INTRODUCTION

Bone defects impose a persistent clinical and socioeconomic burden across orthopedics, oral and maxillofacial surgery, and dental implantology.[1] They result from trauma, tumors, congenital anomalies, chronic periodontitis, and surgical procedures (e.g., tooth extraction), and reconstruction is particularly difficult in load-bearing sites.[2,3] Autografts remain the gold standard yet are limited by donor-site morbidity and finite supply.[4] Allografts reduce harvesting but introduce risks of immune rejection and pathogen transmission.[5,6]

To address these constraints, calcium-phosphate bioceramics have been widely investigated; among them, hydroxyapatite (HA) – the principal inorganic constituent of bone – exhibits excellent biocompatibility, osteoconductivity, and chemical affinity for mineralized tissues, and is used clinically and in tissue-engineering constructs.[7] Growing interest now centers on biogenic HA obtained from marine by-products such as fish bone.[8] Fish bone-derived HA (FHA) offers a sustainable, low-cost route that valorizes waste streams while yielding high-purity material suitable for biomedical use.[9,10] Its natural origin may confer hierarchical architecture, trace-element substitution, and surface chemistries reported to enhance osteoinductive capacity and antibacterial behavior.[11-14]

Hydrogel scaffolds provide hydrated, extracellular matrix-mimetic microenvironments with tunable mechanics suitable for bone graft delivery. Natural polysaccharides are attractive building blocks: Alginate (Al), an anionic copolymer from brown algae, ionically crosslinks with divalent cations (example calcium ions [Ca2+]) under mild, cytocompatible conditions.[15] Agarose (Ag), derived from red algae, contributes mechanical robustness and thermo-reversible gelation that supports three-dimensional cell culture.[16] Blending Al with Ag can enhance stability, mechanical integrity, and swelling control.[17] Incorporating HA nanoparticles into such matrices supplies a bone-mimetic mineral phase that can promote osteogenesis, modulate degradation,[18] and support cellular proliferation and differentiation.[19-21]

This study focuses on the synthesis and rigorous characterization of Al/Ag composite hydrogel beads incorporating HA derived from fish bone (FHA). Beads were produced by ionotropic gelation and examined by scanning emission microscope (SEM) analysis, transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, and dynamic swelling assays to define their structural, chemical, and hydration profiles. Parallel constructs containing conventional synthetic HA provided a source-controlled comparator, enabling direct evaluation of how HA origin modulates bead microarchitecture, nanoparticle dispersion, and water-uptake behavior. These comparative data underpin the assessment of FHA-loaded Al/Ag hydrogels as cost-effective, bioactive scaffolds for bone tissue engineering.

MATERIAL AND METHODS Extraction of HA

HA was extracted from the bones of Sardinella fimbriata (80–150 mm in length), procured from a local supplier in Kuala Terengganu, Malaysia. The fish were transported in a frozen state and stored at –20°C until processing. On thawing, the fish bones were separated from soft tissues by boiling them at 200°C for 36 h. Residual flesh and lipids were removed through successive washing, and the cleaned bones were dried at 110°C for 24 h to eliminate moisture.

The pre-treated bone samples were then subjected to a sequential chemical purification process. First, the dried bones were immersed in 0.8 M sodium chloride (NaCl) to remove soluble proteins. They were then treated with 0.2 M sodium hydroxide (NaOH) at 5°C for 5 h to facilitate deproteinization. After thorough rinsing, the bones were soaked in 0.05 M acetic acid (CH3COOH) solution at a 1:10 (w/v) ratio for 3 h to further eliminate organic content. This was followed by extraction in Milli-Q water under continuous agitation at 40°C for 12 h using a shaking water bath. The HA suspension was centrifuged at 10,000 rpm for 60 min at 15°C, and the resulting sediment was filtered using Whatman No. 5 filter paper. Finally, the purified HA was obtained through freeze-drying and stored in sealed containers for use in hydrogel synthesis.

