Characterization and Biological Performance of Anglerfish Collagen and Bovine Collagen

doi:10.21203/rs.3.rs-6906646/v1

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Characterization and Biological Performance of Anglerfish Collagen and Bovine Collagen

https://doi.org/10.21203/rs.3.rs-6906646/v1

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published 08 Oct, 2025

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Collagen plays a crucial role in maintaining the structural stability and functional integrity of vertebrates. The currently extracted animal-derived collagens include those from mammalian and marine sources. In this study, a comparative analysis was conducted to examine the differences between marine-derived (anglerfish) and mammalian-derived collagen (bovine). Both types of collagen can form a porous fibrous network through in vitro self-assembly. Further analysis using UV-vis, FTIR, CD and SDS-PAGE confirmed that both collagens were typical Type I collagen, maintaining an intact triple-helix structure. DSC results showed that the thermal denaturation temperature of anglerfish collagen was markedly lower than that of bovine collagen. Amino acid composition analysis demonstrated significant differences in proline and serine content. Type I collagenase degrades anglerfish collagen faster than bovine collagen. Both collagens enhanced cell growth and proliferation, showing excellent biocompatibility. In conclusion, marine-derived collagen demonstrates high structural and functional similarity to mammalian collagen. The degradation and biocompatibility of collagen make it promising for hemostasis, wound dressings, and drug delivery. These results provide a scientific foundation for the efficient utilization and sustainable development of marine collagen resources.

Collagen

Self-assembly

Anglerfish skin

Bovine skin

Collagen is a biological macromolecule and a major component of the extracellular matrix in vertebrates [1, 2]. As a functional protein, collagen exhibits remarkable biological activity and plays a crucial role in maintaining the integrity of the structure and function of organisms [3, 4]. Of the 29 distinct types of collagen identified, Type I is the most abundant. Type I collagen is predominantly distributed in mammalian skin, bone, tendons, and ligaments [5, 6], exhibiting exceptional biocompatibility, biodegradability, and low immunogenicity, thereby demonstrating significant application potential [4, 7–9].

Type I collagen consists of two α-chains and one β-chain forming a trimer. Its stable helical structure arises from repetitive (Gly-X-Y)ₙ sequences, with glycine appearing every third residue to maintain the helix's internal geometry [5, 10–12]. The X and Y positions are predominantly occupied by proline (Pro) and hydroxyproline (Hyp) residues [5, 9, 10, 12, 13], rendering the Pro-Hyp-Gly tripeptide the most archetypal sequence motif in collagen [9, 14]. The arrangement of amino acid sequences in collagen ensures that glycine forms bends filling the interior of the helix, while proline and hydroxyproline form smooth bends around the helix, contributing to the stability and tightness of the three-dimensional helical structure [14, 15]. This stable triple helix structure, composed of three polypeptide chains, allows collagen to withstand mechanical stress and provides support for cell growth [11, 16]. The structural integrity of collagen is vital for its biological functions, including the growth, renewal, and repair of tissues [11].

Natural collagen and its derivatives are principally extracted from animal tissues such as bovine and porcine sources [2, 17], owing to their remarkable sequence homology with human collagen which ensures biological compatibility [17–19]. However, collagen derived from mammals may trigger immune responses and lead to the transmission of zoonotic diseases due to its immunogenicity [20]. Notable examples of such diseases include Bovine Spongiform Encephalopathy (BSE), Transmissible Spongiform Encephalopathies (TSE), and Foot-and-Mouth Disease (FMD). As a result, the safety concerns associated with mammalian collagen limit its broader application potential [18, 21, 22]. Additionally, cultural or religious beliefs in some regions further restrict the use of terrestrial mammal collagen and its products [21].

Studies have shown that collagen from marine sources is highly homologous to collagen derived from terrestrial mammals, with only minor differences in amino acid composition [21]. Furthermore, marine collagen exhibits superiority over terrestrial collagen in certain aspects, including its low antigenicity, safety, minimal risk of infectious disease transmission, and excellent biocompatibility [2, 7, 21]. Marine collagen's properties make it both a promising resource for diverse applications and an effective solution for utilizing fish processing byproducts, emerging as an ideal choice for sustainable development [20].

Due to species differences and variations in living environments, collagen derived from different tissue sources exhibits unique structural and performance characteristics. This study aims to explore the differences between marine skin collagen and mammal skin collagen in terms of structure, thermal stability, functional characteristics, and cytocompatibility. A variety of detection methods were employed to examine the characteristics of both collagen types. The goal is to elucidate their structural differences at the molecular level, clarifying the similarities and disparities in their functional properties. Furthermore, this study evaluated the cytocompatibility of both collagen types through cell-based assays. The findings contribute to a deeper understanding of the structure and properties of marine-derived collagen, providing strong scientific evidence for its potential applications.

2.1 Materials and reagents

The marine skin was sourced from anglerfish (purchased from the local market), while the mammal skin was bovine fetal calfskin obtained from Huhhot Caoyuan Hye Bio-Engineering Material Co., Ltd.

