Ronald Bual

Decellularization removes immunogenic intracellular components of fish tissues while keeping the extracellular matrix (dECM) structure, mechanical integrity, and bioactivity. Fish-derived dECM retains native bioactive components, exhibiting high biocompatibility, low immunogenicity, and biodegradability, while supporting cell adhesion, proliferation, and tissue regeneration. Due to its abundance, minimal ethical concerns, and low zoonotic risks, fish wastes are emerging as sustainable sources of dECM, offering an eco-friendly alternative to mammalian biomaterials. This review highlights advances in decellularizing fish wastes such as skin, scales, bones, viscera, and swim bladders from species including tilapia, tuna, milkfish, carp, goldfish, and sturgeon. Physical, chemical, biological, and hybrid decellularization methods are assessed for cell removal, ECM preservation, and mechanical performance. Recent advances in polymer-dECM composites, crosslinking, and 3D bioprinting have significantly improved scaffold performance, making fish-derived dECM applicable for healing of wounds, regeneration of bone and cartilage, and repair of soft tissues. Despite its potential, challenges remain in optimizing perfusion rates, temperature variations, and tissue-specific protocols, as well as developing eco-friendly decellularization techniques using biodegradable reagents. Future perspectives include expanding decellularized fish tissue sources, innovating bio-inks for 3D bioprinting, and refining tissue-specific processing methods to maximize the potential of fish-derived dECM in regenerative medicine and tissue engineering.

This study presents a sustainable method for converting waste Charru mussel shells into high-value biomaterials: calcium oxide (CaO) and hydroxyapatite (HAp). The CaO extraction was optimized by investigating calcination temperatures (750 °C, 850 °C, and 950 °C) and durations (2, 3, and 4 h). Higher temperatures and longer durations increased calcium and decreased carbon content, confirming effective decomposition. Fourier Transform Infrared (FTIR) spectroscopy confirmed the absence of carbonate peaks at 950 °C, identifying 950 °C for 2 h as the optimal condition for complete decomposition without sintering. At this temperature, XRF analysis indicated the highest CaO conversion of approximately 63%. Subsequently, the extracted CaO was used to synthesize HAp via wet chemical precipitation. The researchers optimized the synthesis by varying phosphate precursor concentrations (0.30 M, 0.33 M, and 0.36 M) and aging times (0, 12h, and 24h). According to the two-way ANOVA, the gravimetric HAp yield (0.75–0.88 g HAp/g CaO) was not significantly impacted by variations in phosphate concentration (p = 0.097) or aging time (p = 0.109), indicating these parameters are more critical for controlling HAp quality than the quantity synthesized. Analysis using SEM-EDX revealed fine, spherical-shaped and small aggregates HAp particles. X-Ray Diffraction (XRD) confirmed the presence of characteristic peaks of HAp and its crystallite size. FTIR confirmed the characteristic HAp functional groups. Treatments with phosphate precursor concentrations of 0.33 M and 0.36 M aged for 24 h produced Ca/P molar ratios of 1.70 and 1.93, respectively, close to the ideal stoichiometric ratio (1.67). Moreover, these treatments produced crystallinities of 78.30% and 77.14%, respectively, which are considered suitable for drug delivery and bone scaffold applications. This research highlights the potential of Charru mussel shell waste for producing high-value biomaterials for biomedical applications, as well as addresses the ecological threat of invasive species in Philippine aquaculture, providing a method for population control and waste valorization that directly supports UN Sustainable Development Goal 14, Life Below Water.

The exploration of alternative sources of extracellular matrix (ECM) is driven by increasing demand and the need for sustainable biomaterials. Tuna (Thunnus albacares) bones, a by-product of the fishing industry, represent a potential ECM source due to their abundance, collagen-rich composition, and biocompatibility. This study investigated the effects of demineralization using hydrochloric acid (HCl) at concentrations of 0.5 M and 1.0 M for varying durations (0.5, 1, 6, 12, and 24 h), and decellularization using sodium dodecyl sulfate (SDS) and Triton X-100 (TX100) at concentrations of 0.1%, 0.5%, and 1.0%. Demineralization effectively reduced inorganic content, with residual calcium levels dropping below 5% after 6 h. Kinetic analysis indicated a second-order reaction, and ATR–FTIR spectra confirmed the disappearance of phosphate and carbonate peaks alongside the preservation of amide bands. Demineralized bone matrix (dBM) retained good thermal stability and mechanical properties. Decellularization efficiency was assessed through H&E staining, dsDNA quantification, and SDS-PAGE analysis. Decellularized extracellular bone matrix (dEBM) treated with 1.0% SDS showed the lowest dsDNA levels (14.00 ± 7.94 ng/mg) and absence of cellular material. SDS-PAGE confirmed type I collagen preservation, particularly in samples treated with ≤0.5% SDS or TX100. This study establishes that tuna fish bones can be effectively processed into ECM, supporting their potential as a sustainable biomaterial for advanced biomedical applications.

