Silymarin-functionalized nanohydroxyapatite-chitosan nanocomposite: a promising biomaterial for oral health applications

Abstract

Background

Nanohydroxyapatite (nHAP) is widely recognized for its potential biomedical applications, particularly in bone regeneration and periodontal therapy. Green synthesis methods, which are eco-friendly and non-toxic, have gained attention for the production of nanocomposites. Silymarin, a bioactive compound, can serve as both a reducing and stabilizing agent in such synthetic processes. In this study, we aimed to develop a nanohydroxyapatite nanocomposite using silymarin (SL) and chitosan (CH) and evaluate its antioxidant, antibacterial, and anti-inflammatory properties for oral health applications.

Methods

Nanohydroxyapatite nanocomposites were synthesized using a green synthesis technique, with silymarin acting as both reducing and stabilizing agent. Chitosan was incorporated to form the polymer-based nanocomposites. The synthesized materials (silymarin-chitosan, silymarin-nHAP, and nanocomposite) were characterized by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Fourier Transform Infrared Spectroscopy (FTIR) to assess their morphology and functional groups. Antioxidant activity was evaluated using the DPPH assay, and the antibacterial activity was tested against common oral pathogens. Biocompatibility was assessed using human gingival fibroblast (HGF) cells and cell viability was measured via live/dead cell assays using fluorescence imaging and in vitro migration assays. Wettability analysis was performed using a contact angle measurement technique to evaluate the surface hydrophilicity of the nanocomposite, a crucial factor for biointegration and tissue adhesion. Additionally, the anti-inflammatory potential was examined using the human red blood cell (HRBC) membrane stabilization assay, where the nanocomposite was evaluated for its ability to inhibit heat-induced hemolysis.

Results

The characterization revealed the successful formation of nanoHA nanocomposites with distinct morphological features. Antioxidant assays indicated significant free radical scavenging activity, whereas antibacterial testing demonstrated effective inhibition of oral pathogens, including Streptococcus mutans and Porphyromonas gingivalis. Wettability analysis revealed a favorable contact angle, indicating enhanced surface hydrophilicity, which is beneficial for cell attachment and biointegration. Biocompatibility studies revealed that the nanocomposites exhibited minimal cytotoxicity and enhanced cell viability. Migration assays revealed favorable activity in promoting fibroblast migration, suggesting its potential for tissue regeneration. Furthermore, the HRBC membrane stabilization assay confirmed the anti-inflammatory potential of the nanocomposite, indicating its ability to protect erythrocytes against heat-induced hemolysis.

Conclusion

The silymarin-chitosan-nanohydroxyapatite nanocomposite synthesized using green methods demonstrated high crystallinity, improved wettability, excellent biocompatibility, and multifunctional bioactivity, including antioxidant, antibacterial, and anti-inflammatory effects. The ability of the nanocomposite to support cell migration, inhibit oral pathogens, enhance surface hydrophilicity, and stabilize cell membranes suggests its potential application in oral health, particularly in bone regeneration, periodontal therapy, and inflammation management.

Introduction

Chitosan (CS), derived from chitin via deacetylation, is the second most widely used natural polymer. Its exceptional nontoxicity, biocompatibility, and biodegradability have driven extensive research into diverse applications [1]. Its intrinsic properties include mucoadhesion, permeation enhancement, and antimicrobial activity, which stem from its amino and hydroxyl groups [2]. These properties have positioned chitosan as a versatile material in biomedical and pharmaceutical fields [3]. However, their limited solubility poses challenges, necessitating chemical modification to improve their physicochemical properties and broaden their utility. Chitosan derivatives synthesized through such modifications exhibit enhanced stability, solubility, and bioactivity, thereby expanding their potential for advanced applications [4]. In particular, chitosan nanoparticles (CSNPs) have emerged as safe and efficient nanocarriers capable of controlled and targeted drug delivery with diverse biomedical applications [5]. Despite these advantages, nanoparticle aggregation remains a challenge. To address this, nanocomposites (NCs) have been utilized as flexible tools to improve the stability and functional properties of nanoparticles [6]. The bio-adhesive properties of chitosan allow extended contact with treatment sites, enhancing the localized availability and effectiveness of active agents [7, 8]. Their antimicrobial, antioxidant and anti-inflammatory properties further contribute to their potential as biomaterials for therapeutic applications [9].

