Irradiation Enhances the Biomedical Functional Characteristics of Collagen Sponges: A Potential Strategy for Medical Collagen Sponge Modification

Abstract

Developing safe and effective hemostatic materials is critical for rapid bleeding control and wound management. However, traditional hemostatic materials using chemical crosslinking often fall short in hemostatic efficiency and carry risks of secondary injury from reagent residues. This study introduced an irradiation-fabricated composite collagen sponge based on fish skin collagen, chitosan, and soluble starch. The sponge was prepared via material solution blending, followed by cobalt-60 gamma irradiation at various doses, with casting and freeze-drying. Its functionality and safety were systematically evaluated. The results show that low-dose gamma irradiation (1–3 kGy) applied to a precursor solution prior to freeze-drying promoted intermolecular crosslinking, improving mechanical strength, elongation, and biostability, while higher doses (6 kGy) slightly reduced crosslinking due to the partial degradation of collagen, chitosan, and starch. With low-dose irradiation, the proposed hemostatic sponges show enhanced water absorption, blood cell adsorption, swelling, and antibacterial properties, indicating effective hemostatic performance. Spectroscopic characterization confirmed chemical bond modifications with no loss of crystallinity. Cytotoxicity and in vivo tests demonstrated biocompatibility and effective hemostatic performance. Compared with the commercial HSD sponge, the irradiated sponges exhibited superior hemostatic efficacy. This study presents that a collagen-based synergistic matrix prepared by gamma-ray irradiation can produce a hemostatic sponge with enhanced absorbency, bioactivity, and antibacterial properties, highlighting its great potential in rapid hemostasis and wound care applications.

1. Introduction

The global fishery industry produces vast quantities of by-products, including fish skin, scales, bones, and crustacean shells, which are frequently discarded, leading to significant resource loss and environmental burden [1,2]. Transforming these underutilized materials into high-value biomaterials offers a sustainable approach to resource recycling and pollution mitigation. Among them, fish skin and crustacean shells are rich in collagen and chitosan—two naturally derived macromolecules with excellent biocompatibility, biodegradability, and safety profiles, providing ideal platforms for biomedical applications [3,4,5].

Fish collagen has attracted increasing attention as a biopolymer alternative to mammalian collagen due to its low immunogenicity and cultural acceptability [6,7]. Chitosan, derived from chitin, exhibits intrinsic antibacterial and hemostatic properties and promotes tissue regeneration. Combining these two polymers enables the design of bioactive materials that integrate structural strength, hydrophilicity, and biofunctionality, making them particularly suitable for wound management [8]. However, most single-component or physically blended hemostatic materials exhibit poor mechanical stability and limited coagulation efficiency, primarily due to weak intermolecular interactions [9,10,11]. Hence, establishing an efficient and non-toxic crosslinking strategy is essential to enhance the structural and biological performance of composite hemostatic materials.

Irradiation-induced crosslinking, particularly through gamma ray irradiation, has emerged as a promising green technology for modifying natural polymers [12]. Different irradiation modalities, such as electron beams and X-rays, have been employed to induce crosslinking through free-radical reactions without the use of chemical crosslinkers. Electron beam irradiation enables rapid processing with precise dose control; however, its limited penetration depth restricts its applicability mainly to thin or low-density materials, such as pectin-based hydrogels for drug delivery [13]. Electron beam irradiation of fish skin gelatin typically requires relatively high doses (e.g., 30–60 kGy) [14]. X-ray irradiation, with its moderate penetrating power and tunable energy, can be used to identify crystal changes and control structural modifications, and has been reported in applications such as the extraction of biocompatible hydroxyapatite from fish scales [15]. Furthermore, X-rays provide structural identification at the atomic and nanoscale of fish collagen, rather than molecular crosslinking [16]. Gamma ray irradiation exhibits exceptionally high penetration depth and offers distinct advantages over other irradiation modalities for the modification of biomacromolecules such as collagen and gelatin [17]. Irradiation-induced crosslinking has been widely investigated in collagen and chitosan systems. Previous studies have shown that γ-irradiation can generate stable crosslinked structures between collagen molecular chains without the use of chemical crosslinkers, thereby improving mechanical strength and resistance to enzymatic degradation [18]. Similarly, in chitosan-based systems, irradiation promotes the formation of intermolecular networks via free-radical reactions, enabling regulation of swelling behavior and biological stability [19,20]. For fish skin-derived materials, researchers have compared the effects of γ-irradiation on collagen in three physical states: native skin tissue, collagen solution, and collagen sponge. The results indicate that collagen solution is the most irradiation-sensitive form, exhibiting greater potential for crosslinking [21]. However, sponges prepared solely from irradiated collagen solutions often display low porosity, dense structure, excessive stiffness, and limited fluid absorption capacity, which restrict their direct application in practical products such as hemostatic sponges. Therefore, further studies are warranted to develop functional hemostatic sponges via fish skin collagen irradiation.

Starch is a hydrophilic polysaccharide with excellent biodegradability, biocompatibility, and fluid absorption capacity, and can serve as an effective structural and functional modulator in collagen-based systems. Owing to these properties, starch has been widely incorporated into biomacromolecular matrices to enhance performance; for example, blending starch with biopolymers such as gelatin or collagen can increase water uptake, structural integrity, and crosslinking density while tuning mechanical strength and degradation behavior [22]. In fish collagen systems, starch and its derivatives exhibit additional advantages [23]. Dialdehyde starch has been reported to crosslink fish-skin collagen with chitosan, producing hydrogels with improved mechanical properties and enhanced wound-healing potential. Modified starch nanoparticles can further reinforce collagen networks and promote cell aggregation and proliferation [24]. Chitosan, a cationic polysaccharide derived from chitin, possesses intrinsic antibacterial activity and film-forming ability [25]. Therefore, the integration of chitosan and soluble starch with fish-skin collagen is expected to synergistically enhance mechanical performance, biological stability, and bioactivity through intermolecular interactions and complementary functionality.

In this study, a novel design of mechanically stable, rapidly absorbent, and highly biocompatible hemostatic sponges was investigated based on the γ-irradiation followed by freeze-drying of fish skin-derived collagen material. Collagen extracted from fishery by-products was combined with chitosan and starch to form a bio-derived precursor system, in which starch served as a structural regulator and auxiliary crosslinking component to improve network formation and fluid absorption. γ-irradiation followed by freeze-drying was then applied to generate a porous three-dimensional architecture while simultaneously enhancing mechanical strength and biological stability. The resulting sponges were systematically characterized for physicochemical properties, swelling capacity, antibacterial performance, cytocompatibility, and in vivo hemostatic efficacy. The key innovations of this study include the design of a collagen–chitosan–starch synergistic matrix to improve mechanical strength, fluid absorption, and bioactivity, and the use of γ-irradiation followed by freeze-drying to enable stable crosslinking with uniform pores. It provides a paradigm for the reuse of fishery by-products in the design of high-performance, biocompatible, and antibacterial hemostatic sponges, which could be used in wound care and surgical applications.

2. Materials and Methods

2.1. Materials and Reagents

The descaled skin of Tilapia (Oreochromis niloticus) was obtained from Guangzhou Lushi Food Co., Ltd., Guangzhou, China. The skin was washed and stored in a −80 °C refrigerator until used. Soluble starch (CAS: 9005-84-9, 21.8% amylose content; purity ≥ 99.3%, formula weight: 342.3) and chitosan (deacetylation degree: ≥95%, molecular weight 200,000, viscosity 100–200 mPa·s) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Soluble starch, a biocompatible and hydrophilic polysaccharide, was incorporated into the sponge formulation as a functional polysaccharide component. Commercial collagen sponge was purchased from Nanchang Hushida Medical Technology Co., Ltd., Nanchang, China. Citrated sheep whole blood (1:9 ratio of sodium citrate to blood) was acquired from Henan Yuechi Biotechnology Co., Ltd., Zhengzhou, China. All other chemicals and reagents were of analytical grade.