CaCl₂, NaH₂PO₄·2H₂O, CH₃COOH, NaCl, and NaOH were purchased from Hushi Chemical Company (Shanghai, China); HCl from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); pepsin from Solarbio (Beijing, China); collagenase I (from Clostridium histolyticum) from BasalMedia (Shanghai, China); genipin from Sigma-Aldrich (Shanghai, China); and the amino acid mixture from Wako Pure Chemical Industries, Ltd. (Japan). SDS-PAGE electrophoresis solution (Tris-Gly, Powder, bag) and SDS-PAGE precast gel (10%) were purchased from Beyotime (Shanghai, China). SDS-PAGE uploading buffer (non-reducing, 5x) was purchased from Cwbio (Beijing, China). Color Prestain Marker (10-180kDa) was purchased from Adamas life (Shanghai, China).

For the cell culture, Phosphate buffer solution (PBS), Dulbecco's modified eagle medium (DMEM), Penicillin-Streptomycin (100×) and trypsin EDTA (0.25%) were purchased from Adamas life (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (California, USA).

All other chemicals were of analytical grade and used as received. The deionized water (18 MΩ cm^− 1) was used in the present experiments.

2.2 Collagen extraction

Anglerfish skin and fetal calfskin was washed, weighed, degreased, and digested with pepsin for 48 hours [4, 23]. After centrifugation, the pH was adjusted to inactivate the enzyme [4, 23]. The collagen was then precipitated via salting-out, dialyzed, and dissolved in acetic acid to a final concentration of 10 mg mL^− 1.

2.3 Preparation of collagen

The 10 mg mL^− 1 anglerfish skin collagen solution and 10 mg mL^− 1 bovine skin collagen solution were adjusted to pH = 7.0 with 3M NaH₂PO₄⋅2H₂O and 2M NaOH, respectively, and adjusted to the appropriate final concentration with deionized water solution.

2.4 Physicochemical characterization

The 5 mg mL⁻¹ anglerfish and bovine collagen solutions were deposited on silicon wafers (self-assembled at 4°C for anglerfish collagen and 37°C for bovine collagen), rinsed with deionized water, and freeze-dried. After gold sputter-coating, the samples were observed by SEM (Tescan Vega3, China), and the diameters of 100 fibrils per collagen type were measured and statistically analyzed using ImageJ software.

UV-Vis spectroscopy (UV-8000) measured the absorption peaks of anglerfish and bovine collagen (1 mg mL⁻¹ in 0.5M acetic acid) from 360 − 190 nm, using PBS as blank.

The anglerfish collagen and bovine collagen were respectively analyzed using an FT-IR spectrometer (IS50, China) with a wavenumber range of 4000 − 400 cm⁻¹ and a scanning resolution of 4.0 cm⁻¹. For secondary structure analysis, the amide I region (1600–1700 cm^− 1) was analyzed using PeakFit version 4.12 software and the Gaussian curve fitting method. The fitting was performed to minimize the standard error, with R² > 0.99.

The secondary structure of collagen was determined using a Circular Dichroism spectrometer (J-1500, Japan). 1 mL of each sample was placed in a quartz cuvette, and an equal amount of deionized water was used as a blank for the CD measurements (at 4 ℃ under a constant nitrogen atmosphere). The CD spectra were recorded in the wavelength range of 250 to 190 nm for three repeats.

The anglerfish and bovine collagen solutions were placed in aluminum pans, sealed, and subjected to Differential Scanning Calorimetry (DSC250, USA) analysis. The samples were heated from 0 ℃ to 60 ℃ at a heating rate of 1 ℃/min under a nitrogen atmosphere (purity ≥ 99.9%). Helium was used as the protective gas, and liquid nitrogen served as the cooling medium during measurements. The denaturation temperature (Td) was measured in triplicate.

The molecular pattern of collagen was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method[24]. Both collagen samples were mixed with 5× Laemmli sample buffer (containing SDS, Tris-HCl, bromophenol blue, glycerol, and DTT) at a 4:1 ratio, vortexed, boiled for 5 minutes, and briefly centrifuged at 1,890 g. The test samples along with protein molecular weight markers (10–180 kDa range) were loaded onto a 10% SDS-PAGE precast gel. Electrophoresis was initially performed at 80 V for 20 minutes, then switched to 120 V for approximately 60 minutes to separate the collagen samples. After electrophoresis, the gels were stained with 0.25% Coomassie Brilliant Blue R-250 solution and destained with a mixture of 20% (v/v) ethanol and 10% (v/v) acetic acid until clear protein bands were visible. The protein bands were then captured using a molecular gel imager (Servicebio, China).

The samples were hydrolyzed with 5 mL 1:1 HCl at 110 ± 1°C for 22 h. After cooling, the hydrolysates were filtered into 10 mL volumetric flasks, diluted to volume, and 0.05 mL aliquots were dried under nitrogen. The residues were reconstituted in 2 mL 0.02 M HCl, filtered (0.22 µm), and analyzed using a calibrated Hitachi Amino Acid Analyzer with mixed amino acid standards for quality control.