Silymarin, a bioactive compound derived from the seeds of milk thistle (Silybum marianum (L.) Gaertn, Asteraceae), is a well-known herbal remedy for various ailments. Its rich antioxidant properties and phenolic composition make it a promising candidate for mitigating oxidative stress-related conditions, including dental and oral health issues [10]. In addition to its hepatoprotective effects, silymarin has potent anti-inflammatory, immunomodulatory, and antimicrobial activities, which further enhance its biomedical potential [11–13]. Their antioxidative properties are particularly noteworthy, as they counteract free radical damage, contribute to UV protection, and help in skin regeneration, making silymarin a valuable ingredient in dermatological and cosmetic formulations [12, 14]. Despite these benefits, biomedical applications of silymarin are hindered by its low water solubility, poor bioavailability, and rapid metabolism and excretion [15]. Numerous strategies to improve the solubility and pharmacokinetics of silymarin have been explored, but they have yet to yield substantial breakthroughs [16]. The integration of nanotechnology offers a promising solution to overcome these limitations by enhancing the bioavailability and therapeutic efficacy of silymarin and similar plant-derived compounds [17]. Dental health issues, such as plaque accumulation on teeth and prosthetics, can lead to serious conditions such as dental caries and periodontitis. Dental caries results from acid production by oral bacteria, particularly Streptococcus mutans, whereas periodontitis is marked by gum infections that cause inflammation and bone loss [18]. Addressing these issues with bioactive materials possessing antioxidant and antimicrobial properties can significantly improve the oral health outcomes. This study aimed to develop a bioactive nanocomposite by combining chitosan gel with hydroxyapatite and silymarin. Although each of these components has been extensively studied for its individual benefits, their combined potential remains largely unexplored. By leveraging the antioxidant, antimicrobial, and regenerative properties of silymarin with the mucoadhesive and biocompatible nature of chitosan and the structural properties of hydroxyapatite, this study sought to evaluate the synergistic effects of the composite material. The nanocomposite was designed to enhance the bioactivity and solubility of silymarin while improving in vitro biocompatibility, offering a novel approach for the development of advanced biomaterials for biomedical applications.

Materials and methods

Materials

Silymarin, chitosan, sodium hydroxide (NaOH), glacial acetic acid, and diammonium phosphate dibasic ((NH₄)₂HPO₄) were purchased from Merck or Sigma-Aldrich. All chemicals used for synthesizing the HAP-chitosan composite were of analytical reagent-grade and were used without further purification. Double-distilled water was used as the solvent in all experiments.

Preparation of nanocomposite

Silymarin (20 mL, 1%) was added to an aqueous solution of Ca(NO₃)₂ (30 mL, 0.3 M). An aqueous solution of NH₄H₂PO₄ (10 mL, 0.3 M) was then added to the mixture and stirred for 30 min. NaOH solution was used to adjust the pH to 11, and the synthesis was performed under continuous stirring at 50 °C for 30 min. The resulting suspension was cooled to room temperature, centrifuged, and washed repeatedly with deionized water and ethanol. The product was dried in a laboratory oven and collected for further analysis. For chitosan nanoparticle preparation (CSNPs), chitosan was dissolved at a concentration of 3 mg/mL in a 1% aqueous solution of glacial acetic acid under continuous stirring at 100 rpm. To prepare the CSNPs, 20 mL of chitosan solution was mixed with 7 mL of silymarin dropwise under continuous stirring at 500 rpm for 30 min. The mixture was then centrifuged at 800 rpm for 30 min to separate nanoparticles. The resulting pellet was collected, washed thoroughly with distilled water, and used for further analyses. To synthesize silymarin/CSNP/HAP, 30 mL of 0.3 M Ca(NO₃)₂ was dissolved in 20 mL silymarin. A solution of NH₄H₂PO₄ (10 mL, 0.3 M) was added and stirred for 30 min in the presence of 10 mg chitosan nanoparticles. The pH of the mixture was adjusted to 11 using a NaOH solution. The same procedure was followed for the preparation of the HAP.

FTIR spectral analysis of nanocomposite

Fourier-transform infrared (FT-IR) spectra of the nanocomposite samples were obtained using an FT-IR spectrometer (Thermo Nicolet, USA). The spectra were recorded over the wavenumber range of 500–4000 cm⁻¹. All measurements were conducted at room temperature and performed in triplicate to ensure the accuracy and reliability of the data.

