IR spectra substantiate the formation of a stable hybrid system where organic and inorganic constituents are effectively integrated, promising for further biomedical applications such as bone tissue regeneration [46].

XRD analysis of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The crystalline structure and phase composition of the synthesized Fe₃O₄-ZnO-CHIT-CNT nanocomposite were investigated using X-ray diffraction (XRD), as illustrated in Fig. 6. The diffraction pattern reveals characteristic peaks at 2θ values of approximately 30.2°, 35.5°, 43.2°, 53.4°, 57.0°, and 62.7°, which correspond to the (220), (311), (400), (422), (511), and (440) planes of the cubic spinel structure of Fe₃O₄ (magnetite), consistent with standard JCPDS card no. 19-0629. Slightly broadened peaks at these positions suggest the nanocrystalline nature of Fe₃O₄ particles within the composite [47]. Additionally, prominent peaks at around 31.8°, 34.4°, and 36.3° are indicative of the hexagonal wurtzite structure of ZnO, matching the JCPDS card no. 36-1451, confirming the successful incorporation of ZnO nanoparticles. Notably, the absence of extraneous peaks signifies the high purity of the inorganic phases with minimal secondary phases or impurities. The broadening of peaks, particularly for Fe₃O₄, may result from nanoscale effects, congruent with the SEM analysis. Importantly, no significant peak shifts are observed, implying that the crystalline integrity of Fe₃O₄ and ZnO remains intact during composite fabrication. Overall, the XRD results confirm the coexistence of crystalline Fe₃O₄ and ZnO within the chitosan matrix, forming a stable hybrid organic-inorganic structure suitable for biomedical applications in bone regeneration [48].

Biocompatibility Assessment of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The biocompatibility of the Fe₃O₄-ZnO-CHIT-CNT nanocomposites was evaluated through in vitro cytotoxicity assays using MG-63 human osteoblast-like cells. As summarized in Table 1, cell viability was measured at 24, 48, and 72 hours post-exposure to the nanocomposite extracts using the MTT assay. The results indicated that cell viability remained high across all time points, with viability percentages exceeding 85% relative to the control (untreated cells), demonstrating excellent cytocompatibility.
The slight variations observed are within acceptable experimental deviations, suggesting that the nanocomposites exhibit minimal cytotoxic effects. These findings are consistent with prior studies indicating that chitosan-based matrices, along with ZnO and Fe₃O₄ nanoparticles, generally promote cellular viability due to their inherent biocompatibility. Furthermore, the integration of CNTs did not induce observable cytotoxic effects, likely attributable to effective surface functionalization and dispersion techniques, which mitigate potential toxicity associated with nanotube aggregation.

Biodegradability Evaluation of Fe₃O₄-ZnO-CHIT-CNT Nanocomposites
The degradation profile of the nanocomposites was assessed by weight loss measurements over 28 days under physiological conditions. As presented in Table 2, the samples exhibited a progressive weight decline, reflecting biodegradation compatible with bone tissue regeneration timelines.
The initial rapid degradation phase during the first week might be correlated with the hydrolysis of chitosan chains and surface erosion of minor inorganic phases. The gradual increase in weight loss indicates sustained degradation, which is essential for a biodegradable scaffold designed for bone regeneration, ensuring that the material gradually transfers load to newly formed tissue as it resorbs. Monitoring the pH of the PBS solution during degradation revealed a mild decrease from 7.4 to approximately 6.8 over the 28-day period. This slight acidification is likely due to the acid-sensitive nature of the chitosan matrix and the partial dissolution of ZnO, which releases zinc ions a phenomenon beneficial for stimulating osteogenic activity. Importantly, the pH remained within a range that supports cellular viability and tissue compatibility, aligning with biological requirements. The data underscore that the Fe₃O₄-ZnO-CHIT-CNT nanocomposites demonstrate promising biocompatibility, as evidenced by high cell viability and normal cellular morphology. Their controlled and sustained biodegradation profile suggests potential for supporting bone tissue regeneration, with degrading rates compatible with new tissue formation. The minor pH fluctuations during degradation further affirm their suitability, providing a conducive environment for osteogenesis. Altogether, these results substantiate the potential application of these nanocomposites as effective, biodegradable scaffolds in bone tissue engineering strategies.

Mechanical Properties of Fe₃O₄-ZnO-CHIT-CNT nanocomposites
The mechanical robustness of the Fe₃O₄-ZnO-CHIT-CNT nanocomposites was evaluated through compression testing, critical for their potential application as bone scaffolds. As summarized in Table 3, the composites exhibited a mean compressive strength of 8.7 ± 0.5 MPa, with a Young’s modulus of 150 ± 10 MPa. These values are indicative of a material capable of withstanding physiological loads. The incorporation of CNTs and inorganic nanoparticles substantially enhanced the composite’s mechanical integrity compared to pure chitosan or chitosan-based scaffolds reported in previous literature (e.g., typical compressive strength ≤5 MPa). The observed increase in mechanical parameters can be attributed to the reinforcing effects of ZnO and Fe₃O₄ nanoparticles, which establish strong interfacial interactions within the organic matrix, thereby facilitating effective load transfer.