Anglerfish collagen was placed at 4°C and bovine collagen at 37°C for gelation, then weighed separately. An equal volume of 5 mM genipin solution was added to AC for cross-linking to modify its denaturation temperature, and the sample was reserved for subsequent use. A 0.5 U mL⁻¹ type I collagenase solution in PBS was prepared and added to both AC and BC, followed by incubation at 37 ℃ for 1, 6, 12, 24, 48 and 72 h. After enzymatic digestion, the samples were weighed again, and their dry weights were recorded post-dehydration. (m₁ : the dry weight before degradation, m₂ : the dry weight after degradation.)

$$\:Degradation\:rate=\frac{m₁-m₂}{m₁}\times\:100\%$$

2.5 Cytocompatibility assays

Collagen solutions were dispensed into a 48-well plate, with anglerfish collagen incubated at 4 ℃ and bovine collagen at 37 ℃ for fibrillogenesis. Collagen samples were soaked in 75% ethanol (6 h), rinsed with PBS, then incubated in serum-free DMEM high-sucrose medium under a 95% air and 5% CO₂ atmosphere for 24 h in a 37 ℃ incubator. For cell culture, DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used to culture the cells at 37 ℃ under 95% air and 5% CO₂. When the cell density reached approximately 80%, the cells were detached using 0.25% (w/v) trypsin-EDTA.

Cell proliferation was assessed via CCK-8 assay (measuring formazan production on hydrogels). MC3T3-E1 cells (1×10⁴/well) were cultured for 1, 3, or 5 days. At each timepoint, a 10:1 mixture of medium and CCK-8 reagent was added, followed by 1 h incubation. Absorbance (450 nm) was measured using an HBS-1069A microplate reader after transferring solutions to a fresh 96-well plate.

Live/dead cell staining was performed to assess the biosafety of the hydrogel scaffolds. After the hydrogels underwent the previously described treatments, cell suspensions were added to 48-well culture plates at a density of 1 × 10⁴ cells/well. At 1, 3, and 5 days, the cells were stained using a live/dead cell double-staining kit (Calcein AM and Propidium Iodide). Cells were stained for 10 minutes under dark conditions. After staining, the cells were washed with PBS to remove excess stain. The stained cells were then observed under an inverted fluorescence microscope (Nikon, Japan) to evaluate cell viability.

2.6 Statistical analyses

All the quantitative data were expressed as mean ± SD. For the determination of statistical significance, an ANOVA test was performed with the GraphPad Prism software. P < 0.05 was used as the criterion of statistical significance.

3.1 Characterization of Collagen

When the pH of their respective solutions was adjusted, both anglerfish collagen (AC) and bovine collagen (BC) formed gel structures through self-assembly. This gel morphology arises from the aggregation and self-assembly of collagen molecules, leading to the formation of a three-dimensional network structure [25]. The slight color differences observed between the two may be attributed to variations in the raw material source [9, 26].

Scanning electron microscopy (SEM) observations at magnifications corresponding to 2 µm and 1 µm scales revealed that both anglerfish-derived and bovine collagen exhibited fibrous porous architectures, forming honeycomb-like networks (Fig. 1A and B). Comparative analysis revealed that the collagen fiber distribution in bovine collagen (BC) exhibited a denser microstructure. In contrast, anglerfish collagen (AC) exhibited fewer collagen fibrils with a more loosely arranged architecture. Furthermore, measurements indicated that the average diameter of anglerfish collagen fibers was approximately 172 nm, while the average diameter of bovine collagen was approximately 139 nm (Fig. 1E and F), AC displayed collagen fibers with significantly greater diameter.

Ultraviolet (UV) spectrum analysis of colloids is a technique used to study the absorption of molecules with conjugated structures. The UV spectra of both anglerfish collagen and bovine collagen showed light absorption in the 190–320 nm range, with the maximum absorption peaks occurring at around 226 nm for anglerfish collagen and 225 nm for bovine collagen, which are typical absorption bands for collagen (Fig. 2A). Although the UV spectra of the two types of collagen show differences, implying variations in their molecular structures, these differences are primarily determined by the amino acid composition. Therefore, the distinct UV spectra likely reflect differences in the types and quantities of amino acids, but further experimental verification is needed to confirm these variations [9]. Collagen is rich in amino acids such as glycine, proline, and hydroxyproline, which exhibit characteristic UV absorption. The typical absorption peaks of collagen observed at 220–240 nm are attributed to the C = O, —COOH, and CONH₂ groups in the peptide bonds [23, 27, 28].