X – ray diffraction and wettability analysis

XRD analysis

The purity and crystalline nature of Silymarin – Chitosan nanocomposite were analyzed using XRD diffraction, with scanning conducted from 2θ (ranging from 20° to 80°). Cu Kα radiation (λ = 0.1.5406 A°) was employed. The grain size (D) was calculated using the Debye – Scherrer formula: D = K λ/βCosθ, where K = 0.9, λ is the X – ray wavelength, β is the FWHM and θ is the Bragg’s angle.

Wettability analysis

The wettability of the surface was evaluated using a contact angle measurement technique. The angle was determined by analyzing the shape of the pellet at the three-phase contact line.

SEM analysis

The surface morphology of the silymarin/chitosan/hydroxyapatite nanocomposite was investigated using scanning electron microscopy (SEM, TESCAN VEGA3). To prepare the samples, small pieces of each composite were affixed to a specimen holder using a double-sided adhesive tape. Subsequently, the samples were sputter-coated with gold before electron micrographs were captured at an accelerating voltage of 5.0 kV. This process enabled detailed visualization and analysis of the surface characteristics and structure of the nanocomposite, providing valuable insights into its morphology and composition.

TEM analysis

Transmission electron microscopy (TEM) was employed to investigate the ultrastructure and morphology of the silmarin/chitosan/HA nanocomposite. The TEM analysis was performed using a TEM Leo 912 omega instrument (Zeiss, Germany) operating at 120 kV. The Nanocomposite samples were prepared by affixing a lacey carbon-coated copper grid to the collector before observation. Electrons were transmitted through the sample, generating high-resolution images of the internal structure and morphology of the nanocomposite at the nanoscale level. This analysis provided valuable insights into the composition, dispersion, and interactions of the individual components within the nanocomposite structure.

Antioxidant activity

The antioxidant activities of the nanocomposites were evaluated using a DPPH assay. DPPH solution (1 mL of DPPH solution was added to each tube, followed by the addition of silymarin-chitosan, silymarin-nHAP, and nanocomposite pellet solutions at volumes of 10, 20, 30, 40, and 50 µg/mL. A 50% methanol solution was then added to adjust the total volume to 2 mL, using 1990, 1980, 1970, 1960, and 1950 µL. The tubes were incubated in the dark for 15–20 min. The absorbance was measured at 517 nm using a spectrophotometer, and readings were recorded [19].

where: Ac - Control reaction absorbance; As - Testing specimen absorbance.

Antibacterial activity by agar disc diffusion

The agar disc diffusion method, as described by Chung et al. [20], was used to evaluate the antibacterial activities of silymarin-chitosan, silymarin-nHAP, and nanocomposite extracts against S. mutans and P. gingivalis. Nutrient agar plates were prepared and inoculated with 0.1 mL of a broth culture of the test bacteria. The inoculum was evenly spread across the agar surface using a sterile L-shaped spreader, and allowed to settle. Sterile paper discs (10 mm in diameter) were impregnated with various concentrations (50, 100, and 150 µg/mL) of the samples (silymarin-chitosan, silymarin-nHAp, and silymarin-nHAp-chitosan-incorporated nanocomposites) and placed on seeded plates. A disc impregnated with 25 mg/mL streptomycin was used as the positive control. Plates were incubated overnight at 37 °C in an inverted position. After incubation, the diameter of the inhibition zone around each disc was measured in millimeters using a ruler as previously described [21]. Each experiment was conducted in triplicate.

Cell viability assay

An in vitro cytocompatibility assessment of the silymarin/chitosan/hydroxyapatite nanocomposite was conducted using human gingival fibroblast cells. The effect of the nanocomposite on fibroblast cells was determined using the MTT assay. Briefly, fibroblast cells (1 × 10⁵ cells/mL) were seeded in a 96-well microtiter plate (100 µL per well) with replicates. The cells were then treated for 24 h with different concentrations (10, 20, 40, 50, 100, and 200 µg/mL) of SL + CH, SL + HA, and SL + CH + HA. Following the treatment period, 20 µL of a 5 mg/mL MTT stock solution was added to each well and incubated for 4 h at 37 °C. Formazan crystals were solubilized in DMSO, and the absorbance was measured at 570 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA). Cell viability (%) was calculated as the ratio of absorbance (A570) of treated cells to the absorbance of control cells (0.1% DMSO) (A570). The IC₅₀ value, representing the concentration of the sample required to reduce the absorbance by 50% %compared with the DMSO-treated control, was also determined. The percentage of viable cells was calculated using the following equation.

