Upcycling waste tilapia (oreochromis niloticus) scales through a decellularization process for extracellular matrix extraction

Tilapia scales, typically regarded as low-value biological waste, possess valuable organic components such as collagen and inorganic components such as hydroxyapatite. Converting these wastes into functional biomaterials like decellularized extracellular matrix (dECM) contributes to long-term fish waste management to achieve sustainable fish consumption and promote a circular economy by creating a product with a high market value. Triton X-100 (TX-100) and Sodium-dodecyl sulfate (SDS) at varying concentrations (0.1%, 0.5%, and 1%) are utilized to extract the extracellular matrix. The study aims to evaluate the effectiveness of these agents in decellularizing the scales and producing a suitable dECM scaffold. Histological analysis using H&E staining revealed a significant decrease in cellular components in the decellularized samples. This was supported by the dsDNA quantification results showing the highest removal rate of 96% in the samples treated with 1% SDS. Attenuated Total Reflectance-Fourier-transform infrared (ATR-FTIR) spectra showed the amide peaks (Amide A, B, I, II, and III) indicating the presence of type I collagen. The confirmation of type I collagen was further supported by the analysis of SDS-PAGE images, which displayed the presence of α1 and α2 chains, β-dimer, and the γ-band. Among the dECM, samples treated with 0.1% TX-100 exhibited the highest protein content, averaging 593.33 ± 17.78 μg mg−1 while 1% SDS showed the lowest protein content averaging 61.33 ± 24.03μg mg−1. All dECM samples demonstrated protein denaturation temperatures ranging from 70–75 °C. This study presents baseline data of the single chemical decellularization treatment method utilized to extract ECM from tilapia scales and its resulting dECM. Further research is recommended to assess the biocompatibility and cytotoxicity of the produced dECM and explore its potential applications.

The use of fish scales to produce extracellular matrix (ECM) as scaffold material is considered a promising route.Fish wastes such as scales are biodegradable waste materials, but the structural similarity to the bone ECM is valuable for tissue engineering applications.Wu et al [5] and Fang et al [23] fabricated fish scale-derived scaffolds that exhibited high cell cytocompatibility and promote cell migration.A three-dimensional, nanofibrous fish scale/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite scaffold as bone-filling material was successfully constructed by Kara et al 2020 [24].Self-curling 3D-oriented scaffolds from tilapia scales were fabricated through the work of Shi et al which provides oriented growth and myogenic differentiation of the C2C12 myoblasts [25].These studies proved the effectiveness of fish scales as ECM scaffolds; however, additional research is required to fully harness its potential.
To successfully produce the ECM from fish scales, demineralization, and decellularization methods must be thoroughly established.Demineralization is a crucial step aimed at removing minerals while decellularization involves the removal of cellular components.Demineralization is carried out by employing strong or weak acids such as hydrochloric acid which convert the inorganic components of bones into monocalcium phosphate and calcium chloride [26,27].On the other hand, decellularization is carried out to remove a substantial amount of cellular and nuclear content (immunogenic materials) leaving behind the three-dimensional (3D) support system of the extracellular matrix (ECM) without significantly affecting the biological and mechanical properties; likewise, the biochemical composition of the remaining ECM [28][29][30].It is carried out by using detergents to remove the cellular structure in the tissue.Each decellularization strategy produces different effects on the ECM proteins; therefore, the technique used must be chosen based on the tissue biomechanics necessary for proper function.
Regarding the detergents used as decellularization reagents, sodium dodecyl sulfate (SDS) and Triton X-100 (TX-100) are commonly used.Both cause some extent of damage to the structure of the matrix, but the mechanical integrity of the matrix is maintained at an acceptable level.The utilization of SDS treatments has consistently met the stringent criteria for successful decellularization, ensuring the complete elimination of cellular components and a minimum 90% reduction in host DNA content across diverse tissue and organ types.Prominent examples include the rat forearm [31], porcine liver [32], porcine myocardium [33], porcine heart valve [34], porcine brain [35], and rat liver [36].TX-100 targets the lipid-lipid, and lipid-protein interactions, but it leaves the protein-protein interaction intact [37], [38].
This study aimed at investigating the potential of tilapia fish scales as a viable source for decellularized extracellular matrix (dECM) production.The primary objective is to assess the feasibility and efficacy of decellularizing agents, Triton X-100 (TX-100) and Sodium Dodecyl Sulfate (SDS), at varying concentrations (0.1%, 0.5%, and 1).To achieve this, characterization techniques are implemented to assess the quality and effectiveness of the single chemical decellularization process.The obtained data will serve as a baseline reference for future studies and contribute to a deeper understanding of the suitability of tilapia scales as a potential dECM source.

