Biogenic nanohydroxyapatite derived from Channa striata fish bones using alkaline hydrolysis and calcination

Biogenic nanohydroxyapatite derived from Channa striata fish bones using alkaline hydrolysis and calcination | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biogenic nanohydroxyapatite derived from Channa striata fish bones using alkaline hydrolysis and calcination Nurdiana Dewi, Meirina Gartika, Dikdik Kurnia, Dwi Gustiono, Nendar Herdianto, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7609723/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Nanohydroxyapatite (nHA) derived from natural resources is an environmentally sustainable alternative to synthetic HA for biomedical applications. In this study, nHA was synthesized from Channa striata fish bones via a modified two-step alkaline hydrolysis process followed by calcination at 550°C. The physicochemical and structural properties of the material were characterized via Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy with energy-dispersive X-ray spectroscopy, transmission electron microscopy, Brunauer–Emmett–Teller analysis, particle size analysis, and thermogravimetric analysis. Functional groups typical of hydroxyapatite, including phosphate, hydroxyl, and carbonate groups, were identified, and X-ray diffraction confirmed a pure crystalline apatite phase with a Ca/P ratio of 1.71, closely matching the stoichiometry of stoichiometric hydroxyapatite. Morphological evaluation revealed rod-shaped nanoparticles of 20–80 nm, and surface analysis revealed nanoscale features with reduced agglomeration following calcination. Thermogravimetric analysis confirmed the complete removal of organic matter at 550°C. Compared with conventional high-temperature calcination methods, the proposed synthesis route requires lower thermal input while maintaining crystallinity and structural stability. These findings demonstrate the potential of Channa striata fish bones as a promising sustainable precursor for the production of high-quality nHA, highlighting their sustainability as a material for dental and biomedical applications. Physical sciences/Chemistry Earth and environmental sciences/Environmental sciences Physical sciences/Materials science Physical sciences/Nanoscience and technology nanohydroxyapatite Channa striata alkaline hydrolysis calcination sustainable biomaterial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Nanohydroxyapatite (nHA) is a bioceramic material that has attracted considerable interest in the dental and biomedical fields because of its excellent biocompatibility, remineralization capacity, and close structural resemblance to natural bone and enamel 1 , 2 . Unlike fluoride, which primarily forms a protective fluorapatite layer, nHA actively promotes enamel repair by remineralizing demineralized regions and improving surface microhardness, making it a promising alternative for caries prevention and management 3 – 5 . Compared with synthetic methods, using natural sources, such as animal bones, offers a sustainable and cost-effective approach to producing nHA 6 . Fish bones, in particular, are rich in calcium and phosphate, the main constituents of hydroxyapatite 7 , 8 . These bones have been utilized for nHA synthesis from species such as salmon, tuna, tilapia, and sardine 9 – 12 . Among these, Channa striata (snakehead fish) is abundant in Southeast Asia and widely cultivated for food and medicinal purposes 13 – 15 . This process generates substantial byproducts, including heads, bones, skin, and viscera 16 . These byproducts, if not properly managed, may pose environmental concerns but also represent valuable resources for high-value biomaterials 17 . Although hydroxyapatite has previously been synthesized from Channa striata bones via alkaline hydrolysis and calcination, challenges remain in terms of optimizing conditions to produce phase-pure nHA with a controlled nanoscale morphology and improved crystallinity 9 , 10 , 18 . In this study, we report a modified alkaline hydrolysis–calcination method employing various sodium hydroxide concentrations and calcination temperatures. The resulting nHA was comprehensively characterized to evaluate its structural and physicochemical properties, highlighting the potential of Channa striata fish bones as a sustainable precursor for high-quality nHA suitable for dental and biomedical applications. Conventional thermal calcination is widely used to remove organic components from bone-derived materials and obtain crystalline hydroxyapatite. In many studies, calcination temperatures between 700 and 1000°C are used to ensure complete decomposition of organic matter and to improve the crystallinity of the resulting hydroxyapatite (HA) phase. However, high-temperature treatments may increase energy consumption, promote particle coarsening, and, in some cases, induce partial phase transformation into secondary calcium phosphate phases, such as tricalcium phosphate (TCP). Previous studies have also reported that excessively high calcination temperatures can lead to grain growth and increased particle agglomeration, thereby compromising the nanoscale characteristics required for biomedical applications 18 – 20 . In the present study, a modified two-step alkaline hydrolysis followed by moderate-temperature calcination at 550°C was employed to obtain nanohydroxyapatite (nHA) from Channa striata fish bones. The calcination temperature of 550°C was selected as a moderate thermal treatment condition based on previous studies reporting that calcination in the range of 500–600°C is sufficient to remove residual organic matter while preserving the hydroxyapatite phase in fish-bone-derived materials 10 , 11 , 20 . Compared with conventional high-temperature calcination protocols, the proposed synthesis route is expected to offer several advantages, including lower thermal input, improved phase stability, reduced particle agglomeration, and preservation of nanostructured morphology, while still achieving high crystallinity and phase purity of hydroxyapatite 18 – 20 . Results Functional group analysis Fourier transform infrared (FTIR) spectroscopy was used to identify the functional groups present in the uncalcined and calcined nHA powders at 550°C, as shown in Fig. 1 . The spectrum shows peaks/intensity (transmittance) at specific wavenumbers associated with the functional groups (ions) contained within the sample. The strongest peaks in the wavenumber range of 1030–1045 cm⁻¹ are attributed to the asymmetric stretching vibration ( v3 ) of the phosphate ions (PO₄³⁻), and the weak absorption band at approximately 569 cm⁻¹ corresponds to the bending vibration ( v4) of the phosphate ions (PO₄³⁻). Additionally, the stretching vibration of the OH⁻ ions is also detected at approximately 3568 cm − 1 . The weak adsorption band at 1630–1637 cm⁻¹ and the broad band at 3100–3500 cm⁻¹ indicate surface-absorbed water (bending vibration H₂O) and crystal water (stretching vibration H₂O), respectively. Moreover, the band at approximately 1430–1472 cm⁻¹ with a very low - intensity peak is associated with the vibration of CO₃²⁻. The weak peak at approximately 2919 cm ¹ typically corresponds to the asymmetric stretching vibration of CH₂ groups, whereas that at 2850 cm⁻¹ is characteristic of symmetric ν (C-H), which is in agreement with the fact that the typical nHA synthesized from fish bone may be caused by reactions with organic compounds 11 . These identified functional groups (PO₄³⁻, CO₃²⁻, and OH⁻) are characteristic of HA compounds. Based on these findings, the sample is indeed HA, with the chemical formula Ca 10 (PO 4 ) 6 (OH) 2 . This result is consistent with other published results for nHA, as shown in Table 1 . Table 1 Functional groups of synthesized nHA based on FTIR Functional Group Wavenumber (cm - 1 ) in the current study Wavenumber (cm⁻¹) based on references ν3 asymmetric stretching PO₄³⁻ 1030–1045 (shoulder and sharp) 1023 1 , 1027 38 1032 39 , 1033 38,40 , 1037 1 1041 41 , 1042 38 , 1046 42 ν1 symmetric stretching PO 4 3- 962 (weak) 962 38,40,43,44 , 960 1,38 ν4 asymmetric bending PO 4 3- 565, 604 (sharp) 570 41 , 574 1 , 576 45 605 1 , 638 1 ν2 bending PO 4 3- 478 1 Stretching mode Hydroxyl (OH - ) 3568 (weak) 3431 1 , 3497 45 3568 41 , 3560 46 3571 1 , 3573 46 Asymmetric Carbonate CO 3 2- 1430–1472 (weak) 1426–1473 1 ,1461 41 2017 1 Out of plane bending mode Carbonate CO 3 2- 874 (weak) 876 1 869 41 Harmonic overtone or combination of v1 and v3 2000 (weak) 2000 39 , 2005-2079 47 Ν asymmetric stretching vibration of CH₂ (C-H) 2919 2919 48 Ν symmetric stretching vibration of CH₂ (C-H) 2843 2850 48 As the calcination temperature increases, the transmittance line decreases, in good agreement with reported research [11], and the organic compound content decreases, as confirmed by thermogravimetric analysis (TGA). Phase and crystallinity analysis Crystalline phase analysis of nHA powders from Channa striata fish bone was conducted via X-ray diffraction (XRD), as illustrated in Fig. 2 . As shown in Fig. 2 , the peaks were sharp, indicating that nHA compounds/phases were affected by the removal of organic components. No other compounds or phases, such as tricalcium phosphate (TCP), are visible. According to Siddarthan et al .’s report, the nHA phase is transformed to the β-tricalcium phosphate (β–TCP) phase at an initiation temperature of 650°C if the produced nHA is deficient in calcium. Therefore, the calcined nHA powder at 550°C remains a pure HA phase in the present study. The XRD patterns of uncalcined and calcined nHA confirmed the crystalline nature of a typical apatite structure with the hexagonal HA phase, aligning with the ICSD ( Inorganic Crystal Structure Database ) card No. 98-016-9498 for pure hydroxyapatite (HA). The values of d-spacing and the 2θ angle of ICSD 98-016-9498 and nHA from Channa Striata fish bone are shown in Table 2 . Table 2 d-spacing of synthesized nHA and comparison with standard HA (ICSD) Crystallo-graphic plane (hkl) ICSD 98-016-9498 nHA Channa striata fish bone Uncalcined Calcined at 550°C Angle (θ) d-Spacing (nm) Angle (θ) d-Spacing (nm) Angle (θ) d-Spacing (nm) 100 10.84 81.58 10.75 82.27 10.82 81.67 002 25.88 34.41 25.86 34.43 25.89 34.39 121 31.78 28.14 31.78 28.14 31.83 28.09 112 32.19 27.78 32.18 27.79 32.22 27.76 300 32.91 27.19 32.91 27.20 32.96 27.15 310 39.81 22.63 39.82 22.62 39.84 22.61 222 46.70 19.43 46.71 19.43 46.75 19.41 123 49.49 18.40 49.49 18.40 49.52 18.39 004 53.20 17.20 53.19 17.21 53.20 17.20 The peaks around (002) indicate that the crystallite size was in the nanometer range 21 . Using the Debye–Scherrer equation (Eq. ( 1 )), the crystallite sizes of uncalcined and calcined nHA along the 002 plane were determined to be 29.47 and 30.46 nm, respectively, as shown in Table 3 . Table 3 Crystallite size, degree of crystallinity, phase content, and line profile parameters of synthesized nHA from Channa striata fish bone compared with those of standard HA (ICSD) nHA Uncalcined Calcined at 550°C Crystallite Size (002) (nm) 29.47 30.46 Degree of Crystallinity (002) (%) 57.33 63.34 Phase content (%) 100 HA (ICSD 98-016-9498) 100 HA (ICSD 98-016-9498) R profile (Rp) 4.55 4.98 Weighted R profile (wRp) 6.57 7.38 GoF 1.27 1.36 Brunner-Emmett-Teller (BET) results Figure 3 shows the BET analysis of the nHA powder. The data presented in Table 4 compare the characteristics of the nHA before and after calcination. The surface area decreases after calcination, indicating a reduction. Moreover, the average particle radius increases, suggesting that particle growth is due to calcination. Additionally, the average