Synthesis of Al/Ag-HA composite hydrogels

Hydrogel beads were synthesized using six different formulations by varying the HA source while keeping polymer concentrations constant. Al (2% w/v) and Ag (1.5% w/v) solutions were prepared individually in sterile distilled water under constant stirring. Conventional HA (CHA) or FHA was incorporated at 0.5% (w/v), along with 0.5% (w/v) ammonium phosphate dibasic to mimic mineralized environments. The components were thoroughly mixed to ensure homogeneity. For bead formation, the Al-containing solutions were loaded into a peristaltic pump and extruded dropwise through an 18-gauge needle into a 0.3 M calcium chloride (CaCl2) solution under gentle agitation. Ionic cross-linking occurred immediately, forming spherical hydrogel beads. For Ag-only systems, droplets were cast into sterile distilled water instead of CaCl2, relying on thermal gelation. Beads were collected, washed 3 times with sterile distilled water to remove unreacted ions, and stored at room temperature in sealed containers until further use. The six formulations were categorized as follows: G1-G3 used CHA, while G4-G6 used FHA, with variations in polymer composition.

Scanning electron microscope

Surface morphology and microstructure of the hydrogel beads were observed using SEM. Before imaging, the beads were desiccated in a vacuum desiccator for 24 h to preserve surface features and minimize shrinkage. The dried samples were mounted on aluminum stubs and sputter-coated with gold to enhance conductivity. Imaging was performed under low vacuum at accelerating voltages ranging from 10 to 15 kV with magnifications from ×500 to ×20,000. SEM analysis was carried out at the School of Health Sciences, Health Campus, Universiti Sains Malaysia.

FT-IR spectroscopy

To assess chemical functional groups and potential molecular interactions within the hydrogels, FTIR analysis was conducted using a Nicolet iZ10 spectrometer equipped with an attenuated total reflectance module. Beads were lightly dried using lint-free tissues and scanned directly without pellet formation. Spectral data were collected over the range of 4,000–400 cm-1 at a resolution of 4 cm-1. Triplicate scans were performed for each sample to ensure reproducibility. Testing was conducted at the Faculty of Earth Science, Universiti Malaysia Kelantan.

TEM

To evaluate the morphology and particle size of HA nanoparticles embedded within the hydrogel matrix, TEM analysis was conducted. Optimized bead samples were crushed into fine powders and suspended in ethanol. A small droplet of the suspension was deposited onto carbon-coated 400-mesh copper grids and allowed to dry at room temperature. Imaging was performed using a JEOL JEM 1400 TEM operated at 120 kV. Particle diameters were measured using ImageJ software based on at least 80 individual particles per sample. The mean particle size and standard deviation were calculated and expressed in nanometers.

Swelling ratio test

The swelling behavior of the hydrogels was evaluated in phosphate-buffered saline (PBS, pH 7.4) at 37°C to simulate physiological conditions. Dried beads were weighed (Wa) and immersed in 300 µL of PBS in a 24-well plate. At predetermined intervals (10, 20, 30, 40, 50, 60, 90, 120, 180, and 360 min), the beads were removed, gently blotted to eliminate surface water using lint-free paper, and weighed again (Wb). The swelling ratio was calculated using the equation:

Degree of swelling (%) = ([Wb – Wa]/Wa) × 100%

Each formulation was tested in triplicate, and average values were recorded. Swelling curves were plotted using Microsoft Excel to compare hydration kinetics.

RESULTS Morphology by scanning electron microscope

The surface morphology of the hydrogel composites was examined using SEM to evaluate the microstructural differences influenced by the conventional and extracted HA from fish bone. The SEM images of the Al2.0–Ag1.5/CHA0.5 composite hydrogel beads [Figure 1a-f] revealed a compact and dense surface architecture. At lower magnification [Figure 1a], the surface appeared smooth, with uniformly dispersed mineral particulates. On increasing magnification [Figure 1b-f], the images illustrated embedded HA clusters well integrated within the polymer matrix, with nanoscale granular features evident at the 2 µm scale. These fine particles are indicative of crystallized HA, displaying consistent geometric morphology suggestive of uniform particle synthesis and effective polymer-filler interaction.

Figure 1: Scanning emission microscope images at (a) ×1,000, (b) ×5,000, (c and d) ×10,000, (e) ×20,000 and (f) ×50,000 magnifications of the surface morphology and pore structure of Al2.0/Ag1.5 hydrogel loaded with 0.5% conventional hydroxyapatite.

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In contrast, the SEM micrographs of Al2.0–Ag1.5/FHA0.5 composite hydrogel beads [Figure 2a-f] demonstrated a markedly different morphology. The surface appeared more porous, with irregular topography and numerous voids dispersed throughout the structure. Higher magnification images [Figure 2d-f] revealed loosely packed mineral regions and occasional fibrous textures. The diminished compactness of the FHA-based hydrogels may be attributed to the biogenic nature of the HA, which can retain organic remnants and display non-uniform particle morphology due to natural variability in source material.