Sample collection and pretreatment
Fresh tilapia weighing approximately 400 g was obtained from a local fish landing in Iligan City, Philippines.To ensure the quality, the tilapia was placed in a cooling container with ice flakes during transport.Scales were manually harvested and washed with chilled water.Subsequently, the scales were oven-dried for 24 h at 45 °C to remove moisture.The oven-dried tilapia scales were demineralized using 0.5 M Hydrochloric (HCl) Acid solution at 4 °C for 1 h with constant agitation.After demineralization, the scales were washed with distilled water until a neutral pH was reached.

Decellularization condition
The pretreated scales were immersed in a beaker containing TX-100 (Loba Chemie, Mumbai, India) or Sodium Dodecyl Sulfate (Loba Chemie, Mumbai, India) solution with varying concentrations at 4 °C for 72 h with a sample and solvent ratio of 1:10.The different decellularization conditions are shown in table 1.The dECM was subsequently washed three times at 1 h intervals, followed by an additional 24 h wash, with a constant agitation must be carried out to ensure successful removal of detergent.

Proximate analysis of raw tilapia scales
The proximate analysis of moisture, fat, and ash content, was conducted following the methods described by Qiu et al (2019) [39] using the procedures in AOAC with method numbers: 950.46B, 920.153, and 960.39a.

Evaluation of dECM samples 2.4.1. Calcium content
The extent of calcium removal was evaluated by determining the mass percentage of the retained calcium using an energy-dispersive x-ray fluorescence (EDXRF) analyzer (Rigaku NEX QC, Tokyo, Japan).Before analysis, the samples were size-reduced, pelletized, and placed in the EDXRF analyzer.The resulting data were processed and evaluated to identify the mass percentage of the retained calcium.

Histological staining analysis and polarizing microscopy
The Hematoxylin and Eosin (H&E) staining method described by Bual et al (2022) was employed to assess the removal of cellular components from the scales [28].In this method, the samples were fixed in a 10% formaldehyde solution for 72 h and subsequently dehydrated using increasing ethanol concentration (70%, 90%, and 100%).The dehydrated samples were then embedded in paraffin wax, followed by sectioning into 0.4 μm thick ribbons using a microtome (Slee CUT 4062, Nieder-Olm, Germany).These sections were stained with hematoxylin and eosin solutions (Biognost ® , Zagreb, Croatia) and examined using a phase-contrast microscope (Olympus CX43, Tokyo, Japan) to visualize the cellular components and tissue structures.
Polarizing microscopy was employed to examine the surface structure of tilapia scales before and after decellularization.The images were captured using a polarizing microscope (Opto-edu A15.2601-RT, Beijing, China) to determine the effect of the decellularization on the surface structure of the scales.

dsDNA quantification
The dECM samples weighing 25 mg each of dried sample (n = 3) were subjected to DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen ® , Valencia, CA, USA).The extracted DNA was subsequently quantified using the QubitTM 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions and procedures.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy
The chemical composition and functional group of the samples were analyzed using a QATR-10 singlereflection Fourier-transform infrared (ATR-FTIR) instrument (Shimadzu, Kyoto, Japan).The samples were scanned in the wavenumber range of 400 to 4000 cm −1 with a data acquisition of 40 scans per run.The resulting absorbance spectra were recorded using LabSolutions IR software and subsequently analyzed to identify and characterize the presence of specific chemical bonds and functional groups within the samples.

Protein quantification
The protein content of the samples was determined by solubilizing 50 mg of dECM sample (n = 3) in 5 ml of 0.1 M hydrochloric acid containing 10 mg of pepsin (Merk, St Louis, MO, USA) at 4 °C for 48 h.The protein content was determined using Qubit Protein Assay Kit (Thermo Fischer Scientific, Massachusetts, USA) and quantified using a Qubit Fluorometer (Thermo Fischer Scientific, Massachusetts, USA).