Primary hepatocytes (PHs) are indispensable for studying liver function, drug screening, and regenerative medicine. However, freshly isolated PHs only survive for a few hours in non-adherent suspension culture. This study proposes treatment with PEG-GRGDS, a polymer-peptide conjugate comprising polyethylene glycol (PEG) and the pentapeptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS), to sustain the viability of dispersed single PHs under non-adherent conditions. As a proof of concept, PHs treated with the PEG-GRGDS molecule were cultured in a microarray with single-cell-sized microwells. After 24 h of culture, enhanced cell survival was confirmed via esterase activity alongside activity for Cytochrome P450 1A1 (CYP1A1). Some liver-specific functionalities, including albumin secretion, were observed in the treated PHs. Additionally, it was observed that the length of the PEG-chain in the conjugates influenced the maintenance of single-cell dispersion and the levels of polymerized actin in the cells. These findings suggest that treatment with a polymer-peptide like PEG-GRGDS might provide a promising platform for the short-term culture of non-adherent single PHs.

As global demand for fish continues to escalate alongside the growing population, fish production also surges – resulting in an increase in various fish wastes, including fish heads. To address this challenge, this study aims to extract and characterize collagen from milkfish heads, paving the way for upcycling fish heads. The milkfish heads underwent a series of treatments: demineralization, deproteinization, and delipidation. The milkfish head waste samples were processed with 0.5 M acetic acid to extract acid-soluble collagen, resulting in a yield of 1.8%. The visual assessment revealed a soft, spongy texture with an off-white color. Scanning electron microscopy further showed a sheet-like, fibrillar morphology. Additionally, Attenuated total reflectance–Fourier transform infrared analysis confirmed the presence of characteristic collagen absorption bands. Ultraviolet-visible spectroscopy detected a characteristic peak at a wavelength of 235 nm. Moreover, differential scanning calorimetry analysis showed a melting temperature of 113.02 °C. This study contributes to sustainable resource utilization and waste reduction. Further research is recommended to refine extraction techniques and explore the biomedical potential of collagen derived from milkfish.

Tilapia (Oreochromis niloticus), which is extensively farmed globally and ranks as the second most cultivated fish in the Philippines, generates significant amounts of waste that are often underutilized. One specific type of waste material consists of fish heads, which contain a valuable source of extracellular matrix (ECM). This study aims to evaluate the effects of sonication as a viable decellularization method for the extraction of ECM from tilapia fish heads. Particularly, two treatments were tested on the head samples: sonication-assisted decellularization (dWS) using a water bath sonicator, and decellularization without sonication (dNS), each with different contact times (5 min and 10 min). Histological analysis with H and E staining and DNA quantification revealed that sonication-assisted samples (dWS) showed a greater reduction in basophilic components and DNA content, achieving a 93.7% removal rate. These dWS samples also had the highest protein loss, retaining only 33.86% of the original protein. SDS–PAGE analysis indicated that both dWS and dNS samples maintained similar collagen structures, as evidenced by identical subunit bands. ATR–FTIR spectra confirmed the presence of collagen type I in all samples, detecting characteristic amides A, B, I, II, and III. The results revealed that varying treatments and contact times had significant effects on the physical and mechanical properties of the decellularized extracellular matrix (ECM). These findings highlight the effectiveness of sonication in the decellularization process, particularly for utilizing waste tilapia heads.

Tilapia, the second most extensively farmed fish in the Philippines, generates significant amounts of waste that are often underutilized, leading to environmental concerns. One specific waste material is tilapia heads, which contain a valuable extracellular matrix (ECM) and an abundant source of bioactive materials that have a potential application in various fields, including biomaterials for wound healing and nutraceuticals. This study aims to evaluate the effects of sonication as a viable decellularization method for extracting ECM from tilapia fish heads. Histological analyses, employing H&E staining, revealed a higher reduction in basophilic components in the sonication-assisted (dWS) samples compared to those without sonication (dNS), as confirmed by DNA quantification. Notably, dWS samples subjected to 1% SDS sonication for 10 minutes exhibited the lowest DNA content with a removal rate of 93.7%. However, the same dWS sample also manifested the highest protein removal with a retained protein of 33.86%. The SDS-PAGE analysis of both dWS and dNS samples exhibited a uniform pattern, featuring identical four subunit bands—namely, the β-dimer (225-227 kDa), γ bands (228-230 kDa), and two alpha chains (α1 and α2 chains) (116-120 kDa)—indicating that sonication did not introduce discernible alterations in the helical structure of collagen. ATR-FTIR spectra further confirmed the presence of collagen as indicated by the detection of collagen type I indicators, including amides A, B, I, II, and III, in all decellularized samples. The above findings highlight the potential of sonication to aid in the decellularization of waste tilapia heads with future research targeting practical applications in wound healing, nutraceuticals, and sustainable environmental preservation through waste utilization.