Calcein AM staining

Cell viability and live staining were performed using calcein acetoxymethyl ester (Calcein AM). Fibroblasts were seeded in 6-well plates at a density of 1 × 10⁶ cells per well. Following culture, the cells were treated with SL + CH, SL + HA, or SL + CH + HA (200 µg/mL) for 24 h. After the treatment period, the calcein-AM dye was added to the cells and incubated for 30 min. The cells were then washed with 1xPBS and observed under an inverted phase-contrast fluorescence microscope (Invitrogen). Viable cells that exhibited green fluorescence owing to calcein-AM staining were identified. Live and dead cells were manually counted and the ratio of live to dead cells was calculated for each cell condition. This allowed for the assessment of the viability and overall health of fibroblasts following treatment with the nanocomposite.

In-vitro cell migration assay

Human fibroblasts were seeded into 6-well plates at a density of 3 × 10^5 cells per well and allowed to grow into a monolayer for 24 h. Subsequently, a sterile 20–200 µL pipette tip was used to vertically scratch a cross in each well, inducing a wound-like area. The detached cells were then removed by washing with 1X PBS, and fresh culture medium containing the nanocomposite (SL + CH, SL + HA, SL + CH + HA) at a concentration of 200 µg/mL was added to each well along with a control group, and incubated for an additional 24 h. Following incubation, samples were washed and fixed in 4% paraformaldehyde. Images were captured using an Axio Observer Z.1 microscope (Carl Zeiss, Germany), allowing for the visual observation and analysis of cell migration and wound closure.

Anti-inflammatory activity

Preparation of HRBC suspension

Fresh blood (10 mL) was collected and transferred into an anticoagulant (heparin) coated centrifuge tube. The sample was then centrifuged at 3000 rpm for 10 min. The packed red blood cells (RBCs) were subsequently washed three times with phosphate – buffered saline (PBS) at p^H 7.4. Then 20% (v/v) suspension of human red blood cells (HRBCs) was prepared using PBS for further analysis.

Heat-induced hemolysis inhibition

The potential of Silymarin – Chitosan nanocomposite to inhibit HRBC Haemolysis by scavenging its membrane was evaluated using a modified version of the published article. The test solution (2 mL) comprised 1 mL of Silymarin – Chitosin nanocomposite at varying concentrations (50 µg/mL to 250 µg/mL) and 1 mL of 20% HRBS suspension. The reaction mixture was thoroughly mixed and incubated in a hot water bath at 56 ℃ for 30 min. After incubation, the tubes were allowed to cool to room temperature, followed by centrifugation at 2500 rpm for 5 min. The absorbance of the supernatant was measured spectrophotometrically at 560 nm. Phosphate buffered saline was used as the blank and diclofenac sodium serves as the positive control. The RBC membrane stabilization in percentage was checked by executing the given formula:

Here, AbC signifies control absorbance; AbS signifies screened sample absorbance.

Statistical analysis

All sample data were analyzed in three independent triplicate experiments, ensuring the robustness and reliability of the findings. Mean values and standard deviations (± SD) were calculated to accurately represent the data. Each experiment was repeated twice. Statistical significance was assessed at P ≤ 0.05, and mean differences were evaluated using Duncan’s multiple range test (DMRT).

Result and discussion

FTIR spectrum of silymarin, nHap, chitosan incorporated nanocomposite

The FT-IR spectra depicted in Fig. 1 illustrate the characteristic bands of the various components and nanocomposite. The silymarin-chitosan complex exhibited a characteristic band at 546.94 cm⁻¹, indicating the presence of aliphatic compounds, whereas the band at 1406.13 cm⁻¹ corresponds to a carbonyl compound [22]. In the silymarin-HAP spectrum, the maximum band at 2360.91 cm⁻¹ indicates the presence of a strong O = C = O stretching carbon dioxide group [23]. Additional bands at 598.91 cm⁻¹ and 561.30 cm⁻¹ reveal the presence of strong C-I stretching halo compounds. Finally, in the nanocomposite spectrum, characteristic bands were observed at 1640 cm⁻¹, corresponding to the presence of an amide group, and at 1010 cm⁻¹, indicating the presence of a methylene group [24]. These spectral features provide valuable information about the chemical composition and structural characteristics of the silymarin-chitosan complex, silymarin-HAP, and nanocomposite, aiding the characterization of these materials for various applications.

Fig. 1.

