Efficient Hydroxyapatite Extraction from Salmon Bone Waste: An Improved Lab-Scaled Physico-Chemico-Biological Process

Abstract

The demand for novel tissue grafting and regenerative wound care biomaterials is growing as traditional options often fall short in biocompatibility, functional integration with human tissue, associated cost(s), and sustainability. Salmon aquaculture generates significant volumes of waste, offering a sustainable opportunity for biomaterial production, particularly in osteo-conduction/-induction, and de novo clinical/surgical bone regeneration. Henceforth, this study explores re-purposing salmon waste through a standardized pre-treatment process that minimizes the biological waste content, followed by a treatment stage to remove proteins, lipids, and other compounds, resulting in a mineral-rich substrate. Herein, we examined various methods-alkaline hydrolysis, calcination, and NaOH hydrolysis-to better identify and determine the most efficient and effective process for producing bio-functional nano-sized hydroxyapatite. Through comprehensive chemical, physical, and biological assessments, including Raman spectroscopy and X-ray diffraction, we also optimized the extraction process. Our modified and innovative alkaline hydrolysis-calcination method yielded salmon-derived hydroxyapatite with a highly crystalline structure, an optimal Ca/P ratio, and excellent biocompatibility. The attractive nano-scale cellular/tissular properties and favorable molecular characteristics, particularly well-suited for bone repair, are comparable to or even surpass those of synthetic, human, bovine, and porcine hydroxyapatite, positioning it as a promising candidate for use in tissue engineering, wound healing, and regenerative medicine indications.

Keywords: biomaterial; bone repair; hydroxyapatite; osseoregenerate; process; salmon bone; waste.

Figure 1

Prepared control and experimental HA samples (Left) before characterization and analysis. SEM micrographs of control and experimental HA samples (Top Right). Crystal size control and experimental HA samples obtained via XRD and the Ca/P ratio (atomic weight %) for control and experimental HA samples obtained via SEM/EDS (Bottom Right). The crystal size, studied using XRD and further analyzed using the Scherrer equation, reported sizes ranging from 8.8 nm to 60.38 nm. Using SEM/EDS analysis, the Ca/P ratio (atomic weight %) for the samples determined the lowest values for our salmon HA and bovine HA with 1.94 and 1.98, respectively, yet these were similar/close to what is often presented in the literature (the theoretically stoichiometric value of HA was set at 1.67), validating our experimental protocol. It is noteworthy that all other HA yielded values higher than those commonly reported in the literature. The Ca/P ratio (calcium-to-phosphorus ratio) in terms of the atomic weight percentage (%) is calculated by dividing the atomic weight % of calcium (Ca) by the atomic weight % of phosphorus (P). The atomic weights of Ca and P are approximately 40.08 g/mol and 30.97 g/mol, respectively. As mentioned, human HA has a Ca/P ratio close to 1.67 in its ideal form. Any deviations from this ratio can indicate changes in the bone mineral density and quality.

Figure 2

Raman spectrometry: Raman 1 and Raman 2 measurements.

Figure 3

XRD: XRD pattern and sample measurements.

Figure 4

Illustrating the Prep protocol for HA extraction from salmon bone waste and analytics. HA is a calcium phosphate compound, Ca₁₀(PO₄)₆(OH)₂, that serves as the main inorganic element composition in the bone and teeth. HA extracted from fish bone is considered to be an alternative to synthetic HA from chemicals. Different regions have access to various fish species, leading to the utilization of locally abundant fish waste for HA powder production. These species yield HA with distinct morphologies, porosities, and purities, which makes the choice of the fish source crucial in the process. The selection is often influenced by regional availability, the specific properties of the fish bones, and the goals of sustainability and innovation in material synthesis [31,32]. Fish, such as catfish (Pangasius hypophthalmus), cod, tilapia (Oreochromis sp.), seabass (Lates calcarifer), yellowfin tuna (Thunnus albacares), rainbow trout, Whitemouth croaker, and, more recently, red big-eye snapper (Priancanthus tayenus), are among the wide range of aquatic or marine animals used in these studies. This growing interest underscores the dual advantage of converting bio-waste into valuable and adaptable HA while addressing environmental pollution challenges. This approach not only promotes sustainable biomaterial synthesis and environmental management, but it also possibly offers cost-effective and eco-friendly processes with a strong potential for industrial-scale applications, providing a greener alternative to traditional HA production methods and protocols [15,18,31,32].