Hyaluronic Acid Immersion Enhances Gamma-Ray-Irradiation Cross-Linking of the Fish-Derived Type I Collagen Membrane

Abstract

Fish-derived type I collagen is widely used in biomedical implants; however, its limited thermal and enzymatic stability necessitates cross-linking for clinical applications. This study investigated gamma-ray-induced cross-linking of collagen as a shape of membranes, with enhancement via polysaccharide-assisted immersion. Hydrated collagen membranes were irradiated at doses ranging from 15 kDa to 35 kGy to assess their dose-dependent effects. Irradiation at 15 kGy enhanced thermal and enzymatic stability, which declined at higher doses. To further enhance stability, the membranes were immersed in phosphate-buffered saline containing chondroitin sulfate (CS) or hyaluronic acid (HA) during irradiation at 25 kGy. HA-treated membranes showed a synergistic enhancement in thermal stability (T _onset > 50 °C, T _peak ∼ 54 °C) and enzymatic resistance, surpassing CS-treated and nonimmersed controls. MALDI-TOF mass spectrometry revealed the selective loss of irradiation-sensitive amino acid residues, indicating involvement in cross-linking and scission mechanisms. Tensile testing confirmed that the HA-treated membranes retained a tensile strength (∼1.7 MPa) under hydrated conditions, comparable to the irradiated-only controls. These findings demonstrate that HA-assisted gamma-irradiation at 25 kGy effectively enhances collagen cross-linking, improving its thermal and enzymatic stability while preserving its mechanical performance for biomedical applications.

1

Macroscopic and microscopic characterization of collagen membranes before and after gamma-irradiation with or without polysaccharide treatment. (A) Hydrated collagen hydrogels prior to drying, (B) dried collagen membranes that maintain uniform shape and thickness, and (C) scanning electron microscopy images of collagen membranes under various conditions: (i) NXL, (ii) 15 kGy, (iii) 20 kGy, (iv) 25 kGy, (v) 30 kGy, (vi) 35 kGy, (vii) CS + 25 kGy, and (viii) HA + 25 kGy. Scale bar is 1 mm.

2

Attenuated total reflectance Fourier-transform infrared spectra of collagen membranes subjected to different irradiation doses (i–vi) and immersion conditions (vii, viii): (i) NXL, (ii) 15 kGy, (iii) 20 kGy, (iv) 25 kGy, (v) 30 kGy, (vi) 35 kGy, (vii) CS + 25 kGy, and (viii) HA + 25 kGy.

3

MALDI-TOF profile of collagen membranes before and after gamma-irradiation. Distinct peaks corresponding to specific tripeptide fragments (Gly–Pro–X, where X is isoleucine (GPI), threonine (GPT), arginine (GPR), lysine (GPK), or histidine (GPH)) are noted in the figure. Each tripeptide is detected as a positively charged ion, forming various adducts (e.g., H⁺, Na⁺, K⁺, CH₃OH, and NH₄ ⁺), which are indicated in square brackets. The corresponding mass-to-charge ratios (m/z) of the ionized species are given in parentheses.

4

Thermal properties and the temperatures of onset and denaturation of the collagen membrane. (A, C) DSC curve for the collagen membranes irradiated in (A) different doses and in the presence of HA and CS, and in (C) immersion with irradiation and sequential immersion and irradiation. (B, D) Summary of onset and denaturation temperatures for DSC curves in (A, C). n = 3, mean ± standard deviation. Statistical comparisons were performed using a two-tailed Student’s t test, with p < 0.05 considered statistically significant. # and * indicate a statistically significant difference in T _peak and T _onset, respectively, compared to the 25 kGy group (p < 0.05). n.s. is nonsignificant.

5

Enzymatic stability of collagen membranes subjected to gamma-irradiation and polysaccharide treatment. (A) Time-dependent enzymatic degradation of collagen membranes incubated in collagenase A solution. (B) Weight loss at 0.5 h plotted against T _onset fitted with the exponential decay model (where A ₁ = 100%, t ₁ = 2.48 ± 0.23 °C, x ₀ = 38.21 °C ± 0.03 °C, and y ₀ = 5.01 ± 1.07% (R ² = 0.999), and linear model (a = 51.33 ± 10.55 and b = −0.86 ± 0.21, R ² = 0.77). Data are presented as the mean ± standard deviation (n = 3).

6

Mechanical properties of collagen membranes subjected to gamma-irradiation and polysaccharide treatment. (A) Representative stress–strain curve of the collagen membrane. (B) Young’s modulus and (C) ultimate tensile strength (UTS) of collagen membranes in hydrated conditions. Data were expressed as mean ± standard deviation, n = 5. Statistical comparisons were performed.