The structures of collagen were studied using Fourier transform-infrared spectroscopy. In the Fig. 2B, all samples exhibited characteristic peaks of typical collagen amide A, B, I, II, and III bands. The infrared spectra of the two types of collagen were similar but showed certain differences.The absorption peaks observed at 3000–3500 cm⁻¹ are attributed to the stretching vibration of the N-H band in Amide A [1, 29]. The specific amide A peaks for anglerfish and bovine collagen were located at 3290 cm⁻¹ and 3303 cm⁻¹, respectively, indicating the presence of hydrogen bonding within the collagen molecules [1]. The amide B band, associated with the asymmetric stretching vibration of CH₂ groups and reflecting the tertiary structure of collagen, showed absorption peaks for anglerfish and bovine collagen at 3076 cm⁻¹ and 3079 cm⁻¹, respectively [1, 30]. The absorption peak at 1600 ~ 1690 cm^− 1 corresponds to the C-O stretching vibration of Amide I, which generally had the highest transmittance at 1639 cm^− 1. It is the most prominent absorption band in the infrared spectrum of collagen. In the triple helix structure of collagen, the presence and position of the Amide I band can reflect the conformation and stability of collagen. The absorption peaks at 1480 ~ 1575 cm ^− 1 correspond to the N-H bending vibration and C-N stretching vibration of amide II, and the transmittance is generally highest at 1542 and 1546 cm^− 1, which is helpful for further confirming the triple-helical structure of collagen [29].The absorption peak at 1229 ~ 1301 cm^− 1 corresponded to the C - N stretching vibration of Amide III. Amide III had the highest transmittance at 1237 cm^− 1, and its position and intensity can also provide additional evidence for the triple helix structure of collagen [26].The differences between anglerfish collagen and bovine collagen in the amide I, II, and III bands are not significant. The amide I, II, and III of anglerfish collagen were 1636, 1543, and 1238 cm^− 1, respectively, while the amide I, II, and III of bovine collagen were located at 1633, 1549, and 1240 cm^− 1, respectively. Amide I, II, and III bands correspond to different vibration modes and types of chemical bonds. The presence and characteristics of these amide bands can be used to confirm the complete triple helix structure of collagen [31].

Furthermore, to further analyze the secondary structure differences between AC and BC, the secondary structure patterns of collagen were determined using PeakFit 4.12 software and Gaussian peak fitting algorithms based on the data (Fig. 2C and D). The amide I band corresponds to the stretching vibration of C = O, which can maintain the structural pattern that stabilized the collagen triple helix [23, 26]. This spectral region is used for the analysis of collagen secondary structures, including β-sheet (1600–1640 cm^− 1), random coil (1640–1650 cm^− 1), α-helix (1680–1700 cm^− 1), and β-turn (1660–1700 cm^− 1). The fitting data showed that anglerfish collagen and bovine collagen contained 15.6% and 16% α-helix, and 37.5% and 42.9% β-sheet, respectively. Due to their high stability and regularity, α-helix and β-sheets are considered ordered secondary structures [32]. AC and BC contained 26.9% and 20.3% β-turn, as well as 20% and 20.8% random coil, respectively. β-turn and random coil, due to their relatively flexible and unordered characteristics, are generally considered to be disordered secondary structures [32]. These structures together determine the shape and function of the protein.

Circular dichroism (CD) spectroscopy characterizes protein secondary structures by quantifying differential absorption of left- and right-handed circularly polarized light [33, 34]. The triple-helical structure of native collagen exhibits unique optical activity, with its secondary structural characteristics being highly similar to the polyproline type II (PPII) helix [35–37]. As shown in Fig. 2E and F, the secondary structure of type I collagen exhibited a clear CD transition. Anglerfish collagen showed a positive absorption band at 219 nm and a negative absorption band at 200 nm, with an Rpn of 0.20. For bovine collagen, the maximum and minimum CD spectra were observed at 221 nm (positive absorption band) and 201 nm (negative absorption band), with an Rpn of 0.19. Both AC and BC exhibit characteristic CD spectra, confirming their triple-helical structures [36].

DSC was utilized to measure the thermal denaturation temperatures of both anglerfish collagen and bovine collagen. When the temperature exceeds the thermal denaturation temperature (Tm) of collagen, the hydrogen bonds stabilizing the native triple helix rupture, leading to structural unwinding and dissociation into individual α-chains or partially folded β-chain aggregates [31]. The DSC curves of the two collagens, shown in Fig. 3A and B, revealed that anglerfish collagen had a thermal denaturation temperature peaking at 21.1 ℃, while bovine collagen's thermal denaturation temperature was approximately 38.1 ℃. Previous studies have shown that the thermal denaturation temperature of collagen is positively correlated with its imino acid content, including proline and hydroxyproline [38, 39]. Hydroxyproline, in particular, plays a critical role in stabilizing the collagen triple helix, and the higher the degree of hydroxylation, the higher the collagen denaturation temperature [28, 38, 40]. Bovine collagen exhibited superior thermal properties compared to anglerfish collagen, likely due to its higher proline content. Furthermore, the difference in thermal denaturation temperatures between the two types of collagen may be attributed to variations in the source location of the raw materials, moisture content, and the environmental and physiological temperatures of the organisms [31, 38]. The subsequent change in the heat absorption peak observed in the DSC analysis may indicate the melting absorption peak of collagen. During the heating process, collagen’s molecular chains begin to undergo significant flow and destruction, resulting in the loss of its original structure and properties. This ultimately leads to the loss of its biological activity, mechanical properties, and chemical stability [29].