Figure 2: Scanning emission microscope images at (a) ×1,000, (b) ×5,000, (c and d) ×10,000, (e) ×20,000 and (f) ×50,000 magnifications of the surface morphology and pore structure of Al2.0/Ag1.5 hydrogel loaded with 0.5% fish bone extract which highlighted the porous architecture and distribution of the incorporated particles.

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Elucidation of FT-IR spectra analysis of Al-Ag composite hydrogels

FT-IR spectroscopy was employed to investigate the molecular interactions and chemical bonding within the composite hydrogel matrix. The FT-IR spectra of four samples with the formulations: Ag1.5/FHA0.5 (yellow line), Al2.0/FHA0.5 (red line), Al2.0/Ag1.5/FHA0.5 (blue line), and Al2.0/ Ag1.5/CHA0.5 (black line) are displayed in Figure 3.

Figure 3: Fourier-transform infrared spectra of four samples; Ag1.5/(a) FHA0.5, (b) Al2.0/FHA0.5 (red line), (c) Al2.0/Ag1.5/FHA0.5, and (d) Al2.0/Ag1.5/CHA0.5 at the range of 4,000 cm-1–400 cm-1 displayed the characteristic peaks which confirm the functional groups present in the biopolymer network and interactions with the embedded hydroxyapatite.

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All spectra exhibited broad absorption bands in the range of 3,200–3,400 cm-1, corresponding to the O-H stretching vibrations from hydroxyl groups present in both Al and Ag. Notably, the precise peak positions varied slightly among the samples: 3,313 cm-1 (Ag1.5/FHA0.5), 3,298 cm-1 (Al2.0/ FHA0.5), 3,296 cm-1 (Al2.0/Ag1.5/FHA0.5), and 3,294 cm-1 (Al2.0/Ag1.5/CHA0.5). This progressive red shift indicates enhanced hydrogen bonding, potentially due to increased molecular interactions facilitated by both polymer blending and mineral incorporation. The shift was more pronounced in samples containing CHA, suggesting a slightly different hydrogen-bonding environment compared to FHA.

Characteristic peaks observed near 1,633–1,658 cm-1 correspond to the asymmetric stretching of carboxylate (C=O) groups in Al. The intensity of this band was slightly elevated in FHA groups, possibly reflecting interactions with residual organic matter or variations in mineral purity.

The region between 1,034 and 1,093 cm-1, associated with phosphate (PO43-) asymmetric stretching vibrations, provided further insight into HA integration. The CHA-containing sample (Al2.0/Ag1.5/CHA0.5) displayed a distinct peak at 1,038 cm-1, while the FHA-based samples showed broader and shifted peaks: 1,047 cm-1 (Ag1.5/FHA0.5) and 1,034 cm-1 (Al2.0/FHA0.5). This variation suggests that biogenic HA exhibits lower crystallinity and greater structural disorder compared to its synthetic counterpart.

TEM analysis of HA nanoparticles in Al-Ag composite hydrogels

TEM was employed to assess the morphology, particle size, and distribution of HA nanoparticles within the composite hydrogel matrix. Two representative formulations were analyzed: 2% Al incorporating 0.5% CHA, and 2% Al incorporating 0.5% FHA.

In the CHA group, a total of 94 individual nanoparticles were measured [Figure 4]. The particle size distribution ranged from approximately 15 to 120 nm, with the majority concentrated between 40 and 70 nm. The histogram followed a unimodal Gaussian distribution, yielding an average diameter of 57.14 ± 20.37 nm. A slight right-skew suggested the presence of some larger aggregates (>80 nm), potentially arising from secondary agglomeration or heterogeneous nucleation during synthesis. Morphologically, the particles were generally spherical or near-spherical, exhibiting uniform dispersion and minimal overlap, thereby enabling accurate size characterization.

Figure 4: Transmission electron microscope images of (a) Al2.0/CHA0.5 exhibited spherical structure of nanoparticles which uniformly dispersed and (b) particle size distribution histogram fitted with a Gaussian curve showed an average diameter of 57.14 ± 20.37 nm.

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In the FHA group, a total of 79 individual particles were analyzed using TEM micrographs [Figure 5]. The measured particle sizes ranged from 34 nm to 475 nm, with the majority of particles distributed between 80 and 250 nm. The average particle diameter was calculated as 170.07 ± 95.27 nm, indicating a markedly polydisperse system. The histogram exhibited a unimodal distribution with a pronounced right-skewed tail, primarily due to the presence of several large particles exceeding 300 nm. This size heterogeneity may arise from non-uniform nucleation kinetics or localized particle fusion during synthesis or calcination.