FTIR spectrum of silymarin chitosan (Sily-Chitosan), silymarin nHap (Sily-HAP), silymarin nHap chitosan incorporated nanocomposite (Nanocomposite). FTIR spectrum indicates the nanocomposite with characteristic absorption bands silymarin, nHap, and chitosan

X – ray diffraction & wettability

XRD analysis

The XRD diffraction patterns confirmed the presence of chitosan and HAp phases, as illustrated in Fig. 2(A). The HAp phase was identified using JCPDS data 09–0432. The major diffraction peaks at 2θ ≈ 25.8° (002), 31.7° (211), 32.2° (112), and 32.9° (300) are indicative of the hexagonal phase of HAp, matching the standard JCPDS card for hydroxyapatite. Additional peaks at 2θ ≈ 46.7° (222), 49.5° (213), 53.2° (004), and 63.9° (304) further validate the crystalline nature of HAp. The broad background at lower angles suggests the presence of chitosan, which is typically amorphous. Incorporating chitosan does not significantly alter the crystallinity of HAp but may contribute to a reduction in peak intensity, indicating an interaction between the polymer matrix and HAp. This XRD analysis confirms the successful formation of the HAp-chitosan composite [25].

Fig. 2.

A XRD spectrum of silymarin chitosan (Sily-Chitosan), silymarin nHap (Sily-HAP), silymarin nHap chitosan incorporated nanocomposite (Nanocomposite). B Contact angle of silymarin chitosan (Sily-Chitosan), silymarin nHap (Sily-HAP), silymarin nHap chitosan incorporated nanocomposite (Nanocomposite)

Wettability

The wettability analysis of the HAp and chitosan composite, as shown in the image (Fig. 2B), reveals a contact angle of 27.6°, indicating a highly hydrophilic surface. This suggests that the composite material has excellent water absorption and spreading properties, which can enhance protein adsorption, cell adhesion, and bioactivity, making it highly suitable for bone regeneration and wound healing applications. The hydrophilic nature of the composite is primarily attributed to the intrinsic polarity of chitosan and the porous structure of HAp, which facilitates water penetration and improves surface interactions with biological fluids. The lower contact angle also indicates the potential for better integration with surrounding tissues when used in medical implants [26].

Scanning electron microscopy (SEM) analysis

The morphological characteristics of the silymarin–chitosan complexes were examined using scanning electron microscopy. The nanocomposite image revealed minor aggregation within the silymarin-chitosan complex, along with a consistent dispersion of the chitosan polymer (Fig. 3a1). Subsequently, the structural morphology of nanohydroxyapatite (nHAP) nanoparticles exhibited increased agglomeration compared to the silymarin-chitosan complex (Fig. 3b1) [27]. Upon incorporation into the silymarin-nHAP-chitosan nanocomposite, the significant reduction in agglomeration surpassed that of both the silymarin-chitosan and nHAP samples(Fig. 3c1) [28]. Furthermore, the dispersion of nHAP nanoparticles and intercalation of silymarin-nHAP within the chitosan polymer matrix were investigated.

Fig. 3.

Transmission electron microscopy (TEM) images of silymarin-chitosan (a), nHAP nanoparticles (b) silymarin-nHAP-chitosan incorporated nanocomposite (c). Scanning electron microscopy (SEM) images indicating microstructure of silymarin-chitosan (a1), nHAP nanoparticles (b1), silymarin-nHAP-chitosan incorporated nanocomposite (c1)

Transmission electron microscopy (TEM) analysis

The TEM images in Fig. 3, TEM images depict spherical particles of the silymarin complex within the nanohydroxyapatite with sizes ranging from 15 to 30 nm. Chitosan polymer is instrumental in controlling the structure, size, and shape of nanoparticles by providing nucleation sites. Figure 3b shows images of pure nanohydroxyapatite, exhibiting rod-shaped aggregates with a width of approximately 10 nm and length of up to 15–20 nm [23]. Figure 3c shows the branched structure of silymarin, which allows it to be distinguished from the other components within the nanocomposite. Although the nanocomposite components are not fully separated, they can still be distinguished by a relative size of approximately 25–30 nm [29]. These TEM images offer valuable insights into the morphology, size, and distribution of nanoparticles within the nanocomposite, shedding light on the role of each component in its structure and formation.