Differential scanning calorimetry (DSC)
The thermograms were generated using a Perkin Elmer DSC 4000 instrument (Waltham, Massachusetts, USA) with a heating rate of 10 °C min −1 and a temperature range of 30 °C to 300 °C with samples weighing 5 mg each.The thermograms were evaluated to determine the denaturation temperatures of the samples.

Residual detergent
The quantification of the residual detergents in the dECM was performed using a Victor Nivo Alpha F Multimode plate reader (Perkin Elmer, USA) [28].The concentration of TX-100 was quantified following the protocol described by Pavlović et al 2016 [41], with slight modifications.Briefly, 1 g of lyophilized tissue was soaked in 10 ml of 50% methanol.The sample was then centrifuged, the supernatant was decanted, and the resulting supernatant underwent analysis.
The assessment of residual SDS was performed using a methylene blue assay examination, adapted from the study conducted by Alizadeh et al 2019 [42].The sample was soaked in water and mixed with absolute ethanol.The supernatant was filtered and mixed with methylene blue solution and chloroform.The resulting solution was then analyzed.

Statistical analysis
All sample experiments were independently performed in three replicates, and the experimental data are presented as means ± standard deviations (SD).The analysis of differences among groups was conducted using the One-way ANOVA test, and a p-value of p < 0.05 indicates that the observed differences were statistically significant at the 0.05 significance level.

Proximate composition of scales
In this study, the raw tilapia scales exhibited a moisture content of 36.29 ± 0.52%, a fat content of 1.82 ± 0.14%, and an ash content of 16.74 ± 0.34%.These findings demonstrate a distinct difference in comparison to the scales of other fish species.The proximate composition of different fish scales from various studies is outlined in table 2.

Calcium content
Tilapia scales primarily consist of hydroxyapatite, a calcium phosphate mineral.The results of the XRF analysis showed that the oven-dried tilapia scales initially contained approximately 12% calcium by weight.However, following the demineralization process, significant removal of calcium from the scales was observed.The resulting calcium content was found to be below the detection limit of the XRF equipment, indicating a level below 0.01%

Histological staining and surface imaging
The H&E staining images as shown in figures 1(a)-(g), confirmed the presence of cellular nuclei in the raw tilapia scales as evidenced by the blue/purple stains.All the decellularized samples showed an absence of cells, by the lack of any discernible blue or purple stains within the tissue sections.No apparent signs of structural damage to the tissue were observed in either the TX-100 or SDS decellularization methods.
In the polarized microscopy images, the surface structure of the dECM samples was evaluated in comparison with the raw samples.Remarkably, no visual evidence of structural alteration was observed in either the raw or decellularized samples, as depicted in figures 1(h)-(u).

dsDNA quantification
The results demonstrated a significant reduction in DNA content across all decellularized samples, as depicted in figure 2. The DNA content of the raw tilapia scale was determined to be 124 ± 16.09 μg mg −1 .
Comparing the dECM from different decellularization conditions, the samples treated with 1% SDS exhibited the most effective removal of DNA, with a removal of 96% (5.33 ± 4.16 ng mg −1 DNA content).On the other hand, the use of 0.1% TX-100 resulted in the lowest DNA removal of 84.41% (8.50 ng mg −1 DNA content).Significantly, the quantification of DNA content in all decellularized samples revealed levels below the

Attenuated total reflectance-fourier transform infrared (ATR_FTIR) spectroscopy
In the ATR-FTIR spectrum of the raw tilapia scales, the presence of hydroxyapatite was confirmed by the characteristic peak of the phosphate (PO 4 ) functional group.Interestingly, in the demineralized and dECM samples, the intensity of the PO 4 peak noticeably decreased, indicating the removal of calcium, as shown in figure 3.