As shown in Fig. 4, the electrophoretic patterns of anglerfish collagen and bovine collagen after high-temperature treatment clearly display the composition of their collagen subunits. When compared with the Marker (0-180 kDa), the electrophoresis results of both types of collagen indicated the presence of different α-chains (specifically α₁ and α₂ chains), which are the fundamental building blocks of collagen. Additionally, dimeric β-chain, formed by intramolecular cross-linking of collagen, were observed in the electrophoretic profiles of both anglerfish and bovine collagen [38]. The presence of the characteristic β-chain further confirmed that both types of collagen belong to type I collagen. Upon high-temperature treatment, the triple helix structure is disrupted, allowing the α chains and β chain formed by their cross-linking to be separated and observed in the electrophoresis analysis. The protein bands in the electrophoresis results were clear, with no trailing bands, indicating that no collagen fragments with molecular weights lower than those of the α and β chains were present. This observation suggests that during the extraction, purification, and high-temperature treatment of collagen, the collagen molecules maintained their structural integrity without significant degradation [9]. This finding further verifies the purity of the extracted collagen [31]. It is also worth noting that the positions of the α₁ and α₂ chains of anglerfish collagen (AC) are close to each other, indicating that the molecular weights of the two peptide chains are similar [2]. In contrast, the positions of the α₁ and α₂ chains of bovine collagen (BC) are somewhat further apart, indicating that the molecular weights of these peptide chains are slightly different.

The amino acid composition and content of anglerfish collagen and bovine collagen were systematically analyzed (Fig. 5), and both samples had similar amino acid compositions, although the proportions were slightly different. Both collagen samples had the highest percentage of glycine, which aligns with previous studies in the literature [38, 41, 42]. Other amino acids found in significant proportions included alanine, proline, and alanine [20]. These hydrophilic amino acids can form hydrogen bonds, which help prevent protein molecules from aggregating and undergoing condensation or denaturation [20]. This is crucial for maintaining the triple-helix structure of collagen [41]. Notably, hydroxyproline is formed by the hydroxylation of proline. Thus, in this experiment, the proline content corresponds to the sum of proline and hydroxyproline [38]. It is well established that the content of subamino acids, such as proline and hydroxyproline, is vital for maintaining the structural integrity of collagen and enhancing the stability of its triple helix [16]. This structure is a key marker of collagen's stability and bioactivity, and the content of these subamino acids is often used as an indicator of collagen quality [27]. These amino acids contribute to collagen’s hydrogen bonding and increased thermal stability [38, 41]. Given that the content of subamino acids is closely linked to collagen's thermal stability, the lower thermal stability observed in anglerfish collagen compared to bovine collagen suggests that the living environment of the organisms plays an important role in determining the proportion of these subamino acids in collagen [27, 41]. Additionally, AC contains a higher proportion of serine, while BC is richer in isoleucine and leucine. The presence of serine may enhance collagen's adaptability to cold-water environments, as it can maintain hydrogen bond integrity while conferring greater structural flexibility to the collagen [43–45]. Additionally, anglerfish collagen contains significantly lower levels of isoleucine than bovine collagen, a difference that may be related to their distinct collagen sources.

BC demonstrated greater resistance to type I collagenase degradation than AC under identical 37 ℃ treatment conditions (Fig. 6), as evidenced by significantly lower degradation rates. This differential stability stems from distinct amino acid profiles - the elevated proline and hydroxyproline content in BC reinforces triple-helical stability via hydrogen-bonding networks and conformational constraints, while reduced hydroxyproline in AC decreases helical stability, promoting unwinding and cleavage site exposure that accelerates degradation [46, 47].

3.2 Cytocompatibility assays

The proliferation of MC3T3-E1 cells on both types of collagen was assessed using the CCK-8 assay, and the results are shown in the Fig. 7A. Both anglerfish collagen and bovine collagen significantly promoted cell proliferation, demonstrating their excellent ability to support cell growth. Notably, anglerfish collagen may exhibit superior cell proliferation under certain conditions, potentially due to its unique properties that favor cellular interactions and growth dynamics. The live/dead cell staining results (Fig. 7B - D) demonstrated that after 1, 3, and 5 days of culture, all groups exhibited abundant green-fluorescent live cells with minimal red-fluorescent dead cells. These findings indicate that both AC and BC groups maintained high cell viability rates, confirming the effective support of cell survival and growth by both materials. With prolonged culture duration, both materials showed favorable proliferation trends, further verifying their excellent biocompatibility. These results corroborate the conclusions drawn from the cell proliferation assays.

This study successfully extracted collagen from anglerfish skin (AC) and bovine skin (BC) and conducted a comparative analysis. Experimental results showed that both AC and BC are fibrous networks and exhibited the characteristic triple-helix conformation of type I collagen. While their amino acid compositions show differences in proline and serine content, AC displays a higher degradation rate and better biocompatibility compared with BC. With advancements in technology and increasing environmental awareness, anglerfish collagen is expected to become a suitable material in a wide range of biomedical fields in the future.

Author contributions

H.X. Song: Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation, Conceptualization. B.H. Yin: Visualization, Methodology, Investigation. B. Jiang: Visualization, Methodology. L. Mi: Investigation. Conceptualization. C. Cui: Methodology, Data curation. W. Su: Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization. N. Bai: Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization.