Figure 5: Transmission electron microscope images of (a) Al2.0/FHA0.5 displayed cube-spherical structure of nanoparticles which homogenously distributed and (b) particle size distribution histogram fitted with a Gaussian curve showed an average diameter of 170.07 ± 95.27 nm.

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Morphologically, the FHA nanoparticles displayed irregular polygonal to near-spherical shapes, with distinct boundaries and moderate aggregation in localized regions. The observed structural variation likely reflects the influence of the biogenic fish bone precursor and residual organic content modulating crystal growth.

Swelling behavior of Al-Ag composite hydrogels

Swelling capacity is a critical parameter in hydrogel design, as it reflects internal porosity, cross-linking density, and water retention – key attributes for tissue engineering and drug delivery. In this study, the swelling behavior of six AlAg hydrogel formulations was monitored over 360 min to evaluate the effects of polymer ratio and HA source. Data are summarized in Table 1 and shown in Figure 6. The groups were as follows: G1, Al2.0-CHA0.5; G2, Ag1.5-CHA0.5; G3, Al2.0–Ag1.5-CHA0.5; G4, Al2.0-FHA0.5; G5, Ag 1.5-FHA0.5; and G6, Al2.0 –Ag1.5-FHA0.5. The experimental results [Table 1 and Figure 6] show clear distinctions among formulations.

Table 1: The swelling time of each proportion represent the Al/Ag beads (%) versus time (min).

Group Swelling time 10 min (%) 20 min (%) 30 min (%) 40 min (%) 50 min (%) 60 min (%) 90 min (%) 120 min (%) 180 min (%) 360 min (%) G1 155 215 373 485 530 650 1,055 1,513 1,880 2,480 G2 145 159 159 159 164 168 173 173 177 177 G3 53 89 89 142 158 189 279 326 353 374 G4 165 200 235 385 429 518 791 1,197 1,665 2,168 G5 119 131 131 131 150 150 150 150 150 156 G6 47 84 142 211 226 274 353 458 558 558 Figure 6: Graph of swelling behavior for different proportions Al/Ag hydrogel composite beads in percentage (%) over time (minutes) displayed the water uptake capacity for each composition has reflecting the hydrogel’s structural integrity and porosity.

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G1 (Al-CHA) displayed the greatest swelling, reaching 2,480% at 360 min, consistent with Al’s hydrophilicity and its open Ca2+-mediated ionic network; the low HA loading likely preserved free volume that facilitated rapid hydration. G4 (Al-FHA) also swelled strongly (2,168%), though early swelling was more gradual, suggesting that residual organic components or morphological irregularities associated with biogenic FHA may initially slow water diffusion but ultimately enhance retention by increasing surface area and porosity.

Intermediate swelling was observed in the dual-polymer systems. G6 (Al-Ag-FHA) reached 558%, whereas the CHA analog G3 plateaued at 374%. These findings indicate that Ag’s semi-rigid, double-helical network constrains excessive expansion yet maintains sufficient hydration; the somewhat higher final value in G6 also implies that FHA may promote greater water interaction and nanoparticle integration than CHA under comparable polymer conditions.

The Ag-dominant groups swelled least. G2 (Ag-CHA) and G5 (Ag-FHA) reached only 177% and 156%, respectively, and both saturated within 30 min. Limited free volume and the relatively rigid Ag matrix likely restricted fluid ingress. Such restrained swelling may be advantageous where dimensional stability is required – for example, confined defect sites or post-implantation environments in which excessive expansion could disrupt tissue integration.

DISCUSSION

The surface morphology revealed by SEM analysis highlights the critical influence of HA origin on hydrogel structure. CHA-based hydrogels (G3) exhibited a smoother and more compact surface morphology, indicating well-dispersed mineral particles embedded within the polymer network. This could be attributed to the synthetic purity and higher crystallinity of CHA, which promotes more uniform ionic cross-linking with Al and Ag chains, thus strengthening the interfacial bonding.[22] Conversely, FHA-based hydrogels (G6) displayed a rougher surface with increased porosity and particle heterogeneity. This can be linked to the natural biological origin of FHA, which often contains residual collagen and organic carbonates. These organic residues may interfere with mineral crystallization during calcination, resulting in irregular morphologies and enhanced porosity. Interestingly, this porosity may be beneficial for cell infiltration and vascularization, enhancing biological integration in vivo, despite a potential compromise in mechanical strength.[23]