pore radius increased slightly, which may be attributed to structural changes during calcination that led to an increase in micropores. Table 4 Characteristics of the nHA based on BET analysis No Parameter Before calcination After calcination 1 Surface area (m 2 /g) 82.83 62.81 2 Particle radius (nm) 16.46 21.71 3 Pore radius (nm) 5.54 5.60 Morphology and surface element composition of the HA powder Figure 4 shows scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of Channa striata fish bone nHA powder samples synthesized via the alkaline hydrolysis method (Fig. 4 a, 4 b, and 4 c) and alkaline hydrolysis technique followed by the calcination method at 550°C (Fig. 4 d, 4 e, and 4 f). Figure 4 a, 4 b, 4 c, and 4 d are SEM images showing that the HA powder particles are spherical and form agglomerates of varying sizes. The particle sizes of these powders range from the nanoscale (Fig. 4 b and 4 e) to the micrometer scale (Fig. 4 a and 4 d). Figure 4 c and 4 f are TEM images showing that the single nHA particles synthesized via the alkaline hydrolysis technique and the alkaline hydrolysis technique followed by calcination have a rod-like shape, with a width (or diameter) and length of approximately 20 and 80 nm, respectively. Moreover, nHA synthesized via the alkaline hydrolysis method has a larger width (Figs. 4 b and 4 c) than nHA synthesized via the alkaline hydrolysis method followed by the calcination method (Fig. 4 e and 4 f). The reduction in the width of the nHA particles is thought to be due to a decrease in organic material as the temperature increases to 550°C. There is no organic material, and only the inorganic material remains, namely, HA, as proven by the TGA characterization results below and the XRD characterization results above. Particle size analysis Figure 5 shows the energy-dispersive X-ray spectroscopy ( EDS) characterization results related to the elemental contents of O, Ca, P, Cd, Hg, As and Pb in the Channa striata fish bone nHA powder samples synthesized via the alkaline hydrolysis method (Fig. 5 a) and the alkaline hydrolysis method followed by the calcination method at 550°C (Fig. 5 b). The values of the Ca and P elemental compositions and the Ca/P ratio of HA synthesized via the two methods mentioned above are 16.3% Ca, 9.3% P, and Ca/P ratio = 1.78, and 17.6% Ca, 10.3% P, and Ca/P ratio = 1.71. Figure 6 shows the distribution of the size of the group (agglomerate) of nHA particles derived from the bones of Channa striata before and after calcining at 550°C. In the sample before (not) calcination, the range of particle sizes varied from 700 to 2500 nm, and the number of particles measuring between 1000 and 1600 nm was the greatest. While the size distribution of the HA particles that had been calcined at 550°C ranged from approximately 60 to 1600 nm, the number of particles measuring between 300 nm and 600 nm was the greatest. The results of this particle size analysis (PSA) characterization show that calcination at 550°C reduces the agglomeration of nHA particles, which is thought to be due to the loss of inorganic material that covers and binds between particles, as shown by the results of the TGA characterization, where inorganic material is no longer present starting at a temperature of approximately 530°C. Variations in particle size from tens to thousands of nanometers were also observed in the SEM characterization results, where the increase in particle size was caused by agglomeration between individual nHA particles during the growth and bone formation process. The left image in Fig. 7 shows the TGA curve of the nHA sample before calcination. The TGA curve shows that up to a heating/calcination temperature of approximately 550°C, the sample mass decreases by 11.5%. This indicates that the organic material is completely removed at approximately 550°C. The calcination process from 550 to 1000°C did not result in any further reduction in sample mass. In conclusion, this study’s calcination temperature of 550°C is appropriate to ensure that the resulting sample is pure inorganic nHA. Discussion nHA can be obtained through two main approaches: chemical synthesis and extraction from biological sources 22 . Compared with its synthetic counterpart, nHA derived from animal bones offers several benefits, including a chemical composition and structural properties similar to those of human bone, lower production costs, and enhanced biocompatibility 6 . Organic matter removal from bone is typically achieved through alkaline treatment with NaOH, which hydrolyzes organic components, leaving calcium phosphate, which is subsequently washed and filtered 11 . To ensure the complete elimination of organic residues, calcination is performed, producing a purified nHA material 19 . Calcination plays a critical role in determining the crystallinity, phase composition, and microstructure of hydroxyapatite derived from biological sources. Conventional extraction methods often employ calcination temperatures above 700°C to eliminate residual organic matter and enhance crystallinity. Nevertheless, several studies have shown that excessively high calcination temperatures can promote particle coarsening and may induce a phase transformation of hydroxyapatite to tricalcium phosphate when calcium deficiency occurs 18 , 19 . The modified synthesis route used in the present study combines two-step alkaline hydrolysis with moderate-temperature calcination at 550°C, offering several advantages over conventional high-temperature protocols. First, alkaline hydrolysis effectively removes organic components prior to calcination, allowing the thermal treatment to be performed at a lower temperature 10 . Second, calcination at 550°C is sufficient to eliminate residual organic residues, as confirmed by TGA analysis, while preventing excessive grain growth and maintaining nanoscale particle morphology 11 , 19 . Third, the moderate calcination temperature helps preserve the hydroxyapatite phase without forming secondary phases such as tricalcium phosphate (TCP) 20 . These results suggest that the proposed method provides an energy-efficient and structurally controlled approach for producing phase-pure nanohydroxyapatite from Channa striata bone waste. This study demonstrated that nanohydroxyapatite (nHA) can be effectively synthesized from Channa striata fish bones via two-step alkaline hydrolysis followed by calcination at 550°C. The TGA results confirm that the major mass loss associated with organic decomposition occurred below approximately 550°C, indicating that higher calcination temperatures are not necessary for organic removal. This supports selecting a moderate calcination temperature that minimizes energy consumption while preserving nanoscale morphology and phase stability. The combined treatment successfully removed organic matter, preserved the mineral phase, and promoted the formation of rod-shaped, crystalline nHA particles. Alkaline hydrolysis is essential in the preparation of nHA, as it effectively removes organic components such as collagen, proteins, and lipids from the fishbone 11 . The use of an alkaline solution, typically NaOH, facilitates the breakdown of organic matter while preserving the mineral phase, which is predominantly composed of calcium phosphate. This step ensures that the resulting material retains high purity, which is crucial for biomedical applications 23 . Calcination plays a critical role in the crystallization and phase transformation of nHA. Heating at 550°C for 2 hours effectively eliminates any remaining organic content while promoting the development of a well-ordered crystalline structure. The FTIR and XRD results confirmed the presence of characteristic phosphate, hydroxyl, and carbonate groups, as well as the formation of a pure hydroxyapatite phase with improved crystallinity. Compared with previous reports, the synthesis conditions applied here yielded nHA with higher crystallinity and phase stability at relatively moderate calcination temperatures. In contrast, Herpandi et al . reported that synthesis at higher temperatures (900°C) led to partial decomposition of HA into TCP, whereas our method preserved a stable HA phase 18 . According to Siddarthan et al ., calcium-deficient HA tends to transform into the β-TCP phase at 650°C 24 . The absence of secondary phases such as calcium oxide (CaO) or TCP in this study indicates that the applied synthesis conditions were effective 23 . Uncalcined nHA from Channa striata fish bone resulted in a slightly larger particle size than nHA obtained from tilapia fish scales (24.6 nm) 25 . In contrast, HA synthesized from devilfish (Loricariidae) bone and calcined at 550°C had a particle size of 28.62 nm, which is close to that of the calcined nHA in this study 26 . The crystallinity percentage of uncalcined nHA was calculated to be 57.33%. The crystallinity of uncalcined nHA was 57.33%, increasing to 63.34% after calcination at 550°C. The calcination process induced nucleation and crystal growth of HA, as indicated by the narrowing of the diffraction peaks, suggesting higher crystallinity and larger crystallite sizes. This improvement, reflected by narrower diffraction peaks, indicates enhanced nucleation and crystal growth. The increase in crystallinity is consistent with previous findings on porcine bone-derived nHA and exceeds the values reported for Channa striata nHA synthesized via precipitation methods 27 , 28 . SEM and TEM analyses revealed that the synthesized nHA exhibited a rod-like morphology, which is a typical characteristic of biogenic HA 29 . Previous studies have attributed the transition from needle-like to rod-like morphology to the substitution of CO₃²⁻ for PO₄³⁻ and Na⁺ for Ca²⁺ 1 . BET analysis revealed a high surface area, which may enhance bioactivity and support potential applications in biomedical and dental materials 30 , 31 . The Ca/P ratio is a critical parameter for evaluating HA bioactivity. The nHA synthesized from Channa striata fish bone in this study presented a Ca/P ratio of 1.71, which is close to the stoichiometric value of 1.67 for natural bone minerals. Elemental analysis by EDS confirmed high purity with minimal contaminants. Previous studies have reported varying Ca/P ratios for Channa striata nHA depending on the synthesis conditions, ranging from 1.13 (alkaline hydrolysis alone) to 1.72 (alkaline hydrolysis with calcination at 600°C) 18 . Fatmawati et al . reported that the Ca/P ratio of Channa striata fish bone nHA synthesized via the alkaline hydrolysis technique alone was 1.13 13 . Similar variability has been observed in other fish species, with values ranging from 1.35 to 1.69 depending on the processing method used 32 . Importantly, the heavy metal content (0.0% Cd, 0.1% Hg, 0.2% As, and 0.0% Pb) remained below the permissible threshold defined by ISO 13779-6, underscoring the suitability of Channa striata as a safe precursor for biomedical applications 33 . Stoichiometric hydroxyapatite has a Ca/P ratio of approximately 1.67 34 . In contrast, biological sources such as fish scales and animal bones generally produce nonstoichiometric HA due to the incorporation of carbonate groups and trace ions (HPO₄²⁻, Na⁺, Mg²⁺, Sr²⁺, K⁺, Cl⁻, and F⁻) in the crystal lattice 7 . The nHA synthesized in this study had a Ca/P ratio lower than those reported by Hariani et al . (2.21) and Pon-on et al . (2.01), suggesting that calcination influences the final composition 35 , 36 . Our results are consistent with those of Herpandi et al ., who reported Ca/P ratios of 1.69–1.72 for Channa striata bone-derived nHA obtained via alkaline hydrolysis followed by calcination at 600°C 18 . Although the physicochemical and structural properties of the synthesized nHA were comprehensively characterized, this study did not include in vitro or in vivo biological evaluations. Further investigations focusing on cytocompatibility, bioactivity, and long-term stability are necessary to fully assess its suitability for clinical dental and biomedical applications. Conclusions This study demonstrates a sustainable approach for producing nanohydroxyapatite (nHA) from Channa striata fish bones using a modified two-step alkaline hydrolysis followed by calcination at 550°C. The applied synthesis route effectively removed organic components while preserving the hydroxyapatite phase and nanoscale morphology. Structural and compositional analyses confirmed the formation of phase-pure hydroxyapatite with rod-shaped nanoparticles and a Ca/P ratio close to the stoichiometric value. The moderate calcination temperature enabled the production of crystalline nHA while limiting particle agglomeration and maintaining nanoscale structural characteristics. Compared with conventional high-temperature calcination methods, the proposed approach provides a more energy-efficient route for producing biogenic nanohydroxyapatite. These findings highlight the potential of Channa striata bone waste as an environmentally sustainable and cost-effective precursor for biomedical and dental material applications. Methods Sample collection The Channa striata fish bones were sourced from a Channa striata cracker factory in Banjarmasin, Indonesia. All chemicals used in this study were of analytical grade. Preparation of the fish bones A wooden hammer and a bladed cutter were used to delicately remove the fish bones from the collected samples. The bones were carefully boiled at approximately 100°C in 2 L of distilled water to remove any leftover flesh or skin. To eliminate any remaining proteins, lipids, oils, or other organic contaminants, the cleaned bones were boiled for 1 hour after being submerged in a 500 mL solution containing 2% sodium hydroxide (NaOH, HiMedia Laboratories Pvt. Ltd., Mumbai, India) and 10 mL of acetone (Merck, Darmstadt, Germany). To remove any remaining moisture, the treated bones were cleaned and dried in an oven set to 100°C for 3 hours. Finally, a mortar and pestle were used to grind the dried fish bones into a fine powder. Synthesis of nHA The alkaline hydrolysis method in this study was adapted with slight modifications from the research of Surya et al . 11 . Similarly, the calcination process was modified from the study conducted by Bee et al . 20 . The modifications mainly involved applying a two-step alkaline hydrolysis process with varying NaOH concentrations, followed by moderate-temperature calcination at 550°C. The procedure is summarized and illustrated in Fig. 8 . Two steps of alkaline hydrolysis were applied to the prepared fish bones. They were first heated to 70°C, agitated for 5 hours at 400 rpm, and submerged in a 5% NaOH solution (HiMedia Laboratories Pvt. Ltd., Mumbai, India) at a 1:14 (w/v, FB:NaOH) ratio. After the treated bones were filtered, the precipitates were carefully cleaned with distilled water and dried for two hours at 100°C. The second phase involved heating the dried precipitate to 100°C for 1 hour after treatment with a 50% NaOH solution at a 1:6 (w/v) precipitate:NaOH solution ratio. After the resulting white solution was filtered, the filtrate was rinsed with distilled water until it was clear. Following the suspension of the resulting white precipitate in 200 millilitres of distilled water, 0.1 M phosphoric acid (H₃PO₄, Merck, Darmstadt, Germany) was added to adjust the pH to neutral (pH 7). After an hour of constant stirring, the mixture was filtered. To obtain nHA powder, the finished product was sieved after drying for 2 hours at 100°C in an oven. The samples were subsequently placed in a Carbolite CWF 1100 furnace (Germany) and calcined at 550°C. Over 2 hours, the heating process was conducted at a controlled rate of 5°C per minute. After calcination at 550°C for 2 hours, the sample was allowed to cool naturally inside the furnace. Characterization of nHA powder The synthesized nHA was characterized via multiple analytical techniques to assess its structural and compositional properties. Fourier Transform Infrared Spectroscopy (FTIR) The presence of nHA was verified, and functional groups were identified via Fourier transform infrared (FTIR) spectroscopy. A Thermo Scientific Nicolet iS-10 was used to acquire the spectra via a transmission approach with KBr added at the BRIN. At room temperature, measurements were made between 4000 and 500 cm⁻¹, with a resolution of ± 4 cm⁻¹ and a scan frequency of 16 times. X-ray diffraction (XRD) analysis An X-ray diffractometer (SmartLab, Rigaku Corporation, Japan) equipped with Cu Kα1 radiation at BRIN was used to analyze the crystalline phases and the purity of the nHA powder. To identify the phase, the acquired diffraction patterns were compared with reference data from the Inorganic Crystal Structure Database (ICSD) No. 98-016-9498. The data acquisition was carried out with a step width of 0.01° and within the 2θ range of 10° to 75°. The average crystallite size of a sample was calculated via the Debye–Scherrer equation, and the degree of crystallinity (Xc) was approximated via mathematical relationships 21 , 37 . $${D}_{hkl}=\frac{k\lambda}{\beta\text{cos}\theta}$$ 1 $${X}_{c}=1-{\left(\frac{0.24}{\beta}\right)}^{3}$$ 2 where λ = 1.5405 Å (the wavelength of the X-ray source); β = full width at half maximum (FWHM) of the diffraction peak; Dhkl = crystallite size determined from (hkl) reflections; and k = 0.94 (Scherrer constant). Brunauer–Emmett–Teller (BET) analysis The powder BET surface area, pore size, and pore volume were measured via an N₂ adsorption‒desorption analyzer (Quantachrome, Quadsorbs evo) at BRIN. A 10 mg sample was introduced into the system and pretreated to remove moisture before N₂ adsorption at -196°C. Two measurements were conducted, and the results are presented as the statistical average. Scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) A field emission-scanning electron microscope (FE-SEM) Apreo 2S (Thermo Fisher Scientific, USA) operating at an accelerating voltage of 10 kV was used to examine the microstructures and morphologies of the nHA crystals. The local chemical composition was examined by integrating FE-SEM with energy-dispersive X-ray spectroscopy (EDS) (Thermo Fisher Scientific, USA). Both measurement tools are at the BRIN. Transmission electron microscopy (TEM) analysis Images of the nanostructure were captured via a Talos F200X transmission electron microscope (Thermo Fisher Scientific, USA) operated at 200 kV. The powder particle morphology was examined, and particle size was determined using ImageJ. Thermogravimetric analysis (TGA) The thermal degradation properties of the powders were examined via TGA (TGA Absys Evo Setaram Simultaneous TG DTA) at BRIN. With a heating rate of 5°C·min⁻¹ and a nitrogen flow rate of 70 ml·min⁻¹, a 20 mg sample was heated from room temperature to 700°C in a nitrogen environment. Particle size analysis The dynamic light scattering (DLS) method (CILAS NANO DS Dual Scattering) at BRIN was used to measure the particle size of the nHA powder. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Declarations Acknowledgments The authors express their deepest gratitude to Universitas Padjadjaran and the National Research and Innovation Agency (BRIN) for their full support of this research. The authors also wish to thank the Indonesian Ministry of Education, Culture, Research, and Technology for the Doctoral Dissertation Research Grant, and the National Research and Innovation Agency for Nanotechnology and Materials (BRIN) for its RIIM LPDP research grants. Author contributions statement All authors confirm that they have read and approved the final version of the manuscript and that the author order has been agreed upon by all. Nurdiana Dewi: Writing – original draft; Writing – review & editing; Methodology; Investigation; Data curation. Meirina Gartika: Writing – review & editing; Writing – original draft; Investigation; Conceptualization. Dwi Gustiono: Writing – review & editing; Writing – original draft; Methodology; Investigation; Data curation. Dikdik Kurnia: Writing – review & editing; supervision; conceptualization. Nendar Herdianto: Writing – original draft; visualization; investigation; conceptualization. Riesma Tasomara: Writing – original draft; Writing – review & editing; Methodology; Investigation; Data curation . Winda Rianti: Writing – original draft; visualization; investigation; conceptualization. Nuning Aisah: Writing – original draft; visualization; investigation; conceptualization. Bambang Triwibowo: Writing – original draft; visualization; investigation; conceptualization. Funding This study was supported by the National Research Organization for Nanotechnology and Materials (BRIN) through RIIM LPDP (B-807/II.7.5/FR/6/2022; B-6952/III.10/KS.00.00/6/2022), and by the Indonesian Ministry of Education, Culture, Research, and Technology through the Doctoral Dissertation Research Grant (093/C3/DT.05.00/PL/2025; 1604/UN6.3.1/PT.00/2025). Competing interest The authors declare that they have no competing interests. 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Herpandi, Hanif I, Widiastuti I, Sudirman S. Hydroxyapatite characteristics from snakehead fish ( Channa striata ) bone via alkali treatment followed by calcination method. Tropical Journal of Natural Product Research (TJNPR) 2024; 8 : 6147–6151. Kurzyk A, Szwed-Georgiou A, Pagacz J, Antosik A, Tymowicz-Grzyb P, Gerle A et al. Calcination and ion substitution improve physicochemical and biological properties of nanohydroxyapatite for bone tissue engineering applications. Sci Rep 2023; 13 . doi:10.1038/S41598-023-42271-2. Bee S-L, Noor SNFM, Ul-Hamid A, Hamid ZAA, Bee S-L, Noor SNFM et al. Effect of calcination temperature on the physicochemical properties of natural hydroxyapatite derived from catla fish bone. JPhCS 2024; 2907 : 012010. Padmanabhan VP, Kulandaivelu R, Santhana Panneer D, Vivekananthan S, Sagadevan S, Anita Lett J. Microwave synthesis of hydroxyapatite encumbered with ascorbic acid intended for drug leaching studies. Materials Research Innovations 2020; 24 : 171–178. Mohd Pu’ad NAS, Abdul Haq RH, Mohd Noh H, Abdullah HZ, Idris MI, Lee TC. Synthesis method of hydroxyapatite: A review. Mater Today Proc 2020; 29 : 233–239. Mohd Pu’ad NAS, Koshy P, Abdullah HZ, Idris MI, Lee TC. Syntheses of hydroxyapatite from natural sources. Heliyon 2019; 5 : e01588. Siddharthan A, Seshadri SK, Sampath Kumar TS. Rapid synthesis of calcium deficient hydroxyapatite nanoparticles by microwave irradiation. 2005; 18 : 110–113. Mohd Pu’ad NAS, Abdul Haq RH, Mohd Noh H, Abdullah HZ, Idris MI, Lee TC. Nano-size hydroxyapatite extracted from tilapia scale using alkaline heat treatment method. Mater Today Proc 2020; 29 : 218–222. Cisneros-Ontiveros HG, Zubieta-Otero LF, Medellín-Castillo NA, Flores-Rojas AI, Rodriguez-Garcia ME. Extraction of bio-hydroxyapatite from devilfish (Loricariidae) for the fluoride and cadmium adsorption from water and its feasible photocatalytic properties. Chemosphere 2024; 366 : 143535. Castillo-Paz AM, Londoño-Restrepo SM, Tirado-Mejía L, Mondragón MA, Rodríguez-García ME. Nano to micro size transition of hydroxyapatite in porcine bone during heat treatment with low heating rates. Progress in Natural Science: Materials International 2020; 30 : 494–501. Dewi N, Rahmah RA, Wardhana AS, Puspitasari D, Wasiaturrahmah Y, Gustiono D. Remineralizing potential of natural hydroxyapatite from Snakehead ( Channa striata ) fish bone on remineralization of primary teeth enamel: An in vitro study. European J Gen Dent 2024. doi:10.1055/S-0044-1791706. Wang Z, Jiang S, Zhao Y, Zeng M. Synthesis and characterization of hydroxyapatite nano-rods from oyster shell with exogenous surfactants. Materials Science and Engineering: C 2019; 105 : 110102. Ma Y, Wang A, Li J, li Q, Han Q, Chen Y et al. Preparation of hydroxyapatite with high surface area and dispersity templated on calcium carbonate in dipeptide hydrogels. Colloids Surf A Physicochem Eng Asp 2020; 596 : 124740. Pokhrel S, Pokhrel S. Hydroxyapatite: preparation, properties and its biomedical applications. Advances in Chemical Engineering and Science 2018; 8 : 225–240. Kusumawati P, Triwitono P, Anggrahini S, Pranoto Y. Nano-calcium powder properties from six commercial fish bone waste in Indonesia. Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology 2022; 17 : 1–12. ISO 13779-3:2018 - Implants for surgery - Hydroxyapatite - Part 3: Chemical analysis and characterization of crystallinity ratio and phase purity. https://webstore.ansi.org/standards/iso/iso137792018 (accessed 31 Jul2025). Naubnome V, Prihanto A, Schmahl WW, Pusparizkita YM, Ismail R, Jamari J et al. Chemical precipitation of nanocrystalline hydroxyapatite with calcium carbonate derived from green mussel shell wastes and several phosphorus sources. Case Studies in Chemical and Environmental Engineering 2025; 11 : 101154. Hariani PL, Muryati M, Said M, Salni S. Synthesis of nano-hydroxyapatite from snakehead ( Channa striata ) fish bone and its antibacterial properties. In: Key Engineering Materials . Trans Tech Publications Ltd, 2020, pp 293–299. Pon-On W, Suntornsaratoon P, Charoenphandhu N, Thongbunchoo J, Krishnamra N, Tang IM. Hydroxyapatite from fish scale for potential use as bone scaffold or regenerative material. Materials Science and Engineering: C 2016; 62 : 183–189. Sa Y, Guo Y, Feng X, Wang M, Li P, Gao Y et al. Are different crystallinity-index-calculating methods of hydroxyapatite efficient and consistent? New Journal of Chemistry 2017; 41 : 5723–5731. Gadaleta SJ, Mendelsohn R, Paschalis EL, Camacho NP, Betts F, Boskey AL. Fourier transform infrared spectroscopy of synthetic and biological apatites. Mineral Scale Formation and Inhibition 1995; : 283–294. Kim JH, Kim SH, Kim HK, Akaike T, Kim SC. Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods. J Biomed Mater Res 2002; 62 : 600–612. Fowler BO. Infrared studies of apatites. I. Vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic substitution. Inorg Chem 1974; 13 : 194–207. Chandrasekar A, Sagadevan S, Dakshnamoorthy A. Synthesis and characterization of nano-hydroxyapatite (n-HAP) using the wet chemical technique. International Journal of Physical Sciences Full Length Research Paper 2013; 8 : 1639–1645. Fowler BO. Infrared studies of apatites. I. Vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic substitution. Inorg Chem 1974; 13 : 194–207. Klee WE, Engel G. I.R. spectra of the phosphate ions in various apatites. Journal of Inorganic and Nuclear Chemistry 1970; 32 : 1837–1843. Baddiel CB, Berry EE. Spectra structure correlations in hydroxy and fluorapatite. Spectrochimica Acta 1966; 22 : 1407–1416. Panda NN, Pramanik K, Sukla LB. Extraction and characterization of biocompatible hydroxyapatite from fresh water fish scales for tissue engineering scaffold. Bioprocess Biosyst Eng 2014; 37 : 433–440. Posner AS, Stutman JM, Lippincott ER. Hydrogen-bonding in calcium-deficient hydroxyapatities. Nature 1960; 188 : 486–487. Panda S, Biswas CK, Paul S. A comprehensive review on the preparation and application of calcium hydroxyapatite: A special focus on atomic doping methods for bone tissue engineering. Ceram Int 2021; 47 : 28122–28144. Manullang J, Nugroho R, Rohmah M, Rudianto, Qorysuchi A. Plant-extract-mediated biosynthesis of silver nanoparticles using Eleutherine americana bulb extract and its characterization. 2021. doi:10.13057/nusbiosci/n130216. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Apr, 2026 Reviews received at journal 17 Mar, 2026 Reviews received at journal 07 Mar, 2026 Reviewers agreed at journal 06 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers invited by journal 04 Mar, 2026 Submission checks completed at journal 03 Mar, 2026 First submitted to journal 03 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7609723","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":602294020,"identity":"a5abebe6-9124-4308-a5df-de82a0535fb1","order_by":0,"name":"Nurdiana Dewi","email":"","orcid":"","institution":"Universitas Padjadjaran","correspondingAuthor":false,"prefix":"","firstName":"Nurdiana","middleName":"","lastName":"Dewi","suffix":""},{"id":602294022,"identity":"5e491460-6d88-4086-986c-755c890cbdd8","order_by":1,"name":"Meirina Gartika","email":"","orcid":"","institution":"Universitas 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(BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Nuning","middleName":"","lastName":"Aisah","suffix":""},{"id":602294030,"identity":"5fee1f03-a9ab-4715-9996-9b097ab14a02","order_by":8,"name":"Bambang Triwibowo","email":"","orcid":"","institution":"National Research and Innovation Agency (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Bambang","middleName":"","lastName":"Triwibowo","suffix":""}],"badges":[],"createdAt":"2025-09-14 00:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7609723/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7609723/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104213864,"identity":"590f16e5-90a4-4abf-b509-cf38f5789b55","added_by":"auto","created_at":"2026-03-09 08:34:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196441,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared (FTIR) spectra of nanohydroxyapatite (nHA) derived from \u003cem\u003eChanna striata\u003c/em\u003e fish bone before calcination (red line) and after calcination at 550 °C (blue line), showing characteristic vibrational bands of phosphate (PO₄³⁻), hydroxyl (OH⁻), and carbonate (CO₃²⁻) groups.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/e17dc743376ec38bda5849e9.png"},{"id":104213861,"identity":"29d875b2-fb15-4497-a547-18f69a266034","added_by":"auto","created_at":"2026-03-09 08:34:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187910,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction (XRD) patterns of nanohydroxyapatite (nHA) derived from \u003cem\u003eChanna striata\u003c/em\u003e fish bone, indicating the crystalline hydroxyapatite phase and the absence of secondary phases\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/37c5dd93b024751a5af528ed.png"},{"id":104779703,"identity":"7fa3dff5-ba3e-49e5-b6ec-d781b15dcd49","added_by":"auto","created_at":"2026-03-17 07:44:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126293,"visible":true,"origin":"","legend":"\u003cp\u003eBrunauer–Emmett–Teller (BET) nitrogen adsorption–desorption analysis of nanohydroxyapatite (nHA), showing surface area and pore characteristics of the synthesized material\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/950afad9e5177127601378cf.png"},{"id":104405132,"identity":"c56cb293-9010-4dc2-ac2e-c08adb3c93cf","added_by":"auto","created_at":"2026-03-11 12:21:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":773310,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of nanohydroxyapatite (nHA) derived from \u003cem\u003eChanna striata\u003c/em\u003e fish bone synthesized via alkaline hydrolysis (a–c) and alkaline hydrolysis followed by calcination at 550 °C (d–f), illustrating particle morphology and nanoscale structure\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/f9d6b75f2905eb5099b062d5.png"},{"id":104213867,"identity":"60ab3c8a-ca8c-480e-8475-82372b1e026e","added_by":"auto","created_at":"2026-03-09 08:34:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":359446,"visible":true,"origin":"","legend":"\u003cp\u003eEnergy-dispersive X-ray spectroscopy (EDS) spectra of nanohydroxyapatite (nHA) derived from \u003cem\u003eChanna striata\u003c/em\u003e fish bone before calcination (a) and after calcination at 550 °C (b), showing the elemental composition of calcium (Ca), phosphorus (P), and oxygen (O)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/af3ef82e6acb5f13e73796a5.png"},{"id":104213865,"identity":"62587dbe-0e7b-4552-84c1-dbd32ad884f3","added_by":"auto","created_at":"2026-03-09 08:34:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90954,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution of nanohydroxyapatite (nHA) derived from \u003cem\u003eChanna striata\u003c/em\u003efish bone before (a) and after (b) calcination at 550 °C, measured using particle size analysis (PSA)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/555a80d9b490fcbeb6737a1b.png"},{"id":104213866,"identity":"b12ae399-1eb1-4919-8233-5b9294c5c45b","added_by":"auto","created_at":"2026-03-09 08:34:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":67585,"visible":true,"origin":"","legend":"\u003cp\u003eThermogravimetric analysis (TGA) curve of nanohydroxyapatite (nHA) derived from \u003cem\u003eChanna striata\u003c/em\u003e fish bone before calcination, showing mass loss associated with the removal of organic components during heating.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/7b9ca4e63976361bbcff5f61.png"},{"id":104405197,"identity":"bd5e3b4a-b790-414f-aab6-96dce2212bee","added_by":"auto","created_at":"2026-03-11 12:22:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":165086,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the synthesis process of nanohydroxyapatite (nHA) from \u003cem\u003eChanna striata\u003c/em\u003e fish bone. (1) Fishbone preparation; (2) and (3) alkaline hydrolysis process; (4) calcination process\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/d44096c56d90f5d9c1864321.png"},{"id":104785640,"identity":"1208f6f1-20f2-4d53-ad1d-8e3ce912d6d9","added_by":"auto","created_at":"2026-03-17 08:12:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3183450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7609723/v1/2e45f8c1-ee92-4256-ba54-431b5e52c1b7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biogenic nanohydroxyapatite derived from Channa striata fish bones using alkaline hydrolysis and calcination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanohydroxyapatite (nHA) is a bioceramic material that has attracted considerable interest in the dental and biomedical fields because of its excellent biocompatibility, remineralization capacity, and close structural resemblance to natural bone and enamel\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Unlike fluoride, which primarily forms a protective fluorapatite layer, nHA actively promotes enamel repair by remineralizing demineralized regions and improving surface microhardness, making it a promising alternative for caries prevention and management\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCompared with synthetic methods, using natural sources, such as animal bones, offers a sustainable and cost-effective approach to producing nHA\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Fish bones, in particular, are rich in calcium and phosphate, the main constituents of hydroxyapatite\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These bones have been utilized for nHA synthesis from species such as salmon, tuna, tilapia, and sardine \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Among these, \u003cem\u003eChanna striata\u003c/em\u003e (snakehead fish) is abundant in Southeast Asia and widely cultivated for food and medicinal purposes\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This process generates substantial byproducts, including heads, bones, skin, and viscera\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These byproducts, if not properly managed, may pose environmental concerns but also represent valuable resources for high-value biomaterials\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough hydroxyapatite has previously been synthesized from \u003cem\u003eChanna striata\u003c/em\u003e bones via alkaline hydrolysis and calcination, challenges remain in terms of optimizing conditions to produce phase-pure nHA with a controlled nanoscale morphology and improved crystallinity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this study, we report a modified alkaline hydrolysis\u0026ndash;calcination method employing various sodium hydroxide concentrations and calcination temperatures. The resulting nHA was comprehensively characterized to evaluate its structural and physicochemical properties, highlighting the potential of \u003cem\u003eChanna striata\u003c/em\u003e fish bones as a sustainable precursor for high-quality nHA suitable for dental and biomedical applications.\u003c/p\u003e \u003cp\u003eConventional thermal calcination is widely used to remove organic components from bone-derived materials and obtain crystalline hydroxyapatite. In many studies, calcination temperatures between 700 and 1000\u0026deg;C are used to ensure complete decomposition of organic matter and to improve the crystallinity of the resulting hydroxyapatite (HA) phase. However, high-temperature treatments may increase energy consumption, promote particle coarsening, and, in some cases, induce partial phase transformation into secondary calcium phosphate phases, such as tricalcium phosphate (TCP). Previous studies have also reported that excessively high calcination temperatures can lead to grain growth and increased particle agglomeration, thereby compromising the nanoscale characteristics required for biomedical applications\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, a modified two-step alkaline hydrolysis followed by moderate-temperature calcination at 550\u0026deg;C was employed to obtain nanohydroxyapatite (nHA) from \u003cem\u003eChanna striata\u003c/em\u003e fish bones. The calcination temperature of 550\u0026deg;C was selected as a moderate thermal treatment condition based on previous studies reporting that calcination in the range of 500\u0026ndash;600\u0026deg;C is sufficient to remove residual organic matter while preserving the hydroxyapatite phase in fish-bone-derived materials\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Compared with conventional high-temperature calcination protocols, the proposed synthesis route is expected to offer several advantages, including lower thermal input, improved phase stability, reduced particle agglomeration, and preservation of nanostructured morphology, while still achieving high crystallinity and phase purity of hydroxyapatite\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eFunctional group analysis\u003c/h2\u003e\n \u003cp\u003eFourier transform infrared (FTIR) spectroscopy was used to identify the functional groups present in the uncalcined and calcined nHA powders at 550\u0026deg;C, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The spectrum shows peaks/intensity (transmittance) at specific wavenumbers associated with the functional groups (ions) contained within the sample. The strongest peaks in the wavenumber range of 1030\u0026ndash;1045 cm⁻\u0026sup1; are attributed to the asymmetric stretching vibration (\u003cem\u003ev3\u003c/em\u003e) of the phosphate ions (PO₄\u0026sup3;⁻), and the weak absorption band at approximately 569 cm⁻\u0026sup1; corresponds to the bending vibration (\u003cem\u003ev4)\u003c/em\u003e of the phosphate ions (PO₄\u0026sup3;⁻).\u003c/p\u003e\n \u003cp\u003eAdditionally, the stretching vibration of the OH⁻ ions is also detected at approximately 3568 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The weak adsorption band at 1630\u0026ndash;1637 cm⁻\u0026sup1; and the broad band at 3100\u0026ndash;3500 cm⁻\u0026sup1; indicate surface-absorbed water (bending vibration H₂O) and crystal water (stretching vibration H₂O), respectively. Moreover, the band at approximately 1430\u0026ndash;1472 cm⁻\u0026sup1; with a very low\u003cstrong\u003e-\u003c/strong\u003eintensity peak is associated with the vibration of CO₃\u0026sup2;⁻. The weak peak at approximately 2919 cm \u0026sup1; typically corresponds to the asymmetric stretching vibration of CH₂ groups, whereas that at 2850 cm⁻\u0026sup1; is characteristic of symmetric \u003cem\u003e\u0026nu;\u003c/em\u003e (C-H), which is in agreement with the fact that the typical nHA synthesized from fish bone may be caused by reactions with organic compounds \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These identified functional groups (PO₄\u0026sup3;⁻, CO₃\u0026sup2;⁻, and OH⁻) are characteristic of HA compounds. Based on these findings, the sample is indeed HA, with the chemical formula Ca\u003csub\u003e10\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e. This result is consistent with other published results for nHA, as shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFunctional groups of synthesized nHA based on FTIR\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFunctional Group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWavenumber (cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) in the current study\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWavenumber (cm⁻\u0026sup1;) based on references\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nu;3\u003c/em\u003e asymmetric stretching PO₄\u0026sup3;⁻\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e1030\u0026ndash;1045 (shoulder and sharp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1023\u003csup\u003e1\u003c/sup\u003e, 1027\u003csup\u003e38\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1032\u003csup\u003e39\u003c/sup\u003e, 1033\u003csup\u003e38,40\u003c/sup\u003e, 1037\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1041\u003csup\u003e41\u003c/sup\u003e, 1042\u003csup\u003e38\u003c/sup\u003e, 1046\u003csup\u003e42\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nu;1\u003c/em\u003e symmetric stretching PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e962 (weak)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e962 \u003csup\u003e38,40,43,44\u003c/sup\u003e, 960\u003csup\u003e1,38\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026nu;4 asymmetric bending PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e565, 604 (sharp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e570\u003csup\u003e41\u003c/sup\u003e, 574\u003csup\u003e1\u003c/sup\u003e, 576\u003csup\u003e45\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e605\u003csup\u003e1\u003c/sup\u003e, 638\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026nu;2 bending PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e478\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eStretching mode Hydroxyl (OH\u003csup\u003e-\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e3568 (weak)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3431\u003csup\u003e1\u003c/sup\u003e, 3497\u003csup\u003e45\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3568\u003csup\u003e41\u003c/sup\u003e, 3560\u003csup\u003e46\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3571\u003csup\u003e1\u003c/sup\u003e, 3573\u003csup\u003e46\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAsymmetric Carbonate CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e1430\u0026ndash;1472 (weak)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1426\u0026ndash;1473\u003csup\u003e1\u003c/sup\u003e,1461\u003csup\u003e41\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2017\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eOut of plane bending mode Carbonate CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e874 (weak)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e876\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e869\u003csup\u003e41\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHarmonic overtone or combination of \u003cem\u003ev1\u003c/em\u003e and \u003cem\u003ev3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2000 (weak)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2000\u003csup\u003e39\u003c/sup\u003e, 2005-2079\u003csup\u003e47\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026Nu;\u003c/em\u003e asymmetric stretching vibration of CH₂ (C-H)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2919\u003csup\u003e48\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026Nu;\u003c/em\u003e symmetric stretching vibration of CH₂ (C-H)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2850\u003csup\u003e48\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAs the calcination temperature increases, the transmittance line decreases, in good agreement with reported research [11], and the organic compound content decreases, as confirmed by thermogravimetric analysis (TGA).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePhase and crystallinity analysis\u003c/h3\u003e\n\u003cp\u003eCrystalline phase analysis of nHA powders from \u003cem\u003eChanna striata\u003c/em\u003e fish bone was conducted via X-ray diffraction (XRD), as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the peaks were sharp, indicating that nHA compounds/phases were affected by the removal of organic components. No other compounds or phases, such as tricalcium phosphate (TCP), are visible. According to Siddarthan \u003cem\u003eet al\u003c/em\u003e.\u0026rsquo;s report, the nHA phase is transformed to the \u0026beta;-tricalcium phosphate (\u0026beta;\u0026ndash;TCP) phase at an initiation temperature of 650\u0026deg;C if the produced nHA is deficient in calcium. Therefore, the calcined nHA powder at 550\u0026deg;C remains a pure HA phase in the present study.\u003c/p\u003e\n\u003cp\u003eThe XRD patterns of uncalcined and calcined nHA confirmed the crystalline nature of a typical apatite structure with the hexagonal HA phase, aligning with the ICSD (\u003cem\u003eInorganic Crystal Structure Database\u003c/em\u003e) card No. 98-016-9498 for pure hydroxyapatite (HA). The values of d-spacing and the 2\u0026theta; angle of ICSD 98-016-9498 and nHA from \u003cem\u003eChanna Striata\u003c/em\u003e fish bone are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ed-spacing of synthesized nHA and comparison with standard HA (ICSD)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCrystallo-graphic plane (hkl)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eICSD 98-016-9498\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003enHA \u003cem\u003eChanna striata\u003c/em\u003e fish bone\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eUncalcined\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCalcined at 550\u0026deg;C\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAngle (\u0026theta;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ed-Spacing (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAngle (\u0026theta;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ed-Spacing (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAngle (\u0026theta;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ed-Spacing (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe peaks around (002) indicate that the crystallite size was in the nanometer range\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Using the Debye\u0026ndash;Scherrer equation (Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)), the crystallite sizes of uncalcined and calcined nHA along the 002 plane were determined to be 29.47 and 30.46 nm, respectively, as shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCrystallite size, degree of crystallinity, phase content, and line profile parameters of synthesized nHA from \u003cem\u003eChanna striata\u003c/em\u003e fish bone compared with those of standard HA (ICSD)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003enHA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUncalcined\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCalcined at 550\u0026deg;C\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrystallite Size \u003csub\u003e(002)\u003c/sub\u003e (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDegree of Crystallinity \u003csub\u003e(002)\u003c/sub\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhase content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003eHA (ICSD 98-016-9498)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003eHA (ICSD 98-016-9498)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR profile (Rp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeighted R profile (wRp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eBrunner-Emmett-Teller (BET) results\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the BET analysis of the nHA powder. The data presented in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e compare the characteristics of the nHA before and after calcination. The surface area decreases after calcination, indicating a reduction. Moreover, the average particle radius increases, suggesting that particle growth is due to calcination. Additionally, the average pore radius increased slightly, which may be attributed to structural changes during calcination that led to an increase in micropores.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of the nHA based on BET analysis\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore calcination\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter calcination\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSurface area (m\u003csup\u003e2\u003c/sup\u003e/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e62.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eParticle radius (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePore radius (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eMorphology and surface element composition of the HA powder\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of \u003cem\u003eChanna striata\u003c/em\u003e fish bone nHA powder samples synthesized via the alkaline hydrolysis method (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec) and alkaline hydrolysis technique followed by the calcination method at 550\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec, and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed are SEM images showing that the HA powder particles are spherical and form agglomerates of varying sizes. The particle sizes of these powders range from the nanoscale (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee) to the micrometer scale (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef are TEM images showing that the single nHA particles synthesized via the alkaline hydrolysis technique and the alkaline hydrolysis technique followed by calcination have a rod-like shape, with a width (or diameter) and length of approximately 20 and 80 nm, respectively. Moreover, nHA synthesized via the alkaline hydrolysis method has a larger width (Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec) than nHA synthesized via the alkaline hydrolysis method followed by the calcination method (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). The reduction in the width of the nHA particles is thought to be due to a decrease in organic material as the temperature increases to 550\u0026deg;C. There is no organic material, and only the inorganic material remains, namely, HA, as proven by the TGA characterization results below and the XRD characterization results above.\u003c/p\u003e\n\u003ch3\u003eParticle size analysis\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the energy-dispersive X-ray spectroscopy \u003cstrong\u003e(\u003c/strong\u003eEDS) characterization results related to the elemental contents of O, Ca, P, Cd, Hg, As and Pb in the \u003cem\u003eChanna striata\u003c/em\u003e fish bone nHA powder samples synthesized via the alkaline hydrolysis method (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea) and the alkaline hydrolysis method followed by the calcination method at 550\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). The values of the Ca and P elemental compositions and the Ca/P ratio of HA synthesized via the two methods mentioned above are 16.3% Ca, 9.3% P, and Ca/P ratio\u0026thinsp;=\u0026thinsp;1.78, and 17.6% Ca, 10.3% P, and Ca/P ratio\u0026thinsp;=\u0026thinsp;1.71.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows the distribution of the size of the group (agglomerate) of nHA particles derived from the bones of \u003cem\u003eChanna striata\u003c/em\u003e before and after calcining at 550\u0026deg;C. In the sample before (not) calcination, the range of particle sizes varied from 700 to 2500 nm, and the number of particles measuring between 1000 and 1600 nm was the greatest. While the size distribution of the HA particles that had been calcined at 550\u0026deg;C ranged from approximately 60 to 1600 nm, the number of particles measuring between 300 nm and 600 nm was the greatest. The results of this particle size analysis (PSA) characterization show that calcination at 550\u0026deg;C reduces the agglomeration of nHA particles, which is thought to be due to the loss of inorganic material that covers and binds between particles, as shown by the results of the TGA characterization, where inorganic material is no longer present starting at a temperature of approximately 530\u0026deg;C. Variations in particle size from tens to thousands of nanometers were also observed in the SEM characterization results, where the increase in particle size was caused by agglomeration between individual nHA particles during the growth and bone formation process.\u003c/p\u003e\n\u003cp\u003eThe left image in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e shows the TGA curve of the nHA sample before calcination. The TGA curve shows that up to a heating/calcination temperature of approximately 550\u0026deg;C, the sample mass decreases by 11.5%. This indicates that the organic material is completely removed at approximately 550\u0026deg;C. The calcination process from 550 to 1000\u0026deg;C did not result in any further reduction in sample mass. In conclusion, this study\u0026rsquo;s calcination temperature of 550\u0026deg;C is appropriate to ensure that the resulting sample is pure inorganic nHA.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003enHA can be obtained through two main approaches: chemical synthesis and extraction from biological sources\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Compared with its synthetic counterpart, nHA derived from animal bones offers several benefits, including a chemical composition and structural properties similar to those of human bone, lower production costs, and enhanced biocompatibility\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Organic matter removal from bone is typically achieved through alkaline treatment with NaOH, which hydrolyzes organic components, leaving calcium phosphate, which is subsequently washed and filtered\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To ensure the complete elimination of organic residues, calcination is performed, producing a purified nHA material\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCalcination plays a critical role in determining the crystallinity, phase composition, and microstructure of hydroxyapatite derived from biological sources. Conventional extraction methods often employ calcination temperatures above 700\u0026deg;C to eliminate residual organic matter and enhance crystallinity. Nevertheless, several studies have shown that excessively high calcination temperatures can promote particle coarsening and may induce a phase transformation of hydroxyapatite to tricalcium phosphate when calcium deficiency occurs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe modified synthesis route used in the present study combines two-step alkaline hydrolysis with moderate-temperature calcination at 550\u0026deg;C, offering several advantages over conventional high-temperature protocols. First, alkaline hydrolysis effectively removes organic components prior to calcination, allowing the thermal treatment to be performed at a lower temperature\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Second, calcination at 550\u0026deg;C is sufficient to eliminate residual organic residues, as confirmed by TGA analysis, while preventing excessive grain growth and maintaining nanoscale particle morphology\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Third, the moderate calcination temperature helps preserve the hydroxyapatite phase without forming secondary phases such as tricalcium phosphate (TCP)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These results suggest that the proposed method provides an energy-efficient and structurally controlled approach for producing phase-pure nanohydroxyapatite from \u003cem\u003eChanna striata\u003c/em\u003e bone waste.\u003c/p\u003e \u003cp\u003eThis study demonstrated that nanohydroxyapatite (nHA) can be effectively synthesized from \u003cem\u003eChanna striata\u003c/em\u003e fish bones via two-step alkaline hydrolysis followed by calcination at 550\u0026deg;C. The TGA results confirm that the major mass loss associated with organic decomposition occurred below approximately 550\u0026deg;C, indicating that higher calcination temperatures are not necessary for organic removal. This supports selecting a moderate calcination temperature that minimizes energy consumption while preserving nanoscale morphology and phase stability. The combined treatment successfully removed organic matter, preserved the mineral phase, and promoted the formation of rod-shaped, crystalline nHA particles. Alkaline hydrolysis is essential in the preparation of nHA, as it effectively removes organic components such as collagen, proteins, and lipids from the fishbone\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The use of an alkaline solution, typically NaOH, facilitates the breakdown of organic matter while preserving the mineral phase, which is predominantly composed of calcium phosphate. This step ensures that the resulting material retains high purity, which is crucial for biomedical applications\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Calcination plays a critical role in the crystallization and phase transformation of nHA. Heating at 550\u0026deg;C for 2 hours effectively eliminates any remaining organic content while promoting the development of a well-ordered crystalline structure. The FTIR and XRD results confirmed the presence of characteristic phosphate, hydroxyl, and carbonate groups, as well as the formation of a pure hydroxyapatite phase with improved crystallinity.\u003c/p\u003e \u003cp\u003eCompared with previous reports, the synthesis conditions applied here yielded nHA with higher crystallinity and phase stability at relatively moderate calcination temperatures. In contrast, Herpandi \u003cem\u003eet al\u003c/em\u003e. reported that synthesis at higher temperatures (900\u0026deg;C) led to partial decomposition of HA into TCP, whereas our method preserved a stable HA phase\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. According to Siddarthan \u003cem\u003eet al\u003c/em\u003e., calcium-deficient HA tends to transform into the β-TCP phase at 650\u0026deg;C\u003csup\u003e24\u003c/sup\u003e. The absence of secondary phases such as calcium oxide (CaO) or TCP in this study indicates that the applied synthesis conditions were effective \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUncalcined nHA from \u003cem\u003eChanna striata\u003c/em\u003e fish bone resulted in a slightly larger particle size than nHA obtained from tilapia fish scales (24.6 nm)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In contrast, HA synthesized from devilfish (Loricariidae) bone and calcined at 550\u0026deg;C had a particle size of 28.62 nm, which is close to that of the calcined nHA in this study\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The crystallinity percentage of uncalcined nHA was calculated to be 57.33%. The crystallinity of uncalcined nHA was 57.33%, increasing to 63.34% after calcination at 550\u0026deg;C. The calcination process induced nucleation and crystal growth of HA, as indicated by the narrowing of the diffraction peaks, suggesting higher crystallinity and larger crystallite sizes. This improvement, reflected by narrower diffraction peaks, indicates enhanced nucleation and crystal growth. The increase in crystallinity is consistent with previous findings on porcine bone-derived nHA and exceeds the values reported for \u003cem\u003eChanna striata\u003c/em\u003e nHA synthesized via precipitation methods\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSEM and TEM analyses revealed that the synthesized nHA exhibited a rod-like morphology, which is a typical characteristic of biogenic HA\u003csup\u003e29\u003c/sup\u003e. Previous studies have attributed the transition from needle-like to rod-like morphology to the substitution of CO₃\u0026sup2;⁻ for PO₄\u0026sup3;⁻ and Na⁺ for Ca\u0026sup2;⁺ \u003csup\u003e1\u003c/sup\u003e. BET analysis revealed a high surface area, which may enhance bioactivity and support potential applications in biomedical and dental materials\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Ca/P ratio is a critical parameter for evaluating HA bioactivity. The nHA synthesized from \u003cem\u003eChanna striata\u003c/em\u003e fish bone in this study presented a Ca/P ratio of 1.71, which is close to the stoichiometric value of 1.67 for natural bone minerals. Elemental analysis by EDS confirmed high purity with minimal contaminants. Previous studies have reported varying Ca/P ratios for \u003cem\u003eChanna striata\u003c/em\u003e nHA depending on the synthesis conditions, ranging from 1.13 (alkaline hydrolysis alone) to 1.72 (alkaline hydrolysis with calcination at 600\u0026deg;C)\u003csup\u003e18\u003c/sup\u003e. Fatmawati \u003cem\u003eet al\u003c/em\u003e. reported that the Ca/P ratio of \u003cem\u003eChanna striata\u003c/em\u003e fish bone nHA synthesized via the alkaline hydrolysis technique alone was 1.13\u003csup\u003e13\u003c/sup\u003e. Similar variability has been observed in other fish species, with values ranging from 1.35 to 1.69 depending on the processing method used\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Importantly, the heavy metal content (0.0% Cd, 0.1% Hg, 0.2% As, and 0.0% Pb) remained below the permissible threshold defined by ISO 13779-6, underscoring the suitability of \u003cem\u003eChanna striata\u003c/em\u003e as a safe precursor for biomedical applications\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStoichiometric hydroxyapatite has a Ca/P ratio of approximately 1.67\u003csup\u003e34\u003c/sup\u003e. In contrast, biological sources such as fish scales and animal bones generally produce nonstoichiometric HA due to the incorporation of carbonate groups and trace ions (HPO₄\u0026sup2;⁻, Na⁺, Mg\u0026sup2;⁺, Sr\u0026sup2;⁺, K⁺, Cl⁻, and F⁻) in the crystal lattice\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The nHA synthesized in this study had a Ca/P ratio lower than those reported by Hariani \u003cem\u003eet al\u003c/em\u003e. (2.21) and Pon-on \u003cem\u003eet al\u003c/em\u003e. (2.01), suggesting that calcination influences the final composition\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our results are consistent with \u003cem\u003ethose of\u003c/em\u003e Herpandi \u003cem\u003eet al\u003c/em\u003e., who reported Ca/P ratios of 1.69\u0026ndash;1.72 for \u003cem\u003eChanna striata\u003c/em\u003e bone-derived nHA obtained \u003cem\u003evia\u003c/em\u003e alkaline hydrolysis followed by calcination at 600\u0026deg;C\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough the physicochemical and structural properties of the synthesized nHA were comprehensively characterized, this study did not include in vitro or in vivo biological evaluations. Further investigations focusing on cytocompatibility, bioactivity, and long-term stability are necessary to fully assess its suitability for clinical dental and biomedical applications.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates a sustainable approach for producing nanohydroxyapatite (nHA) from \u003cem\u003eChanna striata\u003c/em\u003e fish bones using a modified two-step alkaline hydrolysis followed by calcination at 550\u0026deg;C. The applied synthesis route effectively removed organic components while preserving the hydroxyapatite phase and nanoscale morphology. Structural and compositional analyses confirmed the formation of phase-pure hydroxyapatite with rod-shaped nanoparticles and a Ca/P ratio close to the stoichiometric value. The moderate calcination temperature enabled the production of crystalline nHA while limiting particle agglomeration and maintaining nanoscale structural characteristics. Compared with conventional high-temperature calcination methods, the proposed approach provides a more energy-efficient route for producing biogenic nanohydroxyapatite. These findings highlight the potential of \u003cem\u003eChanna striata\u003c/em\u003e bone waste as an environmentally sustainable and cost-effective precursor for biomedical and dental material applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eChanna striata\u003c/em\u003e fish bones were sourced from a \u003cem\u003eChanna striata\u003c/em\u003e cracker factory in Banjarmasin, Indonesia. All chemicals used in this study were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the fish bones\u003c/h2\u003e \u003cp\u003eA wooden hammer and a bladed cutter were used to delicately remove the fish bones from the collected samples. The bones were carefully boiled at approximately 100\u0026deg;C in 2 L of distilled water to remove any leftover flesh or skin. To eliminate any remaining proteins, lipids, oils, or other organic contaminants, the cleaned bones were boiled for 1 hour after being submerged in a 500 mL solution containing 2% sodium hydroxide (NaOH, HiMedia Laboratories Pvt. Ltd., Mumbai, India) and 10 mL of acetone (Merck, Darmstadt, Germany). To remove any remaining moisture, the treated bones were cleaned and dried in an oven set to 100\u0026deg;C for 3 hours. Finally, a mortar and pestle were used to grind the dried fish bones into a fine powder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of nHA\u003c/h2\u003e \u003cp\u003eThe alkaline hydrolysis method in this study was adapted with slight modifications from the research of Surya \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e11\u003c/sup\u003e. Similarly, the calcination process was modified from the study conducted by Bee \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e20\u003c/sup\u003e. The modifications mainly involved applying a two-step alkaline hydrolysis process with varying NaOH concentrations, followed by moderate-temperature calcination at 550\u0026deg;C. The procedure is summarized and illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Two steps of alkaline hydrolysis were applied to the prepared fish bones. They were first heated to 70\u0026deg;C, agitated for 5 hours at 400 rpm, and submerged in a 5% NaOH solution (HiMedia Laboratories Pvt. Ltd., Mumbai, India) at a 1:14 (w/v, FB:NaOH) ratio. After the treated bones were filtered, the precipitates were carefully cleaned with distilled water and dried for two hours at 100\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second phase involved heating the dried precipitate to 100\u0026deg;C for 1 hour after treatment with a 50% NaOH solution at a 1:6 (w/v) precipitate:NaOH solution ratio. After the resulting white solution was filtered, the filtrate was rinsed with distilled water until it was clear. Following the suspension of the resulting white precipitate in 200 millilitres of distilled water, 0.1 M phosphoric acid (H₃PO₄, Merck, Darmstadt, Germany) was added to adjust the pH to neutral (pH 7). After an hour of constant stirring, the mixture was filtered. To obtain nHA powder, the finished product was sieved after drying for 2 hours at 100\u0026deg;C in an oven. The samples were subsequently placed in a Carbolite CWF 1100 furnace (Germany) and calcined at 550\u0026deg;C. Over 2 hours, the heating process was conducted at a controlled rate of 5\u0026deg;C per minute. After calcination at 550\u0026deg;C for 2 hours, the sample was allowed to cool naturally inside the furnace.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of nHA powder\u003c/h2\u003e \u003cp\u003eThe synthesized nHA was characterized via multiple analytical techniques to assess its structural and compositional properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eThe presence of nHA was verified, and functional groups were identified via Fourier transform infrared (FTIR) spectroscopy. A Thermo Scientific Nicolet iS-10 was used to acquire the spectra via a transmission approach with KBr added at the BRIN. At room temperature, measurements were made between 4000 and 500 cm⁻\u0026sup1;, with a resolution of \u0026plusmn;\u0026thinsp;4 cm⁻\u0026sup1; and a scan frequency of 16 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eX-ray diffraction (XRD) analysis\u003c/h2\u003e \u003cp\u003eAn X-ray diffractometer (SmartLab, Rigaku Corporation, Japan) equipped with Cu Kα1 radiation at BRIN was used to analyze the crystalline phases and the purity of the nHA powder. To identify the phase, the acquired diffraction patterns were compared with reference data from the Inorganic Crystal Structure Database (ICSD) No. 98-016-9498. The data acquisition was carried out with a step width of 0.01\u0026deg; and within the 2θ range of 10\u0026deg; to 75\u0026deg;.\u003c/p\u003e \u003cp\u003eThe average crystallite size of a sample was calculated via the Debye\u0026ndash;Scherrer equation, and the degree of crystallinity (Xc) was approximated via mathematical relationships\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${D}_{hkl}=\\frac{k\\lambda}{\\beta\\text{cos}\\theta}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$${X}_{c}=1-{\\left(\\frac{0.24}{\\beta}\\right)}^{3}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere λ\u0026thinsp;=\u0026thinsp;1.5405 \u0026Aring; (the wavelength of the X-ray source); β\u0026thinsp;=\u0026thinsp;full width at half maximum (FWHM) of the diffraction peak; Dhkl\u0026thinsp;=\u0026thinsp;crystallite size determined from (hkl) reflections; and k\u0026thinsp;=\u0026thinsp;0.94 (Scherrer constant).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBrunauer\u0026ndash;Emmett\u0026ndash;Teller (BET) analysis\u003c/h2\u003e \u003cp\u003eThe powder BET surface area, pore size, and pore volume were measured via an N₂ adsorption‒desorption analyzer (Quantachrome, Quadsorbs evo) at BRIN. A 10 mg sample was introduced into the system and pretreated to remove moisture before N₂ adsorption at -196\u0026deg;C. Two measurements were conducted, and the results are presented as the statistical average.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS)\u003c/h2\u003e \u003cp\u003eA field emission-scanning electron microscope (FE-SEM) Apreo 2S (Thermo Fisher Scientific, USA) operating at an accelerating voltage of 10 kV was used to examine the microstructures and morphologies of the nHA crystals. The local chemical composition was examined by integrating FE-SEM with energy-dispersive X-ray spectroscopy (EDS) (Thermo Fisher Scientific, USA). Both measurement tools are at the BRIN.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM) analysis\u003c/h2\u003e \u003cp\u003eImages of the nanostructure were captured via a Talos F200X transmission electron microscope (Thermo Fisher Scientific, USA) operated at 200 kV. The powder particle morphology was examined, and particle size was determined using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThermogravimetric analysis (TGA)\u003c/h2\u003e \u003cp\u003eThe thermal degradation properties of the powders were examined via TGA (TGA Absys Evo Setaram Simultaneous TG DTA) at BRIN. With a heating rate of 5\u0026deg;C\u0026middot;min⁻\u0026sup1; and a nitrogen flow rate of 70 ml\u0026middot;min⁻\u0026sup1;, a 20 mg sample was heated from room temperature to 700\u0026deg;C in a nitrogen environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eParticle size analysis\u003c/h2\u003e \u003cp\u003eThe dynamic light scattering (DLS) method (CILAS NANO DS Dual Scattering) at BRIN was used to measure the particle size of the nHA powder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors express their deepest gratitude to Universitas Padjadjaran and the National Research and Innovation Agency (BRIN) for their full support of this research. The authors also wish to thank the Indonesian Ministry of Education, Culture, Research, and Technology for the Doctoral Dissertation Research Grant,\u0026nbsp;and the National Research and Innovation Agency for Nanotechnology and Materials (BRIN) for its RIIM LPDP research grants.\u003c/p\u003e\n\u003cp\u003eAuthor\u0026nbsp;contributions statement\u003c/p\u003e\n\u003cp\u003eAll authors confirm that they have read and approved the final version of the manuscript and that the author order has been agreed upon by all. \u003cstrong\u003eNurdiana Dewi:\u003c/strong\u003e Writing \u0026ndash; original draft; Writing \u0026ndash; review \u0026amp; editing; Methodology; Investigation; Data curation. \u003cstrong\u003eMeirina Gartika:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing; Writing \u0026ndash; original draft; Investigation; Conceptualization. \u003cstrong\u003eDwi Gustiono:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing; Writing \u0026ndash; original draft; Methodology; Investigation; Data curation. \u003cstrong\u003eDikdik Kurnia:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing; supervision; conceptualization. \u003cstrong\u003eNendar Herdianto:\u003c/strong\u003e Writing \u0026ndash; original draft; visualization; investigation; conceptualization. \u003cstrong\u003eRiesma Tasomara:\u003c/strong\u003e Writing \u0026ndash; original draft; Writing \u0026ndash; review \u0026amp; editing; Methodology; Investigation; Data curation\u003cstrong\u003e. \u003cstrong\u003eWinda Rianti:\u003c/strong\u003e\u003c/strong\u003e Writing \u0026ndash; original draft; visualization; investigation; conceptualization. \u003cstrong\u003eNuning Aisah:\u003c/strong\u003e Writing \u0026ndash; original draft; visualization; investigation; conceptualization. \u003cstrong\u003eBambang Triwibowo:\u003c/strong\u003e Writing \u0026ndash; original draft; visualization; investigation; conceptualization.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Research Organization for Nanotechnology and Materials (BRIN) through RIIM LPDP (B-807/II.7.5/FR/6/2022; B-6952/III.10/KS.00.00/6/2022), and by the Indonesian Ministry of Education, Culture, Research, and Technology through the Doctoral Dissertation Research Grant (093/C3/DT.05.00/PL/2025; 1604/UN6.3.1/PT.00/2025).\u003c/p\u003e\n\u003cp\u003eCompeting interest\u003c/p\u003e\n\u003cp\u003eThe authors declare\u0026nbsp;that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to M.G. or D.G.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLe Ho KH, Dao VH, Pham XK, Nguyen PA, Phan BV, Doan TT \u003cem\u003eet al.\u003c/em\u003e Physicochemical properties, acute and subchronic toxicity of nano-hydroxyapatite obtained from Lates calcarifer fish bone. \u003cem\u003eReg Stud Mar Sci\u003c/em\u003e 2022; \u003cstrong\u003e55\u003c/strong\u003e: 102560.\u003c/li\u003e\n \u003cli\u003eMondal S, Park S, Choi J, Vu TTH, Doan VHM, Vo TT \u003cem\u003eet al.\u003c/em\u003e Hydroxyapatite: A journey from biomaterials to advanced functional materials. \u003cem\u003eAdv Colloid Interface Sci\u003c/em\u003e 2023; \u003cstrong\u003e321\u003c/strong\u003e: 103013.\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;Hagan-Wong K, Enax J, Meyer F, Ganss B. 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Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods. \u003cem\u003eJ Biomed Mater Res\u003c/em\u003e 2002; \u003cstrong\u003e62\u003c/strong\u003e: 600\u0026ndash;612.\u003c/li\u003e\n \u003cli\u003eFowler BO. Infrared studies of apatites. I. Vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic substitution. \u003cem\u003eInorg Chem\u003c/em\u003e 1974; \u003cstrong\u003e13\u003c/strong\u003e: 194\u0026ndash;207.\u003c/li\u003e\n \u003cli\u003eChandrasekar A, Sagadevan S, Dakshnamoorthy A. Synthesis and characterization of nano-hydroxyapatite (n-HAP) using the wet chemical technique. \u003cem\u003eInternational Journal of Physical Sciences Full Length Research Paper\u003c/em\u003e 2013; \u003cstrong\u003e8\u003c/strong\u003e: 1639\u0026ndash;1645.\u003c/li\u003e\n \u003cli\u003eFowler BO. Infrared studies of apatites. I. Vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic substitution. \u003cem\u003eInorg Chem\u003c/em\u003e 1974; \u003cstrong\u003e13\u003c/strong\u003e: 194\u0026ndash;207.\u003c/li\u003e\n \u003cli\u003eKlee WE, Engel G. I.R. spectra of the phosphate ions in various apatites. \u003cem\u003eJournal of Inorganic and Nuclear Chemistry\u003c/em\u003e 1970; \u003cstrong\u003e32\u003c/strong\u003e: 1837\u0026ndash;1843.\u003c/li\u003e\n \u003cli\u003eBaddiel CB, Berry EE. Spectra structure correlations in hydroxy and fluorapatite. \u003cem\u003eSpectrochimica Acta\u003c/em\u003e 1966; \u003cstrong\u003e22\u003c/strong\u003e: 1407\u0026ndash;1416.\u003c/li\u003e\n \u003cli\u003ePanda NN, Pramanik K, Sukla LB. Extraction and characterization of biocompatible hydroxyapatite from fresh water fish scales for tissue engineering scaffold. \u003cem\u003eBioprocess Biosyst Eng\u003c/em\u003e 2014; \u003cstrong\u003e37\u003c/strong\u003e: 433\u0026ndash;440.\u003c/li\u003e\n \u003cli\u003ePosner AS, Stutman JM, Lippincott ER. Hydrogen-bonding in calcium-deficient hydroxyapatities. \u003cem\u003eNature\u003c/em\u003e 1960; \u003cstrong\u003e188\u003c/strong\u003e: 486\u0026ndash;487.\u003c/li\u003e\n \u003cli\u003ePanda S, Biswas CK, Paul S. A comprehensive review on the preparation and application of calcium hydroxyapatite: A special focus on atomic doping methods for bone tissue engineering. \u003cem\u003eCeram Int\u003c/em\u003e 2021; \u003cstrong\u003e47\u003c/strong\u003e: 28122\u0026ndash;28144.\u003c/li\u003e\n \u003cli\u003eManullang J, Nugroho R, Rohmah M, Rudianto, Qorysuchi A. Plant-extract-mediated biosynthesis of silver nanoparticles using \u003cem\u003eEleutherine americana\u003c/em\u003e bulb extract and its characterization. 2021. doi:10.13057/nusbiosci/n130216.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"nanohydroxyapatite, Channa striata, alkaline hydrolysis, calcination, sustainable biomaterial","lastPublishedDoi":"10.21203/rs.3.rs-7609723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7609723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanohydroxyapatite (nHA) derived from natural resources is an environmentally sustainable alternative to synthetic HA for biomedical applications. In this study, nHA was synthesized from \u003cem\u003eChanna striata\u003c/em\u003e fish bones via a modified two-step alkaline hydrolysis process followed by calcination at 550\u0026deg;C. The physicochemical and structural properties of the material were characterized via Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy with energy-dispersive X-ray spectroscopy, transmission electron microscopy, Brunauer\u0026ndash;Emmett\u0026ndash;Teller analysis, particle size analysis, and thermogravimetric analysis. Functional groups typical of hydroxyapatite, including phosphate, hydroxyl, and carbonate groups, were identified, and X-ray diffraction confirmed a pure crystalline apatite phase with a Ca/P ratio of 1.71, closely matching the stoichiometry of stoichiometric hydroxyapatite. Morphological evaluation revealed rod-shaped nanoparticles of 20\u0026ndash;80 nm, and surface analysis revealed nanoscale features with reduced agglomeration following calcination. Thermogravimetric analysis confirmed the complete removal of organic matter at 550\u0026deg;C. Compared with conventional high-temperature calcination methods, the proposed synthesis route requires lower thermal input while maintaining crystallinity and structural stability. These findings demonstrate the potential of \u003cem\u003eChanna striata\u003c/em\u003e fish bones as a promising sustainable precursor for the production of high-quality nHA, highlighting their sustainability as a material for dental and biomedical applications.\u003c/p\u003e","manuscriptTitle":"Biogenic nanohydroxyapatite derived from Channa striata fish bones using alkaline hydrolysis and calcination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 08:34:16","doi":"10.21203/rs.3.rs-7609723/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-01T06:48:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-17T10:29:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-07T10:37:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140742304592518067847667772888481801418","date":"2026-03-06T11:09:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112666900553022858497711793000776260997","date":"2026-03-04T14:57:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-04T10:48:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-04T01:19:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-04T01:13:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c9bd5b0-d676-477f-a766-ba70d1252c34","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64098921,"name":"Physical sciences/Chemistry"},{"id":64098922,"name":"Earth and environmental sciences/Environmental sciences"},{"id":64098923,"name":"Physical sciences/Materials science"},{"id":64098924,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2026-05-13T10:56:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 08:34:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7609723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7609723","identity":"rs-7609723","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}