Funding

The National Natural Science Foundation of China (No. 52203173), The Natural Science Foundation of Shandong Province (No: ZR2022QH283), The Application and Basic Research Project of Qingdao West Coast Area (2020-49), Science and Technology Program of Shinan District of Qingdao (Grant No.: 2023-2-005-YY), Clinical Medicine + X Research Project of the Affiliated Hospital of Qingdao University (Grant No.: QDFY+X2023119)

Availability of data and materials

The authors confirm that the data analyzed and generated from these research finding are provided within this article.

Ethics Approval Not applicable.

Consent to Participate Yes. All authors agreed to participate in this research.

Consent for Publication Yes. All authors have approved the last version of the manuscript for its submission.

Competing Interests The authors declare no competing interests.

Shen, Xian-Rong, Xiu-Li Chen, Hai-Xia Xie, Ying He, Wei Chen, Qun Luo, Wei-Hong Yuan, Xue Tang, Deng-Yong Hou, Ding-Wen Jiang, and Qing-Rong Wang. "Beneficial Effects of a Novel Shark-Skin Collagen Dressing for the Promotion of Seawater Immersion Wound Healing." Military Medical Research 4, no. 1 (2017).
Carvalho, Ana M., Alexandra P. Marques, Tiago H. Silva, and Rui L. Reis. "Evaluation of the Potential of Collagen from Codfish Skin as a Biomaterial for Biomedical Applications." Marine Drugs 16, no. 12 (2018).
Arumugam, Gokula Krishnan Sivasundari, Diksha Sharma, Raj Mohan Balakrishnan, and Jagadeesh Babu Ponna Ettiyappan. "Extraction, Optimization and Characterization of Collagen from Sole Fish Skin." Sustainable Chemistry and Pharmacy 9 (2018): 19-26.
Ran, Yaqin, Wen Su, Lei Ma, Yunfei Tan, Zeng Yi, and Xudong Li. "Developing Exquisite Collagen Fibrillar Assemblies in the Presence of Keratin Nanoparticles for Improved Cellular Affinity." International Journal of Biological Macromolecules 189 (2021): 380-90.
Li, Rou, Shiqing Xu, Yanning Guo, Cong Cao, Jingchen Xu, Lijun Hao, Sai Luo, Xinyao Chen, Yuyang Du, Ye Li, Yong Xie, Weitong Gao, Jing Li, and Baohua Xu. "Application of Collagen in Bone Regeneration." Journal of Orthopaedic Translation 50 (2025): 129-43.
Vijayalekha, Ashwathi, Suresh Kumar Anandasadagopan, and Ashok Kumar Pandurangan. "An Overview of Collagen-Based Composite Scaffold for Bone Tissue Engineering." Applied Biochemistry and Biotechnology 195, no. 7 (2023): 4617-36.
Tziveleka, Leto-Aikaterini, Stefanos Kikionis, Labros Karkatzoulis, Kostas Bethanis, Vassilios Roussis, and Efstathia Ioannou. "Valorization of Fish Waste: Isolation and Characterization of Acid- and Pepsin-Soluble Collagen from the Scales of Mediterranean Fish and Fabrication of Collagen-Based Nanofibrous Scaffolds." Marine Drugs 20, no. 11 (2022).
Salvatore, Luca, Nunzia Gallo, Maria Lucia Natali, Lorena Campa, Paola Lunetti, Marta Madaghiele, Federica Stella Blasi, Angelo Corallo, Loredana Capobianco, and Alessandro Sannino. "Marine Collagen and Its Derivatives: Versatile and Sustainable Bio-Resources for Healthcare." Materials Science and Engineering: C 113 (2020).
Li, Yuling, Youxi Tian, Xin Xiong, Huizhi Chen, Yubin Zhou, Yanfang Zhou, and Xinsheng Peng. "Comparison of Collagens Extracted from Swim Bladder and Bovine Achilles Tendon." Materials Research Express 10, no. 5 (2023).
Martins, Eva, Rita Fernandes, Ana L. Alves, Rita O. Sousa, Rui L. Reis, and Tiago H. Silva. "Skin Byproducts of Reinhardtius Hippoglossoides (Greenland Halibut) as Ecosustainable Source of Marine Collagen." Applied Sciences 12, no. 21 (2022).
Geahchan, Sarah, Parnian Baharlouei, and Azizur Rahman. "Marine Collagen: A Promising Biomaterial for Wound Healing, Skin Anti-Aging, and Bone Regeneration." Marine Drugs 20, no. 1 (2022).
Sun, Shanshan, Yahui Gao, Junde Chen, and Rui Liu. "Identification and Release Kinetics of Peptides from Tilapia Skin Collagen During Alcalase Hydrolysis." Food Chemistry 378 (2022).
Sorushanova, Anna, Luis M. Delgado, Zhuning Wu, Naledi Shologu, Aniket Kshirsagar, Rufus Raghunath, Anne M. Mullen, Yves Bayon, Abhay Pandit, Michael Raghunath, and Dimitrios I. Zeugolis. "The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development." Advanced Materials 31, no. 1 (2018).
