2.2. pH-Responsive Swelling Behavior of UV-Crosslinked Collagen Hydrogels

The pH-dependent swelling behavior of UV-irradiated crosslinked collagen hydrogels was investigated over a wide pH range (3–10). At pH ≤ 3, the collagen gel swelled to such an extent that it could not be manipulated using tweezers, precluding quantitative swelling measurements. However, at pH 4–10, the gel maintained sufficient structural integrity for handling and shape retention.

Figure 2a displays photographs of the collagen gel disks (prepared with 40 min of UV irradiation) equilibrated in buffer solutions ranging from a pH of 3 to 10. These images clearly demonstrate that the hydrogel exhibited a larger diameter at a pH of four and a smaller diameter at a pH ≥6. Figure 2b shows the swelling ratio as a function of the pH, revealing significant swelling in buffers with a pH ≤6, particularly at a pH of four. In this range, the swelling ratio exhibited a negative linear relationship with the pH. Above a pH of six, the swelling ratio stabilized at a relatively low value of approximately 0.5.

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Figure 2

The pH-dependent swelling of UV-irradiated collagen hydrogels: (a) representative photographs of collagen gel disks equilibrated in buffers of varying pH levels (UV irradiation time of 40 min) and (b) swelling ratios of collagen gel disks as a function of the buffer pH for two UV irradiation times (40 and 120 min).

This pH-dependent swelling behavior closely mirrors the pH-dependent solubility of the collagen molecules. The observed decrease in swelling at a pH > 6 corresponds to the known increase in turbidity in collagen solutions in this pH range [59]. Notably, the gels crosslinked for 40 and 120 min showed nearly identical pH-dependent swelling trends, indicating that UV irradiation did not impair the pH sensitivity of the gel. This preservation of the pH sensitivity suggests the retention of ionic groups within the gel structure post UV crosslinking.

Interestingly, the gels formed by 120 min of UV irradiation exhibited approximately 1.5 times smaller gel weights (W₁ and W₂) than those formed by 40 min of irradiation. The swelling behavior of polyelectrolyte gels is governed by the balance between the elastic shrinkage force of the gel chains (determined by the Donnan osmotic pressure) and the gel density (determined by the concentration of ionic groups) [62,63,64]. Assuming that UV crosslinking does not alter the concentration of ionic groups, the decrease in gel weight with increasing UV irradiation doses suggests an increase in the crosslinking density within the gel.

Importantly, the UV crosslinking method appeared to preserve these ionizable groups, as evidenced by the maintained pH responsiveness of the hydrogels. This is in contrast with some chemical crosslinking methods which may consume these groups during the crosslinking process, potentially reducing the pH sensitivity of the resulting hydrogels. In contrast to gamma-irradiated collagen gels, which are known to dissolve at a pH ≤ 5, our UV-irradiated gels showed no signs of degradation or dissolution at a pH of four even after 120 min of UV irradiation. This stability at a low pH presents a significant advantage for applications in acidic environments. The enhanced stability of UV-crosslinked gels compared with gamma-irradiated gels may be attributed to different crosslinking mechanisms. Although high-energy gamma radiation can cause both crosslinking and degradation to compete, often resulting in brittle gels [65,66], UV irradiation in the presence of riboflavin appears to promote more controlled crosslinking without significant degradation.

Because the pH responsiveness of UV-crosslinked collagen hydrogels closely resembles the pH dependence of the turbidity of collagen solutions, the pH-responsive behavior of this gel should also be due to the presence of various ionizable groups along the collagen molecules. These include carboxyl groups (–COOH) from aspartic and glutamic acids, as well as amino groups (–NH₂) from lysine and arginine. At low pH values (pH < 6), the amino groups became protonated (–NH₃⁺), increasing the overall positive charge of the hydrogel. This led to electrostatic repulsion between the chains and an increased Donnan osmotic pressure, causing the gel to swell. Conversely, at higher pH values (pH > 6), the carboxyl groups became deprotonated (–COO^-), increasing the negative charge, and the screening effect of the charge reduced the Donnan osmotic pressure, causing the gel to shrink. The isoelectric point of collagen (approximate pH of 6–7) represents the point of minimal net charge, corresponding to the observed minimal swelling in our hydrogels. This behavior is consistent with the pH-induced volume phase transitions observed in other polyelectrolyte hydrogels, such as crosslinked chitosan [67,68]. To confirm whether the pH-responsive change in the swelling degree shown here was a true volume phase transition phenomenon, we investigated the reversibility of the swelling response to a pH change in the next section. This investigation is also important for understanding the potential of these hydrogels in applications which require repeated pH-responsive behaviors, such as controlled drug delivery systems and smart wound dressings.