Antioxidant activity

The antioxidant activities of silymarin-chitosan, silymarin-nHAP, and the HA/CH/SL nanocomposite were evaluated and are presented in Fig. 4, which shows a concentration-dependent increase in the antioxidant activity. As the concentration of individual materials increased, the free radical scavenging ability also improved. Figure 4 highlights the antioxidant activity of the HA/CH/SL nanocomposite, which exhibited significantly higher free radical scavenging activity than silymarin-chitosan and silymarin-nHAP. The percentage inhibition observed for the nanocomposite confirmed its superior antioxidant potential, underscoring the synergistic interactions among hydroxyapatite (HA), chitosan (CH), and silymarin (SL). These findings suggest that the combination of these materials in the nanocomposite formulation enhances their antioxidant efficacy, making them promising candidates for applications requiring potent free-radical scavenging capabilities. These results align with those of previous studies, highlighting the enhanced antioxidant potential of nanocomposites formed by integrating bioactive compounds with biopolymers and inorganic nanoparticles [30]. Khalil et al. demonstrated that combining natural antioxidants with nanostructured materials significantly amplifies their radical scavenging efficiency owing to their improved surface area and electron-donating properties [31]. Similarly, Zagloul et al. emphasized that chitosan-based nanocomposites enhance the stabilization of bioactive compounds, resulting in superior antioxidant efficacy in various applications [32]. These findings underscore the promising application of HA/CH/SL nanocomposites in biomedical and pharmaceutical fields, particularly in contexts requiring effective oxidative stress management. The synergistic interaction among the components not only enhances antioxidant activity but also paves the way for further exploration of drug delivery systems and therapeutic formulations.

Fig. 4.

Antioxidant activity evaluated using the DPPH method for (a) Silymarin-Chitosan (SL + CH), (b) Silymarin-nHAP (SL + nHAP), and (c) HA/CH/SL nanocomposite. Data are presented as means ± SD from three replicates. Different letters indicate a significant difference, while the same letter denotes no significant difference according to Duncan’s multiple range test at P ≤ 0.05

Antibacterial activity

The antibacterial activities of silymarin-chitosan, silymarin-nHAP, and the HA/CH/SL nanocomposite were evaluated against Streptococcus mutans and Porphyromonas gingivalis. The synthesized nanocomposite exhibited significant antibacterial activity against both the tested organisms. At a concentration of 150 µg/mL, the HA/CH/SL nanocomposite displayed the highest zones of inhibition, measuring 28.6 mm and 25.2 mm for S. mutans and P. gingivalis, respectively. Even at lower concentrations, the nanocomposites demonstrated potent antibacterial effects, with inhibition zones of 15.2 mm and 14.5 mm for S. mutans and P. gingivalis, respectively (Fig. 5). The enhanced antibacterial efficacy of the HA/CH/SL nanocomposite could be attributed to the synergistic effects of its components. The polycationic nature of chitosan disrupts bacterial membranes, whereas hydroxyapatite provides stability and supports the controlled release of active agents. Silymarin possesses additional antimicrobial properties, owing to its phenolic compounds. The combined action of these components in the nanocomposite amplifies their individual antibacterial effects, making them more effective than the stand-alone materials. These findings align with those of previous studies, such as the work of Missier et al., which demonstrated that green-synthesized Zn-TiO₂ nanocomposites effectively inhibited oral pathogens, such as S. mutans and S. mitis [33]. Similarly, Zagloul et al. reported that combining bioactive compounds with nanomaterials significantly enhances their antibacterial properties owing to improved penetration and interaction with bacterial cells [32]. These results suggest that the HA/CH/SL nanocomposite has great potential as a powerful antibacterial agent for applications in oral care and dental materials, especially for managing pathogens associated with dental caries and periodontitis.

Fig. 5.

Antimicrobial activity evaluated using the Disc diffusion method for (a) Silymarin-Chitosan (SL + CH), (b) Silymarin-nHAP (SL + nHAP), and (c) HA/CH/SL nanocomposite. Data are presented as means ± SD from three replicates. Different letters indicate a significant difference, while the same letter denotes no significant difference according to Duncan’s multiple range test at P ≤ 0.05