. Proceedings of the National Academy of Sciences.
Minh Thuy, Le Thi, Emiko Okazaki, and Kazufumi Osako. "Isolation and Characterization of Acid-Soluble Collagen from the Scales of Marine Fishes from Japan and Vietnam." Food Chemistry 149 (2014): 264-70.
Zhai, Xiaofei, Xinrong Geng, Wenjun Li, Hongli Cui, Yunqing Wang, and Song Qin. "Comprehensive Review on Application Progress of Marine Collagen Cross-Linking Modification in Bone Repairs." Marine Drugs 23, no. 4 (2025).
Silva, Tiago, Joana Moreira-Silva, Ana Marques, Alberta Domingues, Yves Bayon, and Rui Reis. "Marine Origin Collagens and Its Potential Applications." Marine Drugs 12, no. 12 (2014): 5881-901.
Venkatesan, Jayachandran, Sukumaran Anil, Se-Kwon Kim, and Min Shim. "Marine Fish Proteins and Peptides for Cosmeceuticals: A Review." Marine Drugs 15, no. 5 (2017).
Karakeçili, Ayşe, Serdar Korpayev, and Kaan Orhan. "Optimizing Chitosan/Collagen Type I/Nanohydroxyapatite Cross-Linked Porous Scaffolds for Bone Tissue Engineering." Applied Biochemistry and Biotechnology 194, no. 9 (2022): 3843-59.
Gallo, Nunzia, Alberta Terzi, Teresa Sibillano, Cinzia Giannini, Annalia Masi, Alessandro Sicuro, Federica Stella Blasi, Angelo Corallo, Antonio Pennetta, Giuseppe Egidio De Benedetto, Francesco Montagna, Alfonso Maffezzoli, Alessandro Sannino, and Luca Salvatore. "Age-Related Properties of Aquaponics-Derived Tilapia Skin (Oreochromis Niloticus): A Structural and Compositional Study." International Journal of Molecular Sciences 24, no. 3 (2023).
Coppola, Daniela, Maria Oliviero, Giovanni Andrea Vitale, Chiara Lauritano, Isabella D’Ambra, Salvatore Iannace, and Donatella de Pascale. "Marine Collagen from Alternative and Sustainable Sources: Extraction, Processing and Applications." Marine Drugs 18, no. 4 (2020).
Li, Qing, Lanlan Mu, Fenghua Zhang, Yue Sun, Quan Chen, Cuicui Xie, and Hongmei Wang. "A Novel Fish Collagen Scaffold as Dural Substitute." Materials Science and Engineering: C 80 (2017): 346-51.
Ran, Yaqin, Wen Su, Lei Ma, Xiaoliang Wang, and Xudong Li. "Insight into the Effect of Sulfonated Chitosan on the Structure, Rheology and Fibrillogenesis of Collagen." International Journal of Biological Macromolecules 166 (2021): 1480-90.
"<Sds方法cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4.Pdf>."
Vedhanayagam, Mohan, Anandasadagopan Suresh kumar, Balachandran Unni Nair, and Kalarical Janardhanan Sreeram. "Dendrimer-Functionalized Metal Oxide Nanoparticle-Mediated Self-Assembled Collagen Scaffold for Skin Regenerative Application: Function of Metal in Metal Oxides." Applied Biochemistry and Biotechnology 194, no. 1 (2021): 266-90.
He, Xiong, Lin Lin, Shaotong Jiang, and Jianfeng Lu. "Characterization of Acid-Soluble Collagen (Asc) Derived from Walleye Pollock and Silver Carp Skin and Comparison Them with the Collagen from Pig and Duck Skin." Food Chemistry Advances 4 (2024).
Veeruraj, Anguchamy, Muthuvel Arumugam, Thangappan Ajithkumar, and Thangavel Balasubramanian. "Isolation and Characterization of Collagen from the Outer Skin of Squid (Doryteuthis Singhalensis)." Food Hydrocolloids 43 (2015): 708-16.
Cadenaro, Milena, Luca Fontanive, Chiara Ottavia Navarra, Pietro Gobbi, Annalisa Mazzoni, Roberto Di Lenarda, Franklin R. Tay, David H. Pashley, and Lorenzo Breschi. "Effect of Carboidiimide on Thermal Denaturation Temperature of Dentin Collagen." Dental Materials 32, no. 4 (2016): 492-98.
Charoenchokpanich, Wiriya, Pratchaya Muangrod, Sittiruk Roytrakul, Vilai Rungsardthong, Benjamaporn Wonganu, Sawanya Charoenlappanit, Federico Casanova, and Benjawan Thumthanaruk. "Exploring the Model of Cefazolin Released from Jellyfish Gelatin-Based Hydrogels as Affected by Glutaraldehyde." Gels 10, no. 4 (2024).
Shen, Lirui, Honghong Bu, Huan Yang, Wentao Liu, and Guoying Li. "Investigation on the Behavior of Collagen Self-Assembly in Vitro Via Adding Sodium Silicate." International Journal of Biological Macromolecules 115 (2018): 635-42.
Balikci, Elif, Erkan Türker Baran, Aydin Tahmasebifar, and Bengi Yilmaz. "Characterization of Collagen from Jellyfish Aurelia Aurita and Investigation of Biomaterials Potentials." Applied Biochemistry and Biotechnology (2024).