2.3. Reversibility of pH-Responsive Behavior in UV-Crosslinked Collagen Hydrogels

The reversibility of pH-responsive behavior in UV-crosslinked collagen hydrogels was investigated through cyclic pH changes. Figure 3 illustrates this behavior for the hydrogels prepared with 40 and 120 min of UV irradiation.

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Figure 3

Reversible pH-responsive behavior of UV-crosslinked collagen hydrogels. (a) Representative photographs of collagen gel disks (40 min of UV irradiation) cycled through pH 4 → pH 10 → pH 4. (b) Representative photographs of collagen gel disks (40 min of UV irradiation) cycled through pH 10 → pH 4 → pH 10. (c) Swelling ratios of hydrogels during pH 4 → pH 10 → pH 4 cycle. (d) Swelling ratios of hydrogels during pH 10 → pH 4 → pH 10 cycle.

Figure 3a,b shows the morphological changes of hydrogels cycled through pH 4 → 10 → 4 and pH 10 → 4 → 10, respectively. In both cases, the hydrogels demonstrated a degree of reversibility, nearly returning to their original size when reimmersed in the initial pH buffer.

A quantitative analysis of the swelling behavior is presented in Figure 3c,d. For the pH 4 → 10 → 4 cycle (Figure 3c), both the 40 min and 120 min irradiated gels showed incomplete recovery upon returning to a pH of four. The 40 min irradiated gel reached a swelling ratio of 0.85, while the 120 min irradiated gel only recovered to 0.7.

For the pH 10 → 4 → 10 cycle (Figure 3d), the 40 min irradiated gel achieved a swelling ratio of 0.8 at a pH of four, while the 120 min irradiated gel only reached 0.7. Upon returning to a pH of 10, the 120 min irradiated gel recovered to a swelling ratio of 0.75.

These results demonstrate partially reversible pH-responsive behavior, with some hysteresis observed, particularly after exposure to a pH of 10. The incomplete recovery of the swelling ratio, which was especially pronounced in the gels irradiated for 120 min, suggests irreversible changes in the hydrogel structure during pH cycling.

This behavior can be attributed to the structural changes in collagen at high pH levels. Previous studies have shown that collagen can denature at a pH of 10, leading to reduced gelling properties and water-holding capacity [69,70]. The more significant loss of water-holding capacity in gels irradiated for 120 min suggests that longer UV exposure may result in a more rigid network structure, which is more susceptible to irreversible changes upon pH cycling.

This observed partial reversibility has important implications for potential applications. While these hydrogels demonstrate the ability to respond to pH changes multiple times, the gradual loss of swelling capacity, particularly after exposure to high pH levels, must be considered in their design for specific uses. Future studies should focus on enhancing the reversibility of the pH response, possibly through the incorporation of additional stabilizing agents or optimization of the crosslinking process.

2.4. Mechanical Properties of UV-Crosslinked Collagen Hydrogels at Various pH Levels

The mechanical properties of the UV-crosslinked collagen hydrogels were investigated in different pH environments to further elucidate their pH-responsive behavior. Figure 4a presents the stress–strain curves obtained from the compressive tests on gels immersed in various pH buffer solutions. The curves demonstrate that higher pH values corresponded to steeper slopes, indicating increased stiffness.

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Figure 4

Mechanical properties of UV-crosslinked collagen hydrogels at varying pH levels. (a) Stress–strain curves from compression tests on hydrogels (40 min UV irradiation) equilibrated in different pH buffers. (b) Young’s modulus of hydrogels calculated from the linear regions of stress-strain curves.

Figure 4b quantifies this trend by showing the Young’s modulus, derived from the linear portion of the stress–strain curves. Notably, the elastic modulus of the gel immersed in the buffer with a pH of 6 was approximately 10 times lower than that of the gel immersed in the buffer with a pH of 10. This trend was consistent across different UV irradiation times, supporting the pH-responsive nature of the mechanical properties of these hydrogels in the pH range of 6–10.

The observed increase in both tensile strength and elastic modulus from neutral to alkaline pH levels aligns with the properties of conventional acidic collagen gels which are physically crosslinked [71,72]. This similarity leads to two key conclusions. (1) UV-induced crosslinking preserves the pH-dependent characteristics of collagen, and (2) the increased elastic modulus at high a pH level may suppress swelling deformation, resulting in similar swelling behavior (shrinkage state) at pH levels of 6 and 10.