Protein quantification
The protein content of the dECM samples exhibited a clear decreasing trend as the concentration of the decellularization agent increased, as shown in figure 4. dECM samples treated with TX-100 exhibited the highest protein concentration compared to SDS-treated samples.Specifically, the 0.1% TX-100-treated dECM samples displayed the highest protein concentration of 593.33 ± 20.3 μg mg −1 , with a retention rate of 69.64%.In contrast, the dECM samples treated with 1.0% SDS showed the lowest protein concentration of 61.33 ± 24.03 μg mg −1 , with a retention rate of 7.19%.Among the two detergents, TX-100 demonstrated better preservation of proteins in the dECM samples.The ANOVA and post hoc analysis using Tukey's test (p < 0.05) for dECM protein quantification indicated a significant difference.However, the comparison of means between the 1% TX-100 and 0.5% TX-100 and between the 0.5% SDS and 0.1% SDS did not reveal a statistically significant difference.This means that there is a significant reduction of proteins components to the dECM compared to the raw tilapia scales.

sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The analysis of the SDS-PAGE image, as depicted in figure 5, revealed the presence of distinct protein bands corresponding to the α1 and α2 chains.Furthermore, high molecular weight proteins such as the β-dimer and γ-band were also observed.The electrophoretic patterns of the TX-100-treated samples exhibited well-defined protein bands compared to the SDS-treated samples.Specifically, the α1, α2, and β chains were more prominent in the TX-100-treated samples.In contrast, the SDS-treated samples displayed low-intensity protein bands, particularly at the 0.5% and 1% SDS treatments.

Differential scanning calorimetry
The DSC analysis revealed two distinct endothermic peaks associated with different thermal events, as depicted in figure 6.The first endothermic peak, observed in the temperature range of 70-75 °C, corresponds to the irreversible denaturation of collagen protein present in both the raw scales and dECM samples.It is worth noting that the denaturation temperature of the dECM samples was slightly lower compared to that of the raw scale samples.
The second endothermal peak detected in the temperature range of 200-225 °C is attributed to the release of existing structural moisture trapped within the sample matrix and is not directly related to the denaturation of proteins.

Residual detergent
After washing, the residual detergent analysis of the dECM samples was conducted to assess the retained decellularizing agents.The results revealed a trend of increasing residual detergent content with increasing concentrations of both TX-100 and SDS.These findings indicate that a greater amount of detergent remained within the dECM samples as the concentration of the decellularizing agents increased.
Among the concentrations used in the two detergents, the dECM samples treated with 0.1% TX-100 and SDS showed the lowest residual detergent content of 0.014 ± 0.00 mg ml −1 and 0.02 ± 0.00 mg ml −1 respectively.Moreover, it is observed that the amounts of residual detergent for SDS-treated were found higher as compared to the TX-100-treated samples.