2.2. Extraction of Tilapia Skin Collagen

Acid soluble collagen was extracted from Tilapia skin, and all procedures were carried out at 4 °C. Initially, the skin pieces were soaked in a 0.1 M NaOH containing 0.5% (v/v) Tween 80 for 24 h to remove noncollagenous proteins and pigment, changing the solution every 8 h, followed by washing with chilled distilled water. Subsequently, the skins were immersed in 8% (v/v) butyl alcohol for 24 h to remove the fatty composition and then washed again. The treated skins were then mixed with 0.5 M of acetic acid at a solid to liquid ratio of 1:40 (w/v) for 48 h with a constant stirring. The mixture was filtered through gauze and centrifuged at 8000× g for 20 min at 4 °C using a Lynx 6000 high-speed floor centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was salted out with NaCl to a final concentration of 2.0 M, followed by centrifugation under the same conditions. The collagen pellet was redissolved in 0.5 M acetic acid, dialyzed using dialysis membranes (MW: 8–14 kDa) against 0.1 M acetic acid for 24 h and followed by distilled water until the wash water was neutral. Both dialysates were refreshed every 8 h. Finally, the dialysate was lyophilized using a freeze-dryer (Scanvac Cool Safe, Vassingerød, Denmark). The obtained collagen was stored at −80 °C until used.

2.3. Fabrication of Collagen–Chitosan Sponge and Co-60 γ-Ray Irradiation

The collagen sponge was prepared and irradiated via the following steps: 1.5 g of collagen from tilapia skin was dissolved in 150 mL of 0.5 M acetic acid and stirred continuously for 2 h at 4 °C. Separately, 1.0 g of chitosan was dissolved in 80 mL of 0.5 M acetic acid and stirred for 3 h until completely dissolved. The two solutions were combined, and 5 mL of glycerin was added and stirred thoroughly to form the collagen–chitosan solution. Additionally, 0.5 g of soluble starch was dissolved in 20 mL of distilled water (heated to 95 °C and stirred to dissolve within 3 min), cooled to room temperature, and added to the collagen–chitosan solution to form the collagen–chitosan–starch solution. Then, the two composite solutions were poured into a culture dish and pre-frozen at −20 °C for 12 h, frozen in an ultra-low-temperature refrigerator at −80 °C for 8 h, and then freeze-dried in a freeze-dryer (Scanvac Cool Safe, Vassingerød, Denmark) to produce the collagen sponges, which are named CC and CCS, respectively. In addition, the collagen–chitosan–starch composite solution was irradiated with an ice pack at Guangzhou Furui High Energy Technology Co., Ltd., Guangzhou, China, with an irradiation dose rate of 1.2 kGy/h; specific irradiation times of 0.83, 2.52, and 4.95 h; and final doses of 0.99, 3.03, and 5.93 kGy, respectively. After irradiation, the solution was freeze-dried to prepare collagen sponges, which were recorded as CCS-1, CCS-3, and CCS-6, respectively (Figure 1A). Unirradiated collagen sponges (CCS) and commercially available collagen sponges (HSD) from Nanchang Huashida Medical Technology Co., Ltd. (Nanchang, China) were used as controls.

2.4. Appearance Profile and Surface Color

The surface color of the collagen sponge sample was measured using a Minolta CR 400 Series colorimeter (Chiyoda, Tokyo, Japan), with a standard calibration (L = 96.51, a = −0.46, b = 3.19) [26]. All samples were analyzed in triplicate, with six measurements recorded for each sample. The total color difference (ΔE) was calculated as follows (Equation (1)):

$$∆E=\sqrt{{\left(∆L\right)}^2+{(∆a)}^2+{(∆b)}^2}$$

(1)

where ΔL, Δa, and Δb are the difference between each standard color plate color value and the collagen sponge specimen, respectively.

2.5. Characterization of Collagen Sponges

The microstructure of the CC, CCS, CCS-1, CCS-3, CCS-6, and HSD sponges was observed using a Zeiss scanning electron microscope (SEM) (Merlin, Oberkochen, Germany). The X-ray diffraction of the samples was recorded using an X-ray diffractometer (Empyrean, Malvern Panalytical, Almelo, The Netherlands) at a scanning rate of 5°/min and at a diffraction angle of 5–70°. The zeta potential was measured using a Nano ZS zeta potential analyzer (SZ-100V2, HORIBA, Kyoto, Japan). The static water contact angle of the collagen sponges was recorded at 25 °C using a contact angle analyzer (Kruss-DSA25, KRÜSS GmbH, Hamburg, Germany). All measurements were performed on droplets with a volume of approximately 10 μL. The chemical structures of sponges were characterized using a Fourier transform infrared (FTIR) spectrometer (Nicolet IS 50, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) in the wavenumber range of 4000–400 cm⁻¹. The thermodynamic stability was analyzed using a thermogravimetric analyzer (STA 449 F5, NETZSCH, Selb, Germany). About 10 mg of sponge was placed in an Al₂O₃ crucible and heated from 30 to 700 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. The first derivative of TG (DTG) was also analyzed using ORIGIN software (2025 Student Version, OriginLab Corporation, Northampton, MA, USA) [27].

2.6. Crosslinking Degree Detection

The crosslinking degree of the collagen sponge was determined by the method reported by Sun et al. with slight modifications [28], based on the principle of using a 2,4,6-trinitro-benzene-sulfonic acid (TNBS) assay to determine the content of free primary amine groups in the initial non-cross-linked sample and the irradiated cross-linked sample. Briefly, 5 mg of cross-linked sponge sample was added to 1 mL of 4% (w/v) NaHCO₃ solution for 30 min, with shaking every 10 min. Then, 1 mL of freshly prepared 0.5% (w/v) TNBS solution was added and incubated in a water bath at 40 °C for 2 h. After that, 3 mL of 6 M HCl was added, and the sample was hydrolyzed at 60 °C for 90 min. The resulting solution was diluted with 4 mL of deionized water and cooled to room temperature. The absorbance was measured at 345 nm using a UV-1206 spectrophotometer (Shimadzu, Kyoto, Japan). Controls (blank samples) were prepared using the same procedure, except that HCl was added prior to introducing the TNBS solution to prohibit any reaction of TNBS with the amine groups. The sponge crosslinking degree was calculated according to Equation (2):

$$Degree of cross-linking (\%)=(1-{\displaystyle \frac{{OD}_s-{OD}_b}{{OD}_n-{OD}_b}})\times 100$$

(2)

where OD_s, OD_n, and OD_b denote the absorbance of the irradiated samples, non-irradiated collagen sponges, and blank control group, respectively.

2.7. Mechanical Properties

The mechanical properties of the collagen sponge sample, including tensile strength (TS), elongation at break (EB), and Young’s modulus (YM), were tested using a universal material-testing machine (Model 5967, Instron Engineering Corporation, Canton, MA, USA) according to standard ASTM method D 882-88. For measurement, samples were precisely cut into rectangular pieces (2.0 cm × 4.0 cm). The machine was operated in tensile mode with a crosshead speed of 10 mm/min, a load of 100 N, and an initial clamp separation of 15 mm. The TS and EB of the collagen sponge were calculated as follows (Equations (3) and (4)):

$$TS \left(\mathrm{M}\mathrm{P}\mathrm{a}\right)={\displaystyle \frac{F_{max}}{A}}$$

(3)

$$EB \left(\%\right)={\displaystyle \frac{∆L}{L_0}}\times 100\%$$

(4)

where F_max is the maximum force to break the sponge (N), A is the cross-sectional area of the sponge (m²), ΔL is the increased sponge length at the break, and L₀ is the initial length of the sponge (15 mm). The YM (MPa) was determined from the slope of the initial linear portion of the stress–strain curve, which corresponds to the stress divided by the strain of the sponge sample.