Tilapia, a widely farmed aquaculture fish, produces substantial waste, including viscera that contain extracellular matrix (ECM) utilized as a biomaterial for tissue regeneration applications. Extracting ECM from viscera requires a specific decellularization method, as no standardized protocol exists. This study performed three decellularization methods: sonication, orbital shaking at room temperature, and agitation at 4°C, using SDS and TX100 at concentrations of 0.1% and 0.3%. The effectiveness of each method was assessed through H&E staining, dsDNA quantification, and SEM imaging to verify cellular content removal and ECM structure preservation. Additional analyses, including ATR‐FTIR, SDS‐PAGE, protein quantification, HPLC, and detergent residue tests, were performed to examine functional groups, collagen composition, protein content, amino acid profiles, and detergent residues in the decellularized samples. The results of H&E staining showed a significant reduction in cellular components in all samples, which was confirmed through DNA quantification. Sonication with 0.3% SDS achieved the highest DNA removal rate (96.5 ± 1.1%), while SEM images revealed that agitation at 4°C with 0.3% TX100 better preserved ECM structure. Collagen was present in all samples, as confirmed by ATR‐FTIR analysis, which revealed pronounced spectral peaks in the amide I, II, III, A, and B regions. Samples treated with agitation at 4°C using 0.1% SDS exhibited the highest protein content (875 ± 15 µg/mg), whereas those treated with TX100 had lower detergent residue. Overall, the decellularization methods effectively reduced DNA content while preserving ECM structure and components, highlighting the potential of tilapia viscera as bioscaffolds and offering insights into utilizing fish waste for high‐value products.

Tilapia (Oreochromis niloticus) is a widely cultivated fish in tropical and subtropical regions such as the Philippines, generating substantial waste during processing, including bones that are a valuable source of extracellular matrix (ECM). However, the extraction of ECM from fish bones requires an essential step of demineralization. This study aimed to assess the efficiency of tilapia bone demineralization using 0.5 N HCl at different time durations. By evaluating the residual calcium concentration, reaction kinetics, protein content, and extracellular matrix (ECM) integrity through histological analysis, composition assessment, and thermal analysis, the effectiveness of the process was determined. Results revealed that after 1 h of demineralization, the calcium and protein contents were 1.10 ± 0.12% and 88.7 ± 0.58 μg/mL, respectively. The study found that after 6 h, the calcium content was almost completely removed, but the protein content was only 51.7 ± 1.52 μg/mL compared to 109.0 ± 1.0 μg/mL in native bone tissue. Additionally, the demineralization reaction followed second-order kinetics with an R² value of 0.9964. Histological analysis using H&E staining revealed a gradual disappearance of the basophilic components and the emergence of lacunae, which can be attributed to decellularization and mineral content removal, respectively. As a result, organic components such as collagen remained in the bone samples. ATR-FTIR analysis showed that all demineralized bone samples retained collagen type I markers, including amide I, II, and III, amides A and B, and symmetric and antisymmetric CH₂ bands. These findings provide a route for developing an effective demineralization protocol to extract high-quality ECM from fish bones, which could have important nutraceutical and biomedical applications.

Milkfish (Chanos chanos) is an abundant fish commodity in the Philippines that generates a large number of wastes such as skin, scales, viscera, and bones, which, upon disposal, cause environmental pollution. The abundance of these wastes, such as fish skin, rich in bioactive natural products such as collagen, elicits interest in their conversion into high-market-value products. The decellularization of milkfish skin waste can extract its extracellular matrix (ECM), a potential raw material for biomedical applications such as the repair of damaged skin tissues. In particular, this study characterized the developed decellularized ECM with different concentrations (0.1%, 1.0%) of the decellularizing agents (Triton X-100, SDS) and temperature (4 °C, room temperature) using milkfish skin. The decellularized ECM structure was better preserved using Triton X-100, while SDS was more effective in cell component removal, especially at 1% concentration and 4 °C temperature. There were significant effects of varying the temperatures and concentrations on the physical and mechanical properties of the decellularized ECM. Future studies could explore more variables to further establish protocols and more analyses to better characterize the decellularized milkfish skin.