Discussion
Fish scales have been gaining attention for the development of engineering biomaterials.Its excellent biocompatibility offers great potential in tissue engineering and regenerative medicine [44].Despite its importance and potential, marine wastes, like scales, are often converted into low-quality products such as fish meal, fish oil, and fertilizer, and or directly fed to aquaculture and partially discarded [45].Another way to increase its value and illustrate its potential is to create dECM, a biomaterial with potential tissue engineering and nutraceutical applications.However, it is necessary to establish decellularization methods to ensure the efficacy and quality of the resulting dECM.A single chemical decellularization utilizing TX-100 and SDS is employed in this study to generate a dECM as a possible biomaterial with an advanced application.
Histological evaluation of the dECM, using H&E staining provided insight into the cellular and structural characteristics.The result is evident that the raw sample showed the presence of cellular nuclei, indicated by the blue or purple stains (figure 1(a)).In contrast, the dECM samples showed an absence of cellular nuclei, which shows its removal after decellularization (figures 1(b)-(g)).The blue or purple stains represent the cellular structure, while the proteins appear pink, enabling a clear visualization and differentiation of cellular nuclei and protein structure [46].Upon examination of the dECM images, both TX-100 and SDS detergents, at varying concentrations, effectively decellularized the tilapia scales.These detergents solubilize the cellular membranes and disassociate DNA from proteins [47].
Polarizing microscopy images (figures 1(h)-(u)) provided further insights into the structural characteristics of the investigated samples.Plywood-like surface structures of the scales, composed of tightly packed collagen fiber layers reinforced by a mineral phase of calcium-deficient hydroxyapatite, were seen [5,45].This unique layered structure contributes to the strength and mechanical properties of scales [48].No apparent alterations were observed in the images for its structural integrity or arrangement before and after decellularization.
In addition to the visual evidence provided by the H&E stained images (figure 1), DNA concentration validates the efficiency of the decellularization process.The dECM samples resulted in low DNA levels, with values consistently falling below 50 ng mg −1 (figure 2) confirming our H&E staining results and demonstrating successful decellularization.This is crucial as tissue not thoroughly decellularized may cause chronic inflammation, fibrotic encapsulation, and scar tissue formation response during in-vivo remodeling [49].DNA content results falling within the accepted range reported in previous studies [46], reduce the potential for immunological activation or inflammation if used as a biomaterial scaffold.
The FTIR spectra provide insights into the functional properties of the raw and decellularized waste tilapia scales.In the raw tilapia scale, prominent peaks corresponding to PO 4 groups are observed in the spectra, indicating the presence of hydroxyapatite.Additionally, the intensity of the hydroxyapatite-associated peaks reduces, signifying the removal of calcium ions after the demineralization process.This is due to the HCl's ability to convert major organic substituents into monocalcium phosphate and calcium chloride [26].The intensity of the amide bands, particularly the type I collagen markers was more pronounced in the demineralized and decellularized samples.The increase in the intensity can be related to protein exposure following demineralization as other minerals were removed during the process.Collagen is classified as either mammalian or marine, but both contain a triple-helix structure composed of three polypeptide chains containing repeating units of three amino acids-(Gly-Pro-Xaa)n [19].The preservation of type I collagen in dECM has offered a wide range of potential applications, especially as functional molecules for the cosmetic, pharmaceutical, food, biomaterial, and nutraceutical industries [3].
During decellularization, detergents induce disruption and dissociation of proteins in the ECM, amplify with extended exposure, and vary based on organ subunit, type of tissue, and donor age [47].The retention of protein constituents within dECM is crucial in upholding its structural stability and bioactivity, which are fundamental for its application in tissue engineering.Initially, the protein concentration in the raw tilapia scales as shown in figure 4 was determined to be 852 ± 20.29 μg mg −1 , representing the baseline protein content.Among the dECM, TX-100 at 0.1% concentration had the highest protein concentration, demonstrating preservation during the decellularization.On the other hand, SDS at 1% concentration resulted in the lowest protein concentration, likely due to the denaturing effect of SDS as it targets protein-protein interaction.The protein content in the dECM showed an inverse relationship to the concentration of the decellularization agent, emphasizing the importance of agent choice and concentration in preserving protein components in dECM derived from tilapia scales.This is further supported by analyzing the preservation of the molecular weight of protein present within the dECM samples.The SDS-PAGE results presented in figure 5 confirmed the presence of high-intensity bands corresponding to the α1 and α2 chains.Notably, the α1 chain exhibited a two-fold higher intensity compared to the α2 chain, suggesting that type I collagen is the predominant protein component within the dECM samples [19,50,51].The observed preservation of high molecular weight protein fractions, particularly the α1 and α2 chains, further supports the retention of type I collagen's structural and functional integrity within the dECM.Meanwhile, the decrease in intensity of protein markers in 0.5% and 1% SDS-treated samples can be attributed to the low protein content shown in figure 4 due to the denaturing effect of SDS on the protein [52].
The denaturation temperatures of the dECM samples shown in figure 6 ranged from 70-75 °C, slightly lower than the raw tilapia scales.This decrease is attributed to the reduction in hydroxyproline content during demineralization, which affects the thermal stability of the collagen trimer in the dECM [53].Hydroxyproline is a key amino acid found in collagen, and its presence plays a crucial role in maintaining the thermal stability of the collagen trimer [20].The stability of the collagen trimer significantly contributes to the mechanical strength and durability of the (dECM) to withstand physiological forces and facilitate tissue development.Higher levels of specific amino acids, such as hydroxyproline and proline, are directly associated with increased stability of the helices and heat resistance in collagen [54].The second endothermic peak at 200-225 °C is associated with the release of structural moisture [28,55].On the other hand, a visible difference in the peak of 0.5 and 1% SDS at 200-250 °C, which can be associated with the extent of water loss and denaturation in the protein helical structure [56], proportionate to the low protein count shown in figure 4. Proteins with high water absorption and retention gave a higher proportion of polar residues capable of forming hydrogen bonds with water [57].
The analysis of residual detergent content in the dECM samples presented in figure 7 demonstrated a noticeable increase in residual detergent with higher concentrations of both TX-100 and SDS.SDS exhibited a higher residual detergent level than TX-100, as it is hard to remove due to its ionic nature similar to previous research findings [53,58].The presence of residual detergent in the ECM samples carries important implications for their potential applications, as it can potentially induce cytotoxic effects and compromise the biocompatibility and functionality of the dECM.The residual detergent may hinder or entirely negate the beneficial properties of the dECM, undermining its intended therapeutic or tissue engineering applications [47].Phosphate buffer saline (PBS) and deionized water are two common rinsing agents used in decellularization protocols.However, water is considered a better alternative due to its higher osmotic pressure, facilitating the removal of detergents especially on tilapia skin [59].
The study findings offer significant contributions to the field by demonstrating the potential application of waste tilapia scales to produce a high-quality dECM through a single chemical treatment.It highlights the importance of upcycling fish waste to create valuable biomaterial and promotes adopting circular economy practices in the fish processing industry.However, it is crucial to address environmental concerns, such as potential heavy metal contamination and adherence to sanitary practices, as they can impact the safety and quality of the fish scales.Collaborative efforts among researchers, fish processors, regulatory bodies, and environmental agencies are necessary to develop comprehensive protocols that mitigate environmental risks and ensure ethical and sustainable management of fish biomass waste.
Additionally, future research should focus on assessing the biocompatibility and cytotoxicity of the produced dECM to evaluate its suitability for various applications in tissue engineering and regenerative medicine.Understanding the interactions between the dECM and living cells will provide valuable insights into its efficacy and potential limitations.This knowledge will guide the development of safe and effective approaches for utilizing waste tilapia scales as a valuable resource, contributing to advancing sustainable biomaterials and developing innovative solutions for waste management in the fish processing industry.

