Synthesis and Characterizations of Alginate/Agarose Hydrogel Incorporated with Hydroxyapatite Extract Fish Bone for Potential Bone Healing Applications

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.

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 Al_2.0–Ag_1.5/CHA_0.5 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.

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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 Al_2.0/Ag_1.5 hydrogel loaded with 0.5% conventional hydroxyapatite.

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In contrast, the SEM micrographs of Al_2.0–Ag_1.5/FHA_0.5 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.

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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 Al_2.0/Ag_1.5 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: Ag_1.5/FHA_0.5 (yellow line), Al_2.0/FHA_0.5 (red line), Al_2.0/Ag_1.5/FHA_0.5 (blue line), and Al_2.0/ Ag_1.5/CHA_0.5 (black line) are displayed in Figure 3.

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Figure 3:

Fourier-transform infrared spectra of four samples; Ag_1.5/(a) FHA0.5, (b) Al_2.0/FHA_0.5 (red line), (c) Al_2.0/Ag_1.5/FHA_0.5, and (d) Al_2.0/Ag_1.5/CHA_0.5 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 (Ag_1.5/FHA_0.5), 3,298 cm^-1 (Al_2.0/ FHA_0.5), 3,296 cm^-1 (Al_2.0/Ag_1.5/FHA_0.5), and 3,294 cm^-1 (Al_2.0/Ag_1.5/CHA_0.5). 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 (PO₄^3-) asymmetric stretching vibrations, provided further insight into HA integration. The CHA-containing sample (Al_2.0/Ag_1.5/CHA_0.5) displayed a distinct peak at 1,038 cm^-1, while the FHA-based samples showed broader and shifted peaks: 1,047 cm^-1 (Ag_1.5/FHA_0.5) and 1,034 cm^-1 (Al_2.0/FHA_0.5). 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.

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Figure 4:

Transmission electron microscope images of (a) Al_2.0/CHA_0.5 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.

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Figure 5:

Transmission electron microscope images of (a) Al_2.0/FHA_0.5 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, Al_2.0-CHA_0.5; G2, Ag_1.5-CHA_0.5; G3, Al_2.0–Ag_1.5-CHA_0.5; G4, Al_2.0-FHA_0.5; G5, Ag _1.5-FHA_0.5; and G6, Al_2.0 –Ag_1.5-FHA_0.5. The experimental results [Table 1 and Figure 6] show clear distinctions among formulations.

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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.