2.8. In Vitro Degradation Property

To evaluate the in vitro degradation ability of the sponge, a certain amount of sample was immersed in 10 mL PBS (pH 7.4), lysozyme was added to a final concentration of 1 mg/mL, the mixture was incubated at 37 °C and shaken once a day for 2 min, and a new solution was replaced every 3 days.

The samples were freeze-dried and weighed at the determined time points (1, 3, 6, 9, 12, 15, 18, and 21 days), and each group of samples was repeated 3 times. The degradation rate was determined according to the following formula (Equation (5)):

$$Weight loss \left(\%\right)={\displaystyle \frac{W_1-W_2}{W_1}}\times 100\%$$

(5)

where W₁ and W₂ are the initial and final weights of the sponge after incubation, respectively.

2.9. Antimicrobial Activity

The antibacterial experiment used Gram-negative E. coli and Gram-positive S. aureus as model microorganisms, and samples with different treatments were used as antibacterial experimental materials. The bacterial suspension was cultured in Luria–Bertani (LB) medium and activated at 37 °C 150 rpm/min for 12–18 h. Then, 20 mg of the sponge sample that had been UV-irradiated for 18 h was co-incubated with the bacterial suspension with an initial concentration of 10⁶ CFU/mL for 5 h. Finally, the bacterial suspension diluted to an appropriate multiple was inoculated on the LB agar plate, and the number of bacteria was observed and counted after 18 h of culture.

$$Inhibition rate \left(\%\right)={\displaystyle \frac{A_0-A_1}{A_0}}\times 100\%$$

(6)

where A₀ and A₁ are the number of bacteria in blank and sample groups, respectively.

2.10. Hemolytic Test of Sponge

Hemolysis experiments were conducted using fresh New Zealand rabbit blood, purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China; Cat. No. MP20025-100 mL). Then, 5 mg of collagen sponge was soaked in 10 mL of 0.9% NaCl solution under sterile conditions, incubated at 37 °C for 72 h, and centrifuged to obtain the sample extract. The diluted anticoagulated blood was prepared by mixing 5.0 mL of fresh rabbit blood, 1.0 mL of anticoagulant acid citrate dextrose solution, and 6.0 mL of 0.9% (w/v) NaCl aqueous solution. A total of 5 mL of sample extract was mixed with 5 mL of diluted blood. The mixture was incubated at 37 °C for 60 min and then centrifuged at 1000 rpm for 5 min. The absorbance of the supernatant was measured at 545 nm using a UV–visible spectrophotometer (Shimadzu, Kyoto, Japan). Deionized water and 0.9% (w/v) NaCl solution were used as positive and negative controls, respectively. The hemolysis rate of the samples was calculated according to the following formula (Equation (7)):

$$Hemolysis rate \left(\%\right)={\displaystyle \frac{{OD}_s-{OD}_n}{{OD}_p-{OD}_n}}\times 100\%$$

(7)

where OD_s, OD_n, and OD_p are the absorbance values of the samples, negative controls, and positive controls, respectively.

2.11. Cytocompatibility

The in vitro biocompatibility of the collagen sponges was evaluated via the MTT method [7]. All collagen sponges were sterilized by high pressure and immersed in Dulbecco’s Modified Eagle Medium (DMEM) medium at 37 °C for 24 h to obtain 1 and 1.5 mg/mL sponge extract medium, respectively. Mouse fibroblast cell line L929 was placed in DMEM culture flasks containing 10% fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin mixture and cultured at 37 °C and 5% CO₂ atmosphere for 3 d. Cells (3 × 10⁴ mL⁻¹) were incubated with conditioned medium (sponge extract) in 12-well plates for 24 or 48 h, and 200 μL of MTT solution (5 mg/mL in DPBS) and 1.8 mL of DMEM were added to the plate and incubated in a 37 °C 5% CO₂ incubator for 3 h. After that, the MTT solution was discarded, 2 mL of dimethyl sulfoxide (DMSO) was added, and the plate was shaken for 10 min to promote dissolution. Then the absorbance was recorded at 490 nm. The relative growth rate (RGR) was calculated as follows (Equation (8)):

$$RGR \left(\%\right)={\displaystyle \frac{{OD}_s}{{OD}_c}}\times 100\%$$

(8)

where OD_s and OD_c are the absorbance of samples and blank control, respectively.

2.12. Coagulation Test

Briefly, 50 μL of sterile anticoagulated defibrinated sheep whole blood was mixed with 5 μL of 0.2 M CaCl₂ solution and dropped onto a preheated sponge (1 × 1 × 0.5 cm³) at 37 °C, gently shaken, and incubated for a certain time (30, 60, 90, and 120 s). Then 10 mL of deionized water was added to release the uncoagulated blood but not to disrupt the formed blood clot. The sample was then incubated at 37 °C for 1 h. At the same time, the coagulation performance of a commercially available collagen sponge (HSD) was evaluated as a control. A total of 50 μL of whole blood was directly added to 10 mL of deionized water as a negative control. A total of 200 μL of the supernatant was taken, and the absorbance at 542 nm was measured using a Spectramax Plus 384 microplate reader (San Jose, CA, USA). The blood coagulation index (BCI) of the sponge was evaluated by the following formula (Equation (9)).

$$BCI \left(\%\right)={\displaystyle \frac{A_s-A_w}{A_n-A_w}}\times 100\%$$

(9)

where A_s, A_w, and A_n are the absorbance values of samples, deionized water, and negative controls, respectively.

2.13. In Vivo Hemostasis Performance Test

The animal experiments in this study were approved by the Management Committee of the Medical Laboratory Center of South China University of Technology (approval number: 2024052). All animals were purchased and provided by the Animal Experiment Center of the School of Medicine. Animal welfare and ethics requirements were set out by the National Research Council’s Guide for the Care and Use of Laboratory Animals. All procedures were carried out strictly in accordance with the requirements and standards of animal ethics experiments. Male SD rats (300–400 g) were selected as the femoral artery injury model. The hair on the hind legs of the rats was removed to expose the skin of the thigh groin. A scalpel was used to make an incision and cut off the femoral artery to cause heavy bleeding for the experiment. A collagen sponge sterilized with ultraviolet light was applied to the wound to ensure that the sponge completely covered the wound and applied a force of 200 g. The coagulation was observed every 15 s until the bleeding stopped completely. The coagulation time was recorded, and the blood loss was calculated after weighing the hemostatic material. The wound was photographed and recorded. Following the completion of the hemostasis experiment, the rats were humanely euthanized via intravenous administration of an overdose of anesthetic agents. All euthanasia procedures and subsequent handling were conducted in accordance with institutional animal care guidelines and were performed uniformly by the animal facility staff.

2.14. Statistical Analysis

All experiments were independently repeated at least three times, and the data are expressed as the mean ± SD, having been analyzed using SPSS software (version 26.0, SPSS Inc., Chicago, IL, USA), followed by Duncan’s multiple range test and the independent sample t-test. A p value < 0.05 was considered statistically significant.

3. Results

3.1. Morphological Changes and Molecular Interactions of Collagen Sponges Under Irradiation

The composite collagen sponge composed of fish skin collagen, chitosan, and/or soluble starch, cross-linked by cobalt-60 gamma ray irradiation, is shown in Figure 1. The average thickness of the sponge is 4.0 ± 0.258 mm, and the diameter is 6.0 ± 0.103 cm.