Conclusion
This research highlights the potential of tilapia scales as a source of dECM using a single chemical method.By using detergents, namely TX-100 and SDS, at concentrations of 0.1%, 0.5%, and 1%, a dECM was developed, suggesting its potential as a functional biomaterial for tissue engineering and nutraceutical application.The results obtained from the analysis were favorable, particularly in H&E staining, DNA content, residual detergents, and denaturation temperature.Importantly, primary components like proteins stayed intact.This claim was further backed up by the results from the FTIR test, SDS-PAGE, and protein measurements, showing type-I collagen as the primary component.Among the samples, 0.1% TX-100 dECM exhibits the highest protein concentration, averaging 593.33 ± 20.3 μg mg −1 , a 69.64% retention rate ideal as a potential functional biomaterial.These findings provide valuable baseline data for utilizing single chemical decellularization methods to extract dECM from tilapia scales and pave the way for further research on the biocompatibility, cytotoxicity, and potential applications of the produced dECM in tissue engineering and regenerative medicine.This study contributes to a new source of sustainable biomaterials.It highlights the importance of waste utilization and biomaterial engineering in promoting a circular economy and addressing environmental challenges in the fish processing industry.

− 1 .
The results indicate that the decellularization process effectively removes cell nuclei, minimizing the risk of immunological activation or inflammation.Statistical analysis, utilizing ANOVA and post hoc testing with Tukey's test (p < 0.05) for dECM DNA quantification, indicated a significant difference between the raw sample and all dECM samples.This finding suggests a successful removal of DNA during the decellularization process.

Figure 2 .
Figure 2. DNA quantification comparing the raw and dECM tilapia scales samples on a dry basis.n = 3. Bars represent standard deviation.* p < 0.05.

Figure 3 .
Figure 3. ATR-FTIR spectra of the raw and dECM tilapia scales samples.

Figure 5 .
Figure 5. SDS-PAGE analysis electrophoretic pattern of solubilized raw and dECM tilapia scale samples.

Table 1 .
Decellularization method of the tilapia scales.

Table 2 .
Proximate composition of different scales.
critical threshold of 50 ng mg