FTIR spectra provided further insights into molecular interactions. The observed red shift in O–H stretching vibrations in multicomponent systems suggests stronger hydrogen bonding networks, likely due to the synergistic interplay between Al’s carboxyl groups and Ag’s hydroxyl chains.[24] In particular, the peak shift from 3,313 cm-1 in the Ag1.5/FHA0.5 system to 3,294 cm-1 in the Al2.0/Ag1.5/ CHA0.5 group indicates increased molecular cohesion and denser polymer entanglement. Broader phosphate (PO43-) bands observed in FHA-containing samples reflect lower crystallinity, possibly due to partial carbonate substitution typical of biogenic apatite, which is known to enhance osteoclastic resorption and promote natural bone turnover.

TEM analysis confirmed the successful formation of nanoscale HA particles in both CHA and FHA systems, though notable differences were observed in their particle size distributions. The CHA group exhibited a relatively narrow distribution (57.14 ± 20.37 nm) with predominantly spherical particles, suggesting uniform nucleation and minimal aggregation. In contrast, the FHA group showed a broader distribution (170.07 ± 95.27 nm), with a right-skewed histogram reflecting the presence of larger particles exceeding 300 nm. This heterogeneity may result from the natural variability of the biogenic fish bone precursor and the modulatory influence of residual organic matter during synthesis.

Rather than being a limitation, the diverse particle morphologies and sizes observed in FHA may mimic the structural complexity of native bone tissue. Such microstructural variation could potentially enhance biological signaling and integration during bone remodeling. With further optimization of processing parameters – such as calcination conditions or precursor refinement – FHA holds considerable promise for applications requiring bioactive and naturally derived bone graft materials. The intrinsic advantages of its biological origin, combined with the tunable nature of its synthesis, position FHA as a strong candidate for bone tissue engineering.[25]

Swelling studies highlighted significant differences in hydrogel hydration behavior, which is tightly linked to network architecture and HA source. G1 and G4 groups, which included Al alone with either CHA or FHA, showed the highest swelling ratios (2,480% and 2,168% at 360 min, respectively). This is consistent with Al’s loose ionic cross-linking structure and its pronounced hydrophilicity.[26] The lower early-stage swelling rate of FHA hydrogels may be due to slower water permeation into more disordered and porous matrices.[22] In contrast, dual-polymer systems (G3 and G6) exhibited more moderate and sustained swelling, suggesting that Ag introduces steric hindrance and reinforces structural stability through double-helix hydrogen bonding. This tunable swelling behavior is crucial for applications such as sustained drug release or bone filler matrices, where volume expansion must be balanced against mechanical integrity.

Collectively, the physicochemical characterization supports the conclusion that FHA integration introduces bioinspired complexity into hydrogel systems. The increased porosity, irregular morphology, and slightly reduced crystallinity in FHA samples may provide biological advantages, including improved osteoconductivity and faster remodeling.[27] However, these features require optimization to prevent compromising structural resilience. Thus, the dual-network design of Al and Ag provides an effective platform to fine-tune mechanical and hydration performance while incorporating naturally derived HA for sustainable and cost-effective scaffold development.

CONCLUSION

This study successfully synthesized and characterized Al-Ag hydrogels incorporating either conventional of fish bone extract HA to assess suitability for bone tissue engineering. FHA-based constructs exhibited more porous surface architectures, narrower nanoparticle size distributions, and slightly broader phosphate vibrational bands than CHA systems – features consistent with residual biogenic constituents such as proteins and carbonates that can template mineral formation. TEM and FTIR analyses corroborated these source-dependent distinctions. Swelling studies showed that FHA hydrogels retain adequate hydration while benefiting from improved nanoparticle dispersion and matrix integration. In addition to these physicochemical advantages, FHA offers eco-friendly sourcing and reduced production cost, with potential biomimetic benefits for cellular response. The dual natural-polymer matrix (Al-Ag) provided a tunable platform for balancing structural integrity, swelling control, and mechanical compliance relevant to load-sharing graft environments.

Collectively, the data support FHA-based Al-Ag hydrogels as promising scaffolds for maxillofacial and orthopedic bone regeneration, where porosity, hydration capacity, and particle uniformity are critical for osteoconduction, vascularization, and tissue integration. Future work should pursue in vitro and in vivo biological validation and explore the incorporation of antimicrobial and/or osteoinductive additives to further enhance regenerative performance.