In vitro cytocompatibility

An MTT assay was conducted to assess the cytocompatibility and metabolic activity of the silymarin/chitosan/hydroxyapatite nanocomposite in human gingival fibroblast cells. The cells were exposed to various concentrations of SL + CH, SL + HA, and SL + CH + HA (10–200 µg/mL) for up to 24 h. The graph (Fig. 6) shows that there were no significant changes in cell viability at the tested concentrations (10–200 µg/mL) of SL + CH, SL + HA, and SL + CH + HA nanocomposites. No toxicity was observed, even at the highest nanocomposite concentration of 200 µg/mL. These results suggested that the silymarin-based nanocomposite significantly enhanced the viability of gingival fibroblast cells. Although the number of viable cells in SL + CH slightly decreased to 80%, the differences between SL + CH, SL + HA, and SL + CH + HA were not statistically significant (p > 0.05). Furthermore, the addition of SL + CH + HA improved cell viability, although no statistical differences were observed. Similar to our observations, composite scaffolds composed of castor oil (CO), hydroxyapatite (HA), and modified scaffolds have led to a notable increase in the proliferation of fibroblast cells over time. Interestingly, the results indicate that varying the amount of nHA-CS from 2.5 wt% to 7.5 wt% in the composite does not affect the enhancement of cell viability [34]. The 3T3 murine fibroblast cells treated with chitosan hydrogel loaded with Aloe vera gel and tetrasodium ethylenediaminetetraacetic acid were found to be cytocompatible and promoted cell proliferation after 72 h, which was statistically significant compared to the control group [35]. These findings underscore the biocompatibility and potential benefits of silyl-marin-based nanocomposite for gingival fibroblast cells.

Fig. 6.

The cytotoxic effects of control and experimental groups. Cells were treated with Group-2 (SL + CH), Group-3 (SL + HA), and Group-4 (SL + CH + HA) in respective concentrations 10, 20, 40, 50, 100 and 200 µg along with Group − 1 control for 24 h, and cell viability was evaluated by MTT assay. Different letters indicate a significant difference, while the same letter denotes no significant difference according to Duncan’s multiple range test at P ≤ 0.05

Calcein AM staining

Without observing the cell nucleus, biocompatibility research remains incomplete as it is crucial to determine the cause of cell death with biomaterials, whether it is due to apoptosis or necrosis. After 24 h of incubation with the nanocomposites and their components (200 µg/mL), no apoptotic nuclei were observed in gingival fibroblast cells. The varied characteristics of the cells after exposure to Calcein M dye are shown in Fig. 7. Healthy and viable cells exhibited normal nuclei and a distinct reticular green staining pattern. Live cells consistently appear green. Early apoptotic cells exhibited green staining with bright green spots in their nuclei, due to chromatin condensation and nuclear fragmentation. However, in the present study, silymarin-based nanocomposite did not induce any of these apoptotic characteristics in gingival fibroblasts after 24 h of treatment. In accordance with our findings, injectable hydrogels comprising carrageenan, locust bean gum, and gelatin showed promise for wound healing. The hydrogel exhibited shear-thinning behavior and supported cell growth, suggesting its potential as a delivery vehicle for tissue repair [36]. Shah et al. fabricated biopolymer-based thermoresponsive injectable Chitosan-CMC-g-PF127 hydrogels containing curcumin that exhibited viscoelastic behavior, controlled release, and good biocompatibility, and In vivo studies demonstrated enhanced tissue regeneration and wound repair potential, highlighting their potential for biomedical applications [37].

Fig. 7.

Fluorescent microscopy of Calcein AM (viable cells) staining of gingival fibroblast cells grown in Control, silymarin-chitosan (SL + CH), silymarin-HAP (SL + HA) nanoparticles, silymarin-nHAP-chitosan (SL + CH + HA) incorporated nanocomposite respectively. Corresponding image groups showing the visible fluorescent green from viable cells and F-actin filaments in respective nanocomposites for 24 h. Scale bars represent 100 μm

In vitro cell migration potential

The wound healing assay, also known as the scratch assay, is a widely used in vitro technique for studying cell migration. The ability of the prepared silymarin nanocomposite to accelerate the migration of human gingival fibroblasts was assessed in a time-dependent experiment spanning 0–24 h. Treatment with 200 µg/mL of SL + CH, SL + HA, or SL + CH + HA nanocomposites resulted in significant wound healing. Notably, the SL + CH + HA nanocomposite treatment demonstrated particularly robust cell migration compared with that of the control cells (Fig. 8). However, SL + CH and SL + HA treatments slightly reduced cell migration. Following this study, nano-composite comprising chitosan, riboflavin, and hydroxyapatite was subjected to NIH3T3 cells to riboflavin for 48 h, and the scratch closure reached nearly 100% for all concentrations tested. We deduced that riboflavin does not impede cell migration or metabolic activity. Moreover, it can potentially function as a catalyst for cell proliferation [38]. Sathuvan et al. reported that κ-carrageenan-essential oil (CEO)-loaded composite biomaterial films enhanced mechanosensing and promoted tissue regenerative wound healing. Specifically, κ-carrageenan film containing 1.5% CEO notably increased the cell population after 72 h of incubation, demonstrating a significant impact on fibroblast migration and in vitro wound healing. This suggests that the incorporation of CEO into the κ-carrageenan film positively influences L929 cell migration, indicating its potential effectiveness in promoting wound-healing processes [39]. These findings suggest that an appropriate concentration of silymarin-based nanocomposite could enhance wound healing by promoting the migration of human gingival fibroblast cells.