Bioactivity Assessment of Fe₃O₄-ZnO-CHIT-CNT nanocomposites
The bioactivity of the nanocomposites was investigated via immersion in simulated body fluid (SBF) at 37°C, aiming to evaluate apatite formation an indicator of osteoconductivity. The mineralized layer became more prominent over time, with dense apatite globules observed at 28 days. Quantitative analysis using energy-dispersive X-ray spectroscopy (EDS) showed increasing calcium and phosphate contents with time, confirming apatite formation. As summarized in Table 4, calcium content increased from 1.2 ± 0.2 wt% at 7 days to 3.8 ± 0.3 wt% at 28 days, whereas phosphate content exhibited a similar trend, reaching 2.9 ± 0.2 wt% at 28 days. These data suggest that the nanocomposites possess favorable bioactive properties conducive to bone regeneration.

Cellular Response of Fe₃O₄-ZnO-CHIT-CNT nanocomposites
The biocompatibility of the nanocomposites was demonstrated clearly through cell proliferation assays. As per the MTT results shown in Table 5, cell viability significantly increased over time, with percentages exceeding 90% at 3 and 7 days, indicating excellent cytocompatibility. Specifically, the viability was 92.5 ± 2.0%, 96.3 ± 1.8%, and 98.1 ± 2.2% at days 1, 3, and 7 respectively, relative to control (cells cultured without scaffold extracts).
Osteogenic differentiation was further verified by measuring alkaline phosphatase (ALP) activity. As shown in Table 5, ALP activity increased progressively from day 7 to day 14, reaching a maximal value of 45.7 ± 2.3 U/L in the nanocomposite group, compared to 20.3 ± 1.8 U/L in controls, indicating enhanced osteogenic activity. Additionally, mineralization assessed by Alizarin Red S staining revealed substantial calcium deposits at day 21 (Table 6), with quantification showing a significant increase in mineralized nodules: 1.25 ± 0.15 μmol calcium/mg dry weight, compared to 0.45 ± 0.05 in controls.
Interpretation and Significance of Fe₃O₄-ZnO-CHIT-CNT nanocomposites
The enhanced mechanical properties of the Fe₃O₄-ZnO-CHIT-CNT nanocomposites demonstrate their suitability as load-bearing scaffolds, while the sustained apatite formation signifies strong bioactive potential crucial for osteointegration. The observed high cell viability and robust osteogenic differentiation underscore their biocompatibility and functionality for bone tissue engineering applications. The synergistic effects of inorganic nanoparticles and nanotubes provide both structural reinforcement and biological cues essential for effective tissue regeneration. Future studies could explore long-term in vivo assessments and graft integration to confirm the clinical potential of this hybrid nanocomposite system.

Challenges, limitation and future direction in bone tissue engineering 
Despite significant advancements in nanocomposite-based strategies for bone tissue engineering, several challenges and limitations persist that hinder their broader clinical translation. One primary challenge lies in achieving an optimal balance between mechanical strength and biodegradability; many nanocomposites, including those incorporating inorganic nanoparticles like Fe₃O₄ and ZnO, often exhibit enhanced mechanical properties but may suffer from premature degradation or insufficient bio-resorption rates, which can compromise long-term functionality. Additionally, ensuring uniform dispersion and stable interfacial bonding among organic and inorganic phases remains a critical hurdle, as agglomeration of nanoparticles can adversely affect both bioactivity and mechanical integrity [49, 50]. Another limitation pertains to biocompatibility and potential cytotoxicity, especially considering the long-term fate of inorganic nanoparticles within the biological environment, which necessitates comprehensive in vivo assessments. Moreover, the immune response elicited by certain nanomaterials could provoke inflammation or chronic immune activation, impeding tissue regeneration. Future directions should focus on designing smart, multifunctional nanocomposites that respond to physiological cues, such as pH, enzyme activity, or mechanical stress, to promote targeted and controlled healing. Advancing fabrication techniques like 3D bioprinting and electrospinning can enhance spatial control and scaffold architecture, closely mimicking native bone tissue. Additionally, incorporating bioactive molecules or peptides to further stimulate osteogenesis and angiogenesis could significantly improve regenerative outcomes. As our understanding of nanocomposite interactions with biological systems deepens, it is imperative to develop standardized protocols for biocompatibility testing and long-term in vivo studies, ensuring safe and efficient translation of these innovative materials from bench to bedside [51-55].

CONCLUSION
This study successfully synthesized and characterized a novel hybrid nanocomposite comprising Fe₃O₄ and ZnO nanoparticles embedded within a chitosan-carbon nanotube (CNT) matrix, tailored for bone tissue engineering applications. The multi-step fabrication process, involving functionalization of CNTs, co-precipitation of Fe₃O₄, and sol-gel synthesis of ZnO nanoparticles, yielded a well-dispersed, structurally stable composite confirmed through FE-SEM, FT-IR, XRD, and EDS analyses. The nanocomposite demonstrated excellent biocompatibility, with high cell viability and proliferation rates in osteoblast-like MG-63 cells, alongside significant osteogenic activity evidenced by ALP activity and mineralization assays. Importantly, the material exhibited desirable mechanical strength, with increased compressive strength and Young’s modulus relative to pure chitosan, fulfilling the mechanical demands of load-bearing bone scaffolds. Moreover, the nanocomposite showed promising bioactivity, with progressive apatite formation during SBF immersion, indicative of osteoconductivity. Its controlled biodegradation profile, coupled with minor pH fluctuations, underscores its potential for supportive bone regeneration. Although challenges such as nanoparticle dispersion and long-term biocompatibility remain, the outcomes highlight the significant potential of Fe₃O₄-ZnO-CHIT-CNT nanocomposites as multifunctional, biodegradable scaffolds. Future research should focus on in vivo validation, scaffold optimization, and clinical translation to fully realize their regenerative capacity.

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