De Meutter, Joëlle, and Erik Goormaghtigh. "Searching for a Better Match between Protein Secondary Structure Definitions and Protein Ftir Spectra." Analytical Chemistry 93, no. 3 (2020): 1561-68.
Karabchevsky, Alina. "Single-Shot Circular Dichroism Spectroscopy." Light: Science & Applications 11, no. 1 (2022).
Miles, A. J., and B. A. Wallace. "Circular Dichroism Spectroscopy of Membrane Proteins." Chemical Society Reviews 45, no. 18 (2016): 4859-72.
Sloseris, Daniel, and Nancy R. Forde. "Ageing of Collagen: The Effects of Glycation on Collagen’s Stability, Mechanics and Assembly." Matrix Biology 135 (2025): 153-60.
Thuanthong, Mantaka, Nualpun Sirinupong, and Wirote Youravong. "Triple Helical Structure of Acid‐Soluble Collagen Derived from Nile Tilapia Skin as Affected by Extraction Temperature." Journal of the Science of Food and Agriculture 96, no. 11 (2016): 3795-800.
Drzewiecki, Kathryn E., Daniel R. Grisham, Avanish S. Parmar, Vikas Nanda, and David I. Shreiber. "Circular Dichroism Spectroscopy of Collagen Fibrillogenesis: A New Use for an Old Technique." Biophysical Journal 111, no. 11 (2016): 2377-86.
Zheng, Yan, Yushuang Li, Cong Ke, Xiyuan Gao, Zhiyu Liu, and Junde Chen. "Comparison of Structural and Physicochemical Characteristics of Skin Collagen from Chum Salmon (Cold-Water Fish) and Nile Tilapia (Warm-Water Fish)." Foods 13, no. 8 (2024).
Meshcheryakova, Olga V., Maxim A. Bogdanov, and Alexander V. Efimov. "Relationship between Thermal Stability of Collagens and the Fraction of Hydrophobic Residues in Their Molecules." Journal of Structural Biology 216, no. 3 (2024).
de Vasconcelos, Jamile Maria Pereira Bastos Lira, Robson Coelho de Araújo Neri, Amanda Vieira de Barros, Carlos Eduardo Sales da Silva, Maria Cecília Ferreira Galindo, Bruno Oliveira de Veras, Ranilson Souza Bezerra, and Maria Betânia Melo de Oliveira. "Goat Skin (Capra Aegagruss Erxleben, 1777): A Promising and Sustainable Source of Collagen." Applied Biochemistry and Biotechnology (2025).
Cao, Lin, Julieth Joram Majura, Lu Liu, Wenhong Cao, Zhongqin Chen, Guoping Zhu, Jialong Gao, Huina Zheng, and Haisheng Lin. "The Cryoprotective Activity of Tilapia Skin Collagen Hydrolysate and the Structure Elucidation of Its Antifreeze Peptide." Lwt 179 (2023).
Hassan, Mahjabeen, Tehreem Kanwal, Amna Jabbar Siddiqui, Arslan Ali, Dilshad Hussain, and Syed Ghulam Musharraf. "Authentication of Porcine, Bovine, and Fish Gelatins Based on Quantitative Profile of Amino Acid and Chemometric Analysis." Food Control 168 (2025).
Sheng, Mingzhu, Filippo Silvestrini, Malgorzata Biczysko, and Cristina Puzzarini. "Structural and Vibrational Properties of Amino Acids from Composite Schemes and Double-Hybrid Dft: Hydrogen Bonding in Serine as a Test Case." The Journal of Physical Chemistry A 125, no. 41 (2021): 9099-114.
Akita, Monami, Yuri Nishikawa, Yuya Shigenobu, Daisuke Ambe, Takami Morita, Katsuji Morioka, and Kohsuke Adachi. "Correlation of Proline, Hydroxyproline and Serine Content, Denaturation Temperature and Circular Dichroism Analysis of Type I Collagen with the Physiological Temperature of Marine Teleosts." Food Chemistry 329 (2020).
"<Marinedrugs-23-00190.Pdf>."
Tian, Zhenhua, Wentao Liu, and Guoying Li. "The Microstructure and Stability of Collagen Hydrogel Cross-Linked by Glutaraldehyde." Polymer Degradation and Stability 130 (2016): 264-70.
Fields, Gregg B. "Interstitial Collagen Catabolism." Journal of Biological Chemistry 288, no. 13 (2013): 8785-93.

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Characterization and Biological Performance of Anglerfish Collagen and Bovine Collagen

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Abstract

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1 Introduction

2 Materials and methods

2.1 Materials and reagents

2.2 Collagen extraction

2.3 Preparation of collagen

2.4 Physicochemical characterization

2.5 Cytocompatibility assays

2.6 Statistical analyses

3 Results and disscusion

3.1 Characterization of Collagen

3.2 Cytocompatibility assays

4 Conclusion

Declarations

References

Supplementary Files

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