Fig. 8.

Representative images from in-vitro scratch wound healing assays demonstrate that cell migration is significantly accelerated in the presence of Group-2 (SL + CH), Group-3 (SL + HA), Group-4 (SL + CH + HA) (200 µg/mL) when compared to Group-1 control for 24 h (scalebar:100 μm). Images were obtained using an inverted Phase contrast microscope

Anti-inflammatory activity

In this study, the ability of silymarin - chitosan nanocomposite to inhibit the haomolysis and the stabilization of RBC membrane was investigated. The test was carried out at different concentrations like 50, 100, 150, 200, 250 µg/mL respectively. The Silymarin chitosan nanocomposite were also able to stabilize the RBC membrane by inhibiting the hemolysis process from 48.1 to 82.4%. Diclofenac sodium showed inhibition in the hemolysis process and stabilization of RBC membrane from 28.7 to 73.2% (Fig. 9). With the increase of chitosan concentration and deacetylation degree (DDA), the number of amino groups available for interaction also increases, which improves its ability to inhibit enzymes and its antioxidant [40]. Chitosan with higher DDA and concentration have better efficacy against enzymes such as hyaluronidase providing anti-inflammatory properties. The higher mucoadhesion quality of chitosan an enhanced to retention time at inflammation site resulting better therapeutic effect. High concentration, high DDA chitosan possesses such bioactivity making it suitable for wound healing and oral care applications where oxidative stress and inflammation must be resolved call for this property [41].

Fig. 9.

Anti-inflammatory activity of Silymarin - chitosan nanocomposite

Conclusion

Silymarin-mediated nanohydroxyapatite (nHAP) was successfully synthesized using a green method. XRD analysis confirmed the high crystallinity and purity of the nanocomposite, with grain size calculations supporting its nanoscale features. TEM analysis revealed that the particle size ranged from 25 nm to 30 nm. The SEM results confirmed the dispersion of nHAP nanoparticles and the intercalation of silymarin-nHAP within the chitosan polymer matrix. The FT-IR analysis further verified the presence of phosphate, amide, and carboxylate groups in the nanocomposite. Antioxidant assays indicated the significant free radical-scavenging activity of the silymarin-based nanocomposite. Furthermore, antibacterial testing demonstrated effective inhibition of oral pathogens, including Streptococcus mutans and Porphyromonas gingivalis. Wettability analysis showed a favorable contact angle, suggesting enhanced surface hydrophilicity, which is crucial for cell adhesion and biointegration in biomedical applications. Biocompatibility studies using cell viability assays demonstrated that the silymarin-based nanocomposite significantly improved the viability of gingival fibroblast cells without inducing significant morphological changes compared with the control group. The AO/EtBr dual staining further confirmed the absence of apoptosis in the nanocomposite-treated cells after 24 h of treatment. The scratch wound healing assay highlighted the ability of the nanocomposite to enhance wound healing by promoting migration of human gingival fibroblasts. Furthermore, the HRBC membrane stabilization assay confirmed the nanocomposite’s anti-inflammatory potential, as it effectively protected erythrocytes against heat-induced hemolysis. This property enhances its suitability for biomedical applications requiring inflammation control. Overall, these findings underscore the potential of silymarin-based nanocomposites as biocompatible, multifunctional materials with promising applications in tissue engineering and regenerative medicine. Their high crystallinity, improved wettability, antioxidant, antibacterial, and anti-inflammatory properties make them particularly suitable for oral wound healing, periodontal therapy, and combating oral pathogens.

Authors’ contributions

Aravind Kumar Subramanian and Gautham Sivamurthy: Conceptualization, writing–original draft. Karen Sarkisovich Karapetyan, Ammar AL-Farga, Rashad Saleh and Mohammad Ali Shariati: Writing–review, and editing.

Funding

None.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval and consent to participate

All methods were performed in accordance with the relevant guidelines and regulations. This study did not involve human or animal experiments. The cell lines used were artificially cultured and did not require ethical approval or moral explanations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.