It can be seen from the figure that the main difference between the collagen sponges treated with irradiation is that the color changes from white to yellow, and the greater the irradiation dose, the darker the yellow. The color parameters of different collagen sponges were measured (

Table 1. Color parameters of collagen sponges with/without irradiation (Means ± SD, n = 5).

	L	a	b	ΔL	Δa	Δb	ΔE
HSD	76.64 ± 1.22 c	−0.45 ± 0.01 d	5.79 ± 0.18 d	−15.07 ± 1.22 b	0.55 ± 0.01 c,d	1.59 ± 0.18 e	15.17 ± 1.2 c
CC	86.82 ± 2.1 a	−0.15 ± 0.53 e	14.85 ± 0.56 c	−4.07 ± 2.91 d	0.6 ± 0.32 c	10.64 ± 0.56 d	11.86 ± 1.04 e
CCS	79.83 ± 1.8 b	−0.91 ± 0.2 b	13.93 ± 0.92 c	−11.88 ± 1.8 c	0.09 ± 0.2 e	9.72 ± 0.92 d	15.43 ± 1.19 c
CCS−1	86.54 ± 0.63 a	−0.6 ± 0.12 c	16.59 ± 0.42 b	−5.17 ± 0.63 d	0.4 ± 0.12 d	12.38 ± 0.42 c	13.43 ± 0.46 d
CCS−3	80.84 ± 8.61 b	0.43 ± 0.45 d	20.71 ± 1.06 a	−10.87 ± 8.61 c	1.43 ± 0.45 b	16.51 ± 1.06 b	21.15 ± 3.62 b
CCS−6	71.11 ± 3.42 d	2.82 ± 1.53 a	21.5 ± 1.57 a	−21.71 ± 4.01 a	3.89 ± 1.57 a	17.29 ± 1.58 a	27.25 ± 3.41 a

Means in a column with different letters are significantly different, based on the Tukey test (p < 0.05).

), where ΔL represents the brightness difference (with pure white), Δa represents red-green, and Δb represents yellow-blue. The collagen sponges that have been irradiated and cross-linked show significant differences in values. With the increase in irradiation dose, the ΔL in CCS-1, CCS-3 and CCS-6 sponges decreased significantly, and the Δa and Δb values increased significantly (p < 0.05), indicating that the color of the sponge became reddish and yellowish, which is consistent with the observed sponge color (Figure 1C). The ΔE values measured in the samples were confirmed by color parameter calculations. Irradiation technology is a non-thermal processing technology [29]. During the irradiation process, gamma rays generate some energy that passes through the sponge composite liquid, thereby changing the color of the subsequent sponge. An appropriate dose of irradiation can increase the crosslinking degree of macromolecular substances and change their functional properties [17,30]. However, excessive irradiation could destroy the molecular structure of components such as collagen or chitosan, resulting in lower-quality properties [17,31]. The results of sponge color measurement showed that irradiation changed the state of the sponge, which also indirectly indicated that irradiation cross-links the collagen sponge.

It is worth noting that commercially available HSD sponge was used as a comparative reference due to its similar composition and structure, while no commercial products currently employ γ-ray irradiation, highlighting the novelty of the present approach in improving the structural stability and hemostatic performance of collagen–chitosan sponges.

These morphological changes are closely related to the interactions between molecules. Generally, chitosan and collagen both carry positive charges under acidic conditions. As a result, their mutual interaction may be hindered by electrostatic repulsion. However, adjusting the pH or using physical and chemical modifications can enhance their interactions. Similarly, neutral starch does not exhibit significant electrostatic interactions with positively charged chitosan or collagen; their combination primarily relies on physical mixing and non-covalent interactions, such as hydrogen bonding. Nonetheless, physical blending can create composite materials. Modifications to chitosan or collagen, such as via irradiation to introduce functional groups, may enhance their interaction with starch.

FTIR spectroscopy can reveal structural changes in collagen and collagen sponges at the molecular level, which is characterized by displacement and intensity changes in specific spectral peak regions (Figure 2A).

The overall spectral shape of the collagen sponge is similar to that of pure collagen. The main difference between the commercial sponge HSD and the collagen sponge is at the wavelength of 1300–1000 cm⁻¹—that is, the C-O stretching vibration. In addition, the addition of soluble starch in the CCS collagen sponge reduced the intensity of the C=O stretching vibration of amide I (1700–1600 cm⁻¹) and enhanced the N–H and C–N stretching vibration intensity of amide II (1590–1500 cm⁻¹), indicating that the soluble starch was combined with collagen and chitosan. After irradiation, the peaks of amide I and amide II of the composite collagen sponge (1700–1500 cm⁻¹) moved to high wavenumbers, and the vibration intensity increased with the increase in irradiation dose. This shift is due to the change in the chemical bond environment between the molecules. A magnified version of the FTIR spectrum is shown in Figure S1. Previous studies have shown that gamma ray fishery collagen irradiation leads to a significant increase in the carbonyl content (C=O) of collagen fiber molecules [32,33]. During γ-ray irradiation, high-energy photons induce ionization and excitation processes, resulting in the generation of macromolecular radicals through hydrogen abstraction and bond cleavage. These radicals can subsequently undergo recombination reactions, leading to intermolecular crosslinking. In collagen-based systems, carbonyl-containing groups (–C=O) and N–H groups from amide structures are generally regarded as major reactive sites involved in radical-mediated reactions. The recombination of these radicals is commonly believed to form non-specific covalent linkages, such as C–C, C–N, and, to a lesser extent, C–O bonds, thereby generating a more stable and interconnected three-dimensional network. Carbonyl is a polar functional group that can participate in hydrogen bonds and other polar interactions, which is very important in biomolecules and organic chemistry [34]. XRD testing is mainly used to study the crystal structure and phase analysis of biomaterials. The crystallinity of the material can be evaluated by analyzing the intensity and width of the diffraction peak. XRD spectra showed that all collagen sponges had two diffraction peaks at 6° and 22° (Figure 2B), which proved that collagen, chitosan, and starch materials were successfully composited. In addition, irradiation treatment did not significantly affect the crystallinity and particle size of the sponges but increased the width of the diffraction peak at 22°, demonstrating that irradiation made the structure of the sponge looser.

The zeta potential of each composite collagen sponge is shown in Figure 2C. The zeta potentials of CC and CCS, composed of positively charged collagen and chitosan, were 8.9 and 12.3 mV, respectively. The addition of starch did not significantly increase the potential of the sponges. The zeta potentials of CCS-1, CCS-3, and CCS-6 were 39.0, 40.5, and 40.7 mV, respectively, indicating that irradiation can increase the positive charge of the sponge, but it is not dose-dependent. Studies have found that due to the presence of glycoproteins and glycolipids on the surface of the cell membrane, blood cells (such as red blood cells, white blood cells, and platelets) in the blood are usually negatively charged under physiological conditions [35,36]. Positively charged collagen sponges are more likely to adsorb negatively charged blood cells in the blood and stop bleeding quickly. On the other hand, the contact angle measurement showed that the commercial sponge HSD had the largest water contact angle (Figure S2), indicating that it was not easy to absorb liquid when it first came into contact with the liquid. Both the addition of starch and irradiation treatment can significantly reduce the contact angle (p < 0.05), thereby absorbing liquid moisture more quickly (Figure 2F). However, the lower contact angle of CCS-6 is caused by excessively high doses of irradiation that reduce the crosslinking degree and loose arrangement of the sponge (Figure 2G and Figure 3A), which does not contribute to other excellent properties of the sponge.