Interestingly, the pH dependence of the swelling ratio showed a minimal change between a pH of 6 and 10 (Section 2.2), which appears to contradict the general understanding that the elastic modulus increases with the pH while the swelling ratio remains constant and that hydrogels with higher water contents typically exhibit lower elastic moduli [73]. This apparent contradiction can be resolved by considering that the high elastic modulus at a pH of 10 suppresses gel deformation, resulting in a swelling degree at a pH of 10 which is similar to that at a pH of 6. If the elastic modulus is low near a pH of 10 (near the isoelectric point of acid-soluble collagen (pH of 6–7)), the increased Donnan osmotic pressure due to carboxyl group dissociation would likely lead to higher swelling, but this is counteracted by the increased elastic modulus.

The increase in the elastic modulus at higher pH values can be attributed to several factors. As the pH increases, the net positive charge decreases, owing to deprotonation of the collagen monomer side chains, which improves the hydrogen bonding capacity between the collagen triple helices [74]. This enhanced hydrogen bonding not only stabilizes the collagen structure but also promotes fibril formation, resulting in a more compact and stable fibril arrangement [71,72]. The presence of hydrogen bonds strengthens the interactions between collagen fibrils, leading to a more cohesive and mechanically stable gel [75,76].

Notably, the gels prepared with 120 min of UV irradiation exhibited approximately twice the elastic modulus of those prepared with 40 min of irradiation. This result suggests that prolonged UV crosslinking may promote hydrogen bonding or increase the crosslinking density through the formation of additional chemical crosslinks, although further investigation is needed to confirm these mechanisms.

These findings highlight the complex interplay between the pH, swelling behavior, and mechanical properties of UV-crosslinked collagen hydrogels. The ability to modulate the mechanical properties of these gels by changing the pH while maintaining a relatively constant swelling ratio at higher pH values presents intriguing possibilities for applications requiring precise control of the material stiffness without significant volume changes.

3.2. Preparation of Collagen Hydrogels

A 0.02 mM riboflavin-mixed collagen solution was prepared by combining the commercial collagen solution with riboflavin in a beaker. The mixture was stirred at 7 °C for 12 h using a magnetic stirrer (500 rpm) and then cooled to 4 °C for stabilization prior to UV irradiation. Collagen gels were fabricated via UV-initiated crosslinking. Aliquots of the riboflavin-mixed collagen solution were carefully pipetted into circular depressions (20 mm in diameter and 2 mm deep) on a polytetrafluoroethylene (PTFE) plate. The samples were then subjected to UV irradiation (λ = 366 nm) for either 40 or 120 min using a UV lamp (CAMAG^® UV Lamp 4, 8 W; Camag, Muttenz, Switzerland). The distance between the UV source and the sample surface was maintained at 90 mm throughout the irradiation process, resulting in a light intensity of approximately 3.5 mW/cm² at the sample surface. All procedures were conducted at 25 °C to prevent thermal denaturation of the collagen, with temperature regulation achieved using a Peltier temperature controller (VPE-35 and VTH1.8K-70S; VICS, Tokyo, Japan). To monitor potential water evaporation during the 120 min irradiation period, sample weights were measured before and after UV exposure using an analytical balance (accuracy: ±0.1 mg). No significant weight loss was observed, indicating minimal evaporation of the water. The sample temperature was monitored using a thermographic camera (ARTCAM-320-THERMO-WOM-16, Artray, Tokyo, Japan), which confirmed that the sample temperature did not exceed 27 °C during the irradiation process.

3.3. Preparation of pH Solutions

Eight different pH solutions were prepared for immersion of the gel samples, targeting pH values of 4, 5, 6, 7, 8, 9, and 10. All buffer solutions were made using 0.1 M stock solutions of MES, Tricine, HEPES, CHES, and NaOH. MES buffer was used to prepare a pH 4 solution. A solution of pH 5 was prepared using tricine buffer. For the pH 6 solution, a mixture of 25 mL of MES buffer and 10 mL of NaOH was employed. The pH 7 solution consisted of 25 mL of HEPES buffer and 5 mL of NaOH. The pH 8 solution was prepared by mixing 25 mL of HEPES buffer with 20 mL of NaOH. The pH 9 solution was prepared by combining 25 mL of CHES buffer with 5 mL of NaOH. Finally, a solution with pH 10 was prepared using 25 mL of CHES buffer and 20 mL of NaOH. Each buffer solution was thoroughly mixed, and the pH was verified using a calibrated pH meter to ensure that the target pH values were accurately achieved. Each buffