The use of waste bones of rabbitfish (Siganus sp.) for the synthesis of hydroxyapatite

This study obtained hydroxyapatite (HA) material by extracting waste bones from rabbitfish (Siganus sp.). The fish bones used are obtained from the waste bones of rabbitfish (Siganus sp.); besides being readily available in nature, they also reduce unwanted environmental impacts. X-ray fluorescence (XRF) and scanning electron microscopy (SEM) observations were used to analyze the hydroxyapatite (HA) produced by the sintering process. Samples produced at sintering temperatures of 800 °C, 850 °C, 900 °C, 950 °C, and 1000 °C had stoichiometric molar ratios of (Ca/P) that were, respectively, 1.728, 1.875, 1.759, 1.758, and 1.696. These results are consistent with the hydroxyapatite stoichiometric ratio (Ca/P = 1.67), and SEM was used to examine the measurement morphology of hydroxyapatite. This study reveals that bone waste from rabbitfish can be a source of biomaterials for bone tissue engineering.


Introduction
Improvements in fisheries, aquaculture production, and canning techniques have increased fish waste.The economy and environment are harmed because it is estimated that almost two-thirds of the total fish are discarded.Waste recycling is now a severe problem that has to be addressed [1,2].Therefore, fish bone debris is utilized as a cheap biological hydroxyapatite (HA) source to lessen environmental influence [3].The environmentally sustainable technique of turning fish bones into HA has the potential to save medical expenditures and repair or replace bones.In addition, natural or synthetic hydroxyapatite (HA) is also known to have been used as an implant material for orthopedic and dental applications [4].Hydroxyapatite (HAP) from fish bone waste reduces cytotoxicity in pre-osteoblast cells [5].
Its potential for bioactivity, biocompatibility, osteoconductivity, and its Ca/P molar ratio of 1:67 make HAP (Ca10(PO4)6(OH)2) a bioceramic biomaterial with potential medicinal applications [6].The bones of catfish (Pangasius hypophthalmus), tilapia (Oreochoromis sp.), seabass (Lates calcarifer), and yellowfin tuna (Thunnus albacares), which were calcined at 700 °C for two hours and had a Ca/P ratio of about 1.80, were among those used in studies on HA [7], a bone from a Nile tilapia (Oreochromis niloticus) was calcined for five hours at 800 °C [8], fish bones (Salmo salar, Anoplopoma fimbria, and Sardine) are calcined in a muffle furnace for an hour at various temperatures ranging from 600 °C to 1100 °C [9], the atomic ratio of Ca/P in HA obtained from rainbow trout, cod, and salmon were reported to be 1.47, 1.88, and 1.51, respectively [10].
There are many similarities between the elemental composition samples and the chemical composition of real bones.Regarding weight and atomic percentages, the calculated Ca/P ratios for the 1230 (2023) 012042 IOP Publishing doi:10.1088/1755-1315/1230/1/012042 2 HA-900 sample were 2.04 and 1.58, respectively.At temperatures between 900 and 1100 °C, the Ca/P atomic ratio of the HAp samples in contact was typically 1.58 to 1.94.The HA-900 Ca/P atomic ratio (1.58) came closest to the hydroxyapatite stoichiometric Ca/P ratio (1.67).The melting temperature, which influences the kind and composition of calcium-based compounds that arise in the final HAp bioceramic, is one of the most significant elements that could be to blame for the deviation of the Ca/P ratios from stoichiometry [11].
Pure biocompatibility of HA is by far its most alluring quality.The pH of the substance and the Ca/P ratio must meet specific requirements to produce bioactivity.In order to allow for bone formation, the HAp material also needs to have a Ca/P ratio of roughly 1.67 to enable phosphate stabilization in aqueous solutions.There are numerous additional bioapplications for HA, including increasing and enhancing the biocompatibility of bioceramics and metal composite coatings [12].According to the data, hydroxyapatite comprises purified Ca, P, and O and exhibits the typical Ca/P molar ratio of 1.654 for manufactured hydroxyapatite.The product has a Ca/P ratio of 1.654, which is close to the standard ratio for pure hydroxyapatite of 1.667 and does not include any contaminants according to the elemental analysis [13].
Even after being heated to 600 °C, the elemental compositions of raw fish bones still include significant amounts of organic material and are partially colored grey or black (0.5%) by carbon residue.At temperatures above 800 °C, the powder turns white.The current bone apatite has a somewhat calcium-deficient Ca:P ratio.The main natural impurities are potassium (~0.3%), magnesium (~0.5%), and sodium (~1%), some of which seem to burn off at 1300°C.Lead quantities were inadequate (~1 ppm), and no arsenic, cadmium, or mercury was found (< 0.1 ppm).Chemical investigations point to the existing ceramic made from fish bones having good grades as a mineral resource [14].
As a result of the Ca/P ratio being below 1.67 (the stoichiometric ratio for HA) and almost 1.5 (the stoichiometric ratio for TCP), HA is deficient in calcium in human bones.A ratio of less than 1.67 Ca/P is frequently attained, which results in the synthesis of biphasic material, HAp:β-TCP, whose proportions vary depending on the value of that ratio, even though HAp low in Ca can be created.Therefore, altering the Ca/P ratio can result in either pure HAp or a biphasic material with a specified ratio between the two phases [15].
Fish bones have been used in the development of HAP, with sintering being the most common process for its synthesis.According to Stea and colleagues, sintered HAp reportedly creates coherent connections with bone tissue [11].Subsequently, the composition and morphology of the pore-melted hydroxyapatite were characterized for further applications.This leads to profitable products in biomaterials and an effort to reduce waste production in the fishing industry.

Research Methods
Rabbitfish (Siganus sp.) taken from the waters of Ghai Village, Muna Regency, Southeast Sulawesi, were boiled using distilled water (boiling temperature of 2 liters ~ 200 o C) to separate the meat that was still attached to the fish bones.After thoroughly cleaning, the bones were added to 500 ml of the mixing solution containing 10 ml of acetone and boiled for an hour to eliminate extra protein, fat, oil, and other organic contaminants.The bones were cleaned and dried off in an oven set at 100 o C for three hours.A mortar and pestle were used to grind the dried fish bones into a 200-mesh powder.Then it was annealed for two hours each at 800, 850, 900, 950, and 1000 o C [16], where the color of the powder turned white.Furthermore, the ball mill was used at 1 Hz for 30 minutes.
The elemental composition of the sample is quite similar to that of natural bone in terms of its look, and the Ca/P ratio result for the sintered sample in Table 1 shows this similarity.According to the table, the recovered hydroxyapatite from rabbitfish bones had percentage atomic ratios of Ca/P that were 1.72, 1.876, 1.759, 1.758, and 1.696, respectively.The carbonate substrate may have caused the higher Ca/P ratios of the HAp synthesized from rabbitfish bones, which are approaching stoichiometric HAp (Ca/P = 1.67) [10].Additionally, it was claimed that HAp might break down into CaO or β-tricalcium phosphate (β-TCP; Ca3(PO4)2), increasing the Ca/P ratio during the calcination process of bone [7].The HA-1000 sample had the atomic Ca/P ratio closest to the hydroxyapatite stoichiometric Ca/P ratio (1.67) of all the samples included in this investigation.The sintering temperature, which affects the nature and composition of the calcium-based compounds present in the resultant Hap bioceramics, is one of the critical parameters determining the divergence of the Ca/P ratio from stoichiometry.In general, it has been observed that HAp biomaterials with a Ca/P ratio near the theoretical stoichiometric value of 1.67 have a significant potential to boost osteoinductivity activity for bone healing in substitution and regeneration applications [11].This study also shows several elements during the sintering process (Table 1).The presence of elements also decreases with increasing sintering temperature.However, the percentage content of these elements at each sintering temperature is minimal, so it does not affect the resulting HAp content.
Scanning electron microscopy (SEM), which uses powdered materials attached to a standard copper microgrid-backed conductive adhesive to view the morphology, was used to reveal the morphology of calcified fishbone samples [10].It is crucial to demonstrate the microstructural characteristics of the materials because they clarify the impact of sintering temperature on mechanical properties.Table 2 displays SEM micrographs of sintered HAp at temperatures of 850, 900, and 950 °C.The micrographs demonstrate that, as the temperature rises, the grain structures become closer together and the apertures become smaller.This effect enhances the mechanical properties of natural HA, i.e. a rise in temperature increases the mechanical properties of HA [20].The microporous structure of the biomaterials was visible in the SEM micrographs; as the temperature rose, the porosity decreased, and the mechanical characteristics improved.The average shape of pores changed from being firmly flat to spherical at higher melting temperatures, which was also observed [11].

Conclusion
Hydroxyapatite is present in discarded fishbone material, which can be used in bone tissue engineering.The amount of hydroxyapatite is nearly equal to the stoichiometric ratio of hydroxyapatite from human bone, which is Ca/P = 1.67.Additionally, as the sintering temperature rises, the morphological structure of hydroxyapatite also appears denser.Overall, the results of this study show that rabbitfish (Siganus sp.) bone hydroxyapatite is a reasonably priced and environmentally responsible source of biomaterials.

Table 1 .
Micro XRF analysis was used to assess the oxide composition of the HAp made from fish bone rabbitfish (Siganus sp.) sintered at 800, 850, 900, 950, and 1000 o C for two hours.