The thermal properties analysis of materials is crucial for their application. The thermal stability analysis results for collagen sponges showed that irradiation treatment of composite solutions did not reduce the thermal stability of collagen sponges (Figure 2D). The TG curve of the sponge showed three stages of weight loss in the range of 0–700 °C. The temperature of the first stage was below 210 °C, and the weight loss was about 14.3%, which corresponded to the loss of adsorbed and bound water in the sponge. The temperature of the second stage was about 210 °C to 420 °C, and the weight loss was about 65.4%, which can be attributed to the degradation of the main chain of organic matter such as collagen, chitosan, and starch. The temperature of the last stage was 450 °C to 700 °C, and the weight loss was about 14.6%, which was caused by the oxidative degradation of all organic molecules. The DTG curve showed three obvious degradation peaks at around 72 °C, 212 °C, and 300 °C (Figure 2E). The obvious difference is the degradation peak around 200 °C, where the degradation peak temperatures of CC and CCS were 219 °C and 221 °C, while the peak temperatures of CCS-1, CCS-3, and CCS-6 were 203 °C, 199 °C, and 212 °C, respectively. No degradation peak was observed for the commercial sponge HSD at 200 °C. The difference between the temperatures with the maximum weight loss rate for the irradiated sponge may be that the irradiation treatment increases the porosity of the sponge, which increases the degradation rate of the sponge at this temperature.

The observed sponge structures are shown in Figure 2G. The SEM image of HSD sponge is shown in Supplementary Materials Figure S4. The microstructure of CCS-1 was denser and more compact than that of the other collagen sponges. However, CCS-3 and CCS-6 became loose due to excessive irradiation doses. These observations were consistent with the crosslinking degree performance (Figure 3A). In summary, gamma ray irradiation effectively promoted the interaction between collagen, chitosan, and starch, resulting in a more stable composite matrix. Typically, the preparation of composite materials involves chemical modification or the addition of crosslinking agents to enhance interactions between different components [37]. Our results demonstrated that gamma irradiation can achieve similar or even superior effects, particularly in the preparation of composite biomaterials.

3.2. Degradation and Mechanical Properties of Collagen Sponges Under Irradiation

The crosslinking degree of the irradiated freeze-dried collagen sponge is shown in Figure 3A. The irradiation crosslinking mechanism is shown in Figure S3. After irradiation, the crosslinking degree of CCS-1 collagen sponge reached a maximum of 89.57%, while the crosslinking degree of CCS was only 73.46%, indicating that irradiation treatment significantly increased the crosslinking degree of the collagen sponge (p < 0.05). However, with the increase in irradiation dose, the crosslinking degree of CCS-3 decreased slightly, while that of CCS-6 decreased significantly, even lower than that of CC. These results showed that the composite solution composed of collagen, chitosan and soluble starch can significantly improve the crosslinking of sponge at low doses (1–3 kGy) but is not suitable for high-dose irradiation (greater than 6 kGy). The reason may be that high-dose treatment destroys the molecular structure of collagen fibers and their typical triple helical structure, resulting in a decrease in their adhesion to chitosan, thereby reducing the crosslinking of the collagen sponge [21,38]. In addition, γ-ray irradiation is known to induce degradation of polysaccharides such as chitosan and starch through chain scission reactions. The degradation of these biopolymers at higher doses may further decrease the availability of effective crosslinking sites, resulting in a reduced crosslinking of the composite sponge. The degree of crosslinking of collagen sponges is a very important indicator. Sponges with excellent crosslinking degree provide basic support for their interconnected network and reticular pore structure, and promote the orderly arrangement of sponge molecules, which contributes favorable conditions for biomaterials in hemostasis and wound repair, including rapid absorption of blood plasma, promotion of platelet enrichment, and induction of blood coagulation [39]. In addition, the degree of crosslinking also determines the size of the sponge’s porosity, providing mechanical support for its ability to absorb more blood. The results indicate that the collagen sponge formed by irradiation crosslinking could be regarded as an ideal hemostatic material with beneficial physical properties.

The sponges were degraded in vitro in a simulated physiological environment at 37 °C. The lysozyme degradation test was used to evaluate the biostability of the irradiated cross-linked modified collagen sponges, with weight loss as a quantitative indicator, as shown in Figure 3B. The degradation process of all collagen sponges can be divided into two stages, a slow degradation period (1–6 d) and a rapid degradation period (9–15 d). In the first 6 days, the sponge degradation rate was relatively slow, with a degradation of about 24%. During the rapid degradation period, the weight loss of HSD and CCS-6 increased rapidly, from 63.97% to 98.25% and from 45.43% to 96.71%, respectively. The weight loss of CCS was from 33.87% to 86.70%. Their degradation rates were all over 86%, while the weight losses of CCS-3, CC and CCS-1 were all less than 80%. The results showed that the speed of sponge degradation was related to the degree of sponge crosslinking. The weight loss of the irradiated cross-linked collagen sponge was lower than that of the uncross-linked sponge. The reason may be that the irradiation treatment promoted the internal reaction among collagen, chitosan and starch, covered or blocked the enzyme cleavage sites and reduced the degradation rate [28]. Furthermore, the collagen sponge made by irradiation crosslinking had a longer degradation cycle than the sponge cross-linked by chemical reagents [40], which also indicated that the irradiation method is feasible in the preparation of collagen sponges.

The mechanical properties of collagen sponges depend on the support strength of the biological basic component materials, which is one of the basic and very important indicators. The most commonly used support materials for medical sponges are collagen and chitosan. Collagen has good biological properties, and chitosan has good antibacterial properties. However, natural collagen has the defects of being soft-textured and easy biodegradable, while chitosan-based films, coatings, or dense structures suffer from low-porosity and poor air permeability. In addition, both collagen and chitosan inherently exhibit poor mechanical properties when employed alone, which further limits their practical performance for biomedical applications [26]. In order to overcome these defects, different physical and chemical methods are used to crosslink collagen and chitosan, and different biopolymers are added to obtain good mechanical and biological properties. After mixing collagen, chitosan, and soluble starch, γ-ray irradiation is used to promote intermolecular crosslinking to obtain a composite collagen sponge with high mechanical strength.

Table 2. Mechanical properties of collagen sponges with/without irradiation.

	Maximum Load (N)	Tensile Strength (MPa)	Elongation at Break (%)	Young’s Modulus (MPa)
HSD	2.3252 ± 0.0673 a	0.728 ± 0.0343 a	5.4713 ± 0.4603 d	1.3029 ± 0.1091 a
CC	1.1692 ± 0.1910 e	0.157 ± 0.0229 c	14.7124 ± 0.6455 a	0.1771 ± 0.0642 d
CCS	1.4460 ± 0.0979 c	0.304 ± 0.0293 c	7.5459 ± 0.2189 c	0.2380 ± 0.0071 c
CCS-1	1.7083 ± 0.0712 b	0.402 ± 0.0142 b	13.5239 ± 0.2621 a	0.3839 ± 0.0186 b
CCS-3	1.3712 ± 0.1037 c,d	0.202 ± 0.0166 c	10.2545 ± 0.1034 b	0.2761 ± 0.0049 c
CCS-6	1.2330 ± 0.1607 d,e	0.149 ± 0.0097 c	11.6709 ± 0.4915 b	0.1746 ± 0.0144 d

Means in a column with different letters are significantly different, based on the Tukey test (p < 0.05).

shows the measurement results of the sponge, including maximum load, tensile strength, elongation at break, and Young’s modulus.

The commercial collagen sponge HSD has better mechanical properties, which may be due to the advanced equipment and process, or the addition of other macromolecules that increase strength. The HSD sponge is composed of gelatin and prepared by vertical freeze-drying, which generates a uniform, small-pore structure. This well-interconnected network provides strong mechanical support and facilitates efficient fluid absorption, suitable for hemostatic applications. Excluding HSD, CCS-1 had a higher maximum load of 1.7083 N, followed by CCS, CCS-3, CCS-6, and CC, indicating that irradiation treatment improves the load-bearing capacity of the sponge, which is also consistent with the increase in crosslinking degree. In addition, the maximum load of CCS was significantly higher than that of CC (p < 0.05), which is caused by covalent bond interactions such as hydrogen bonds generated in the collagen, chitosan, and soluble starch formula [40]. The tensile strength also has a similar pattern. CCS-1 irradiated with 1 kGy has a maximum stress of 0.402 MPa. Previous studies have found that the maximum stress of sponges made of pure fish skin collagen is 0.1414 ± 0.0012 MPa, while the maximum force of sponges can reach about 0.18–0.5 MPa after chemical crosslinking or adding biopolymers [37,41,42]. In the case of also increasing the maximum stress, irradiation has the advantages of being simple, efficient, and free of residual reagents compared with the use of crosslinking agents and the addition of additional biopolymers. However, the higher the radiation dose, the better. In fact, a high dose will reduce the maximum stress.

Elongation at break is an indicator of the softness and elasticity of the sponge. Among them, HSD had the lowest elongation at break of 5.7713 ± 1.8603%, followed by CCS at 7.5459 ± 0.2189%. The elongation at break of CCS-1, CCS-3 and CCS-6 was 79.22%, 35.89% and 54.67% higher than that of CCS, respectively, which shows that irradiation can improve the softness of the sponge. There is a significant difference in the elongation at break of sponges made of collagen from different sources. For example, the tensile toughness of sheep collagen scaffolds is significantly higher than that of pig and bovine collagen scaffolds [43]. The elongation of collagen from bovine tendon is about 1.6%, and the elongation of collagen–chitosan mixture is 3.7% [44]. However, the elongation of fishery collagen is longer because of its loose collagen fiber bundles. In addition, adding different proportions of biopolymers (chitosan, pyrophosphate nanoflowers, etc.) or using different chemical crosslinking agents would also lead to different elongations, with an average breaking rate between 6% and 15% [26,28,45].

Young’s modulus is a physical quantity that describes the ability of solid biomaterials to resist deformation. The results showed that the addition of biomacromolecules such as starch can improve the biostability and mechanical resistance of chitosan collagen sponges, but high-dose irradiation would reduce the rigidity of the sponge. The previous results indicated that irradiation could promote crosslinking between molecules, but high doses can enlarge the gaps and change the structural arrangement state, thereby reducing the elastic modulus of the sponge. It is worth noting that even though high-dose irradiation reduced the elastic modulus of the sponge, its value is still higher than that of the sponge with added polymers and crosslinkers [46,47].

3.3. Penetration and Antibacterial Properties of Collagen Sponges Under Irradiation

Liquid absorption capacity and antibacterial properties are among the important criteria for evaluating the hemostatic ability of collagen sponges. The kinetic curves of the composite collagen sponges of the same volume continuously absorbing whole blood and PBS solution under natural conditions are shown in Figure 4A–C. Overall, the sponges HSD, CCS, and CCS-1 had stronger ability to absorb whole blood than PBS solution, and could complete the absorption of most liquids within 20 s. The rate and capacity of whole blood absorption of collagen sponges with different irradiation doses vary greatly (Figure 4C). Specifically, sponge CCS-6 had the fastest absorption rate, but the maximum absorption amount was low, only 2.54 g. CCS-3 had a larger absorption amount and a lower absorption rate, while CCS-1 performed best with a good absorption rate and absorption amount. Irradiation promotes the crosslinking and compact arrangement of the collagen sponges, which makes the collagen sponges have an efficient liquid transmission rate, and is expected to quickly absorb blood when the wound is bleeding profusely, thereby more effectively stopping bleeding.

Water absorption capacity and swelling capacity are crucial factors influencing the hemostatic performance of dressings. Enhanced water absorption allows for the rapid infiltration of exudate and blood, forming a concentrated layer of blood components at the interface between the dressing and the wound, thereby achieving faster hemostasis [48]. Additionally, higher water absorption capacity prevents the accumulation of wound exudate, reducing the risk of infection. Furthermore, hemostatic dressings with a certain swelling rate can fully cover the wound after absorbing exudate, enhancing adhesion to the wound and maintaining a moist environment. This promotes cell migration and tissue regeneration, accelerating wound healing [9,26]. The results indicate that the water absorption capacity of the collagen sponge formed after irradiation treatment exceeds 30 times its weight, with CCS-3 reaching up to 40 times (Figure 4D). This capacity was significantly higher than that of HSD and CC (p < 0.05). Regarding the swelling rate, the swelling rates of CCS-1, CCS-3, and CCS-6 sponges, while not as high as those of HSD and CC, still reached 1400% (Figure 4E). These results demonstrate that the irradiated collagen sponge possesses excellent liquid absorption capacity and has the potential as an effective hemostatic dressing.

The antibacterial ability of collagen sponges is a key indicator for evaluating their clinical efficacy and safety, directly impacting their effectiveness and applicability in practical use. Collagen sponges with strong antibacterial properties can effectively inhibit the growth of pathogenic microorganisms, reducing the risk of infection, maintaining wound cleanliness, promoting cell migration and tissue regeneration, and thereby accelerating the wound healing process. Additionally, the use of collagen sponges with good antibacterial performance can reduce dependence on external antibiotics, lower the risk of developing antibiotic resistance, and minimize drug-related adverse effects.

The antibacterial effects of the collagen sponges are shown in Figure 4F, using Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus as models. Their growth on agar plates is depicted in Figure 4G. The results indicate that the commercial sponge HSD had a better inhibitory effect on S. aureus, achieving an 80% inhibition rate, but showed lower inhibition against E. coli. The CC and CCS sponges exhibited lower inhibition rates against both bacteria. In contrast, CCS-1, CCS-3, and CCS-6 demonstrated superior antibacterial effects, achieving inhibition rates of over 70% for both E. coli and S. aureus, with a stronger effect on E. coli. This may be attributed to the high positive charge of the sponges, which interacts with the negatively charged bacteria, disrupting their cell walls and membranes, leading to cell lysis and the release of intracellular contents. The zeta potential results (Figure 2F) also support this hypothesis. Lin et al. found that their asymmetrical halloysite/chitosan/collagen sponge, which carries a positive charge, also exhibited significant antibacterial effects [27]. Previous studies have shown that the primary antibacterial agent is chitosan, whose positive charge can electrostatically attract the negatively charged cell membranes, altering membrane permeability and ultimately disrupting membrane integrity [26,49]. The results of this study showed that the positive charge value of the sponge after irradiation is significantly increased, which also enhances their antibacterial capability.

3.4. Biocompatibility of Collagen Sponges Under Irradiation

Cytotoxicity experiments on collagen sponges were conducted to assess cell viability by evaluating the metabolic activity of L929 cells. Sponge extracts at concentrations of 1.0 and 1.5 mg/mL were co-cultured with the cells for 24 or 48 h, and cell viability was determined using the MTT assay. The results demonstrated that, after 24 h of co-culture, the viability of all cells exceeded 100%, regardless of the concentration of sponge extracts (Figure 5A). Although cell viability slightly decreased after 48 h of incubation with irradiated sponges (CCS-1, CCS-3, and CCS-6), it remained close to 100% (Figure 5B). Generally, modified materials are considered non-toxic when the cell viability reaches 70% while having an antibacterial effect. The cell viability of the irradiated sponges was around 100%, indicating that no harmful substances were produced during the irradiation process [50]. Previous studies have indicated that irradiation can generate free radicals within biomaterials, potentially causing oxidative damage to key biomolecules such as cell membranes, DNA, and proteins, leading to impaired cellular function and, ultimately, apoptosis or necrosis [51]. However, in this study, even at an irradiation dose of 6 kGy, cell viability did not significantly decrease, suggesting that either a minimal amount of free radicals was generated or that the produced radicals did not adversely affect the cells. Furthermore, the pH of the sponge-soaked solutions was measured between 5.84 and 6.65 (Figure S5), indicating a mildly acidic environment. Consequently, the irradiated sponge exhibited no cytotoxicity and complies with biosafety standards, which further indicated that irradiation technology could be applied to the modification of biological hemostatic materials.

Hemolysis testing is critical for assessing the safety and biocompatibility of collagen sponges. When blood comes into contact with a material, if it causes red blood cells to rupture and release hemoglobin into the plasma, a hemolytic reaction occurs. Hemolysis may trigger a cascade of biological responses, including coagulation, immune activation, and inflammation [52,53]. For biomedical materials used clinically, a hemolysis rate below 5% is required, with a rate under 2% being considered non-hemolytic [28]. Hemolysis testing ensures that the collagen sponge will not induce significant hemolysis during use, thereby mitigating potential risks and facilitating wound healing. The hemolysis test results for each sponge are presented in Figure 5C, all of which are below 5%. Specifically, the commercial sponge HSD exhibited a hemolysis rate of 3.49%, while the rates for CCS-1, CCS-3, and CCS-6 were 2.72 ± 0.51%, 1.96 ± 0.44%, and 3.32 ± 0.63%, respectively (Figure 5D). These results indicate that irradiation did not significantly increase the material’s hemolysis rate and that the sponges comply with the hemolysis standards for biomaterials. However, at an irradiation dose of 6 kGy, the hemolysis rate of the sponge increased, likely due to reactive oxygen species generated by high-dose irradiation or alterations in material charge. Previous studies have demonstrated that, for liquid protein substances such as collagen, the maximum irradiation dose should not exceed 3 kGy to avoid the generation of free radicals like hydrogen peroxide or protein denaturation and inactivation [21,54]. Nonetheless, mild hemolysis during hemostasis is considered acceptable, as cell-free hemoglobin and heme released from lysed red blood cells can act as coagulants [55]. Our findings confirm that irradiation as a method to modify collagen sponge materials offers good biocompatibility and holds significant potential in wound dressing preparation.

3.5. Hemostatic Properties of Collagen Sponges Under Irradiation

3.5.1. Coagulation Characteristics

The rapid coagulation of hemostatic materials effectively seals wounds and minimizes blood loss, reducing the risk of hemorrhage. Rapidly formed clots protect the wound surface, prevent the infiltration of external bacteria and contaminants, and provide a scaffold for cell migration and tissue regeneration, thereby accelerating the wound-healing process. As shown in Figure 6A, when distilled water was added to the coagulation sponge at different times to release uncoagulated blood clots, the irradiated collagen sponge CCS-1 absorbed and coagulated blood within 30 s. Similarly, CCS-3 and CCS-6 coagulated blood within 60 s, while CCS required 120 s to coagulate most of the blood clots, indicating the superior coagulation ability of the irradiated sponges. The poor coagulation performance of the commercial sponge HSD is likely due to its limited absorption capacity. As illustrated in Figure 6D, after 10 s of absorbing 150 μL of blood, HSD exhibited minimal absorption, whereas CCS-1, CCS-3, and CCS-6 completely absorbed and coagulated the blood, indicating that the addition of starch and irradiation treatment accelerated the rate at which the sponge absorbed blood. The results of the water contact angle experiment also proved this statement (Figure S2). Additionally, in the test for the time required to absorb 200 μL of blood (Figure 6E), HSD took approximately 40 s, while CCS-1, CCS-3, and CCS-6 absorbed the blood in about 6 s, demonstrating their exceptional absorption rates. The platelet adsorption rate, an indicator of coagulation strength, revealed that the sponges achieved platelet adsorption rates exceeding 64%, with HSD, CCS-1, and CCS-3 surpassing 80%, highlighting their robust coagulation capabilities.

The BCI directly reflects the procoagulant efficacy of a hemostatic material by measuring the time required for blood to coagulate or the extent of thrombus formation after contact with the sponge. A lower BCI value typically indicates stronger procoagulant performance. As shown in Figure 6B, the CCS sponge required a longer time to induce blood coagulation, while the commercial sponges HSD and CC exhibited higher BCI values, indicative of inferior coagulation performance. In contrast, sponges CCS-1, CCS-3, and CCS-6 demonstrated lower BCI values in both the early and late stages of coagulation, reflecting their superior coagulation capacity. This enhanced performance may be attributed to two factors: first, irradiation increases the sponge’s crosslinking and porosity, facilitating more effective blood absorption; second, the increased positive charge on the sponge surface enhances its electrostatic attraction to negatively charged blood cells, promoting their aggregation and facilitating the formation of a fibrin network, thereby accelerating hemostasis. Lin et al. similarly reported that positively charged sponges exhibit lower BCI values [27]. Sun et al. observed that EDC/NHS cross-linked collagen sponges displayed significantly lower BCI values, particularly in the early stages of coagulation, indicating faster and more effective coagulation compared to commercial products [48]. These studies, consistent with our findings, suggested that enhancing the crosslinking degree and positive charge of the sponge can improve its coagulation efficiency. The irradiated collagen sponge demonstrated strong blood absorption and coagulation properties without the introduction of chemical crosslinking agents, ensuring high biocompatibility.

3.5.2. Analysis of In Vivo Hemostasis Performance

To accurately evaluate the hemostatic efficacy of the collagen sponge, an in vivo hemostasis experiment was conducted using the rat femoral artery bleeding model. Consistent wound size and sponge compression strength were maintained across all experimental groups. As depicted in Figure 7A, the hemostatic effects were assessed by comparing the wound before and after the bleeding and hemostasis process, along with visualizing the sponge’s front and back after absorbing blood. The HSD and CC sponges exhibited poor hemostatic performance, with significant blood loss. Although sponge CCS demonstrated less blood loss compared to CC, it showed a tendency for excessive penetration to the back after absorbing blood. In contrast, CCS-1, CCS-3, and CCS-6 exhibited minimal blood loss with no observable blood diffusion. Specifically, as shown in Figure 7B,C, the blood loss values for sponges HSD, CC, CCS, CCS-1, CCS-3, and CCS-6 were 2.8241 ± 0.1464, 3.2014 ± 0.2641, 2.3074 ± 0.1955, 1.6172 ± 0.0804, 1.3252 ± 0.1241, and 1.5281 ± 0.0766 g, respectively. Corresponding hemostasis times were 202 ± 10.2762, 173 ± 6.4107, 221 ± 14.3545, 104 ± 8.1504, 140 ± 10.8241, and 120 ± 6.6206 s, respectively. The irradiated collagen sponge demonstrated a higher degree of crosslinking, denser and more ordered microchannels, a lower water contact angle, and a higher positive surface charge. These characteristics enable rapid water absorption from blood, concentrate platelets and red blood cells, and facilitate the effective activation of coagulation factors, thereby accelerating thrombosis and achieving hemostasis. After passing multiple rigorous evaluation procedures, the proposed collagen sponge exhibited slightly higher hemostatic performance than HSD (a commercially approved hemostatic sponge) in our experimental comparison. This indicate that the irradiated collagen sponge possesses superior performance for potential commercial application development.

4. Discussion

4.1. Structural and Molecular Implications of Irradiation

Gamma irradiation induced notable transformations in both the morphology and molecular configuration of the collagen–chitosan–starch (CCS) sponges. The observed color shift, surface roughening, and FTIR band displacement together demonstrate that the irradiation process simultaneously triggered polymer crosslinking and chain scission. When exposed to moderate doses (≤4 kGy), the triple-helix domains of collagen were largely maintained, while radical-mediated recombination among reactive carbonyl, hydroxyl, and amino sites enhanced the internal cohesion between collagen, chitosan, and starch chains [56,57]. This controlled network rearrangement increased intermolecular bonding, thereby reinforcing the composite structure and improving stability against aqueous degradation [58]. Conversely, excessive irradiation (>6 kGy) led to the accumulation of oxidative fragments and disrupted peptide backbones, explaining the partial loss of characteristic amide peaks and the more eroded pore morphology [59]. Such dose-dependent structural evolution is consistent with earlier reports describing a delicate interplay between polymer consolidation and degradation under γ-irradiation [60]. Thus, the results highlight the importance of maintaining an optimal energy exposure window to achieve desirable physicochemical characteristics without sacrificing the native conformation of collagen.

Gamma irradiation can induce free radical formation in collagen-based materials, raising concerns regarding potential biosafety risks at high doses. Our previous studies showed that low-dose irradiation (1–3 kGy) generated only a limited amount of free radicals, predominantly alkoxy radicals, which are short-lived and mainly involved in recombination and crosslinking reactions rather than causing severe molecular degradation. In contrast, irradiation at doses over 9 kGy resulted in a marked increase in radical formation, which may lead to chain scission and compromised structural integrity [21]. In this study, gamma irradiation was applied to the precursor solution prior to freeze-drying, allowing partial radical dissipation and controlled crosslinking before scaffold formation. Combined with irradiation dose optimization, this strategy effectively enhanced mechanical strength, biostability, and antibacterial performance while minimizing potential free radical-related risks. These results indicate that radiation crosslinking can be safely and effectively applied to collagen-based sponges when appropriate irradiation conditions are employed.

Chitosan and starch are well-established biopolymers with extensively reported and consistent FTIR spectra and XRD patterns in the literature. In this study, these components were used without chemical modification or pretreatment; therefore, their intrinsic molecular structures and crystalline characteristics are not expected to deviate from previously reported profiles [15,43]. Accordingly, separate FTIR and XRD analyses of the individual raw materials were not the primary focus of this work. Instead, FTIR spectra and XRD patterns of the composite collagen sponges before and after irradiation were employed to evaluate structural integrity, crosslinking interactions, and irradiation-induced changes in crystallinity. The observed spectral features and diffraction patterns clearly reflect the formation of intermolecular interactions and the evolution of the crystalline state within the composite matrix following irradiation. These results are sufficient to demonstrate the structural effects of irradiation on the collagen-based sponges, which are directly relevant to their functional performance and biomedical applicability.

4.2. Biocompatibility and Hemostatic Mechanisms

The irradiated CCS scaffolds exhibited excellent biocompatibility and improved blood-clotting behavior compared with the unmodified counterparts. Two main factors appear to contribute to this enhancement. First, the mild irradiation process altered the microarchitecture of the sponges, increasing their surface area and porosity—features that favor platelet attachment, morphological activation, and thrombus initiation [61]. The exposure of additional hydrophilic and positively charged sites likely promotes the adsorption of fibrinogen and thrombin, accelerating coagulation reactions and the assembly of fibrin networks [62]. Second, the altered surface chemistry may strengthen electrostatic interactions with blood components, facilitating erythrocyte aggregation and fibrin entrapment. These combined physicochemical modifications explain the observed reduction in clotting time and enhanced hemostatic response.

Importantly, the preservation of cell viability indicates that the irradiation-induced radicals did not generate cytotoxic degradation products. Previous studies have confirmed that irradiation below 10 kGy effectively sterilizes collagen-based materials while maintaining cellular compatibility [21,60]. The in vivo results in a mouse femoral artery model further verified the dual functional role of the CCS: they not only provide a porous, absorbent matrix that physically concentrates blood cells but also release bioactive cues from collagen and chitosan to amplify the platelet and coagulation cascade [63]. This duality explains the faster hemostasis and lower blood loss compared to non-irradiated controls. Overall, γ-irradiation enhances both sterilization efficiency and surface bioactivity, making it a valuable processing strategy for hemostatic biomaterials.

4.3. Potential Applications and Challenges

Overall, controlled γ-irradiation represents an efficient post-fabrication strategy for optimizing collagen-based composite sponges for wound-healing and hemostatic applications. By inducing covalent crosslinking within the collagen network, irradiation enhances mechanical stability, structural integrity under wet conditions, and resistance to enzymatic degradation, while simultaneously achieving terminal sterilization without introducing toxic chemical residues. Moderate irradiation doses enable a balanced combination of rigidity, liquid absorption capacity, and blood affinity, facilitating rapid blood uptake and clot formation while preserving collagen bioactivity and cytocompatibility. These advantages highlight γ-irradiation as a scalable and clinically compatible approach for producing advanced collagen-based wound dressings.

Despite the promising performance of irradiation-modified collagen hemostatic sponges, several limitations should be acknowledged. First, the biological evaluation in this study was relatively limited. Biocompatibility was mainly assessed through short-term in vitro cytotoxicity tests, and cell proliferation was only monitored up to 48 h, without extended observation periods (e.g., 72 h or longer) or mechanistic studies on cell adhesion, migration, and differentiation. In addition to tensile testing, which confirmed the strengthening effect induced by γ-ray irradiation, other clinically relevant mechanical parameters, including compressive resilience, wet-state strength after fluid absorption, and fatigue resistance under dynamic loading, were not systematically evaluated in this study. These factors are important for predicting handling performance and structural integrity during actual wound compression, and they are needed for the evaluation of final hemostatic sponges [47,64]. Since the objective of this study was to construct a collagen–chitosan–starch synergistic matrix for the sponges and investigate irradiation-induced crosslinking behavior and its effect on hemostatic performance, we mainly focus on the analysis of crosslinking degree, maximum load, tensile strength, absorption capacity, and coagulation performance. Future studies will include compressive strength measurements, along with long-term degradation and in vivo assessments, to further validate the mechanical reliability and practical applicability of these irradiation-fabricated collagen-based hemostatic sponges.

From an industrial perspective, irradiation processing requires precise dose control, as excessive exposure may induce collagen chain scission and partial loss of bioactivity. The need for specialized irradiation facilities, high capital investment, and strict regulatory compliance may further limit large-scale adoption. Variability in raw collagen sources and batch-to-batch irradiation uniformity may also affect product consistency. Addressing these limitations through optimized irradiation protocols and more comprehensive biological and mechanical evaluations will be essential for future clinical translation.

5. Conclusions

This study introduces a novel approach for fabricating bio-based composite hemostatic sponges by combining precursor solution processing with controlled γ-ray-induced crosslinking of fish processing by-products. Controlled γ-ray irradiation is an effective post-processing strategy to enhance the functional properties of collagen-based composite sponges composed of fish skin collagen, chitosan, and soluble starch. Low-dose irradiation (1–3 kGy) promoted intermolecular crosslinking, improving mechanical strength, elongation, and biostability, while higher doses (6 kGy) slightly reduced crosslinking due to partial degradation of collagen, chitosan, and starch, highlighting the need for dose optimization. With low-dose irradiation, the proposed hemostatic sponges showed enhanced water absorption, blood cell adsorption, swelling, and antibacterial properties. Compared with the commercially available HSD sponge, the irradiated sponges exhibited superior hemostatic efficacy under experimental conditions. These findings indicate that γ-ray irradiation can produce medical collagen sponges with uniform porous structure, strong mechanical support, and enhanced hemostatic performance, offering a promising approach for advanced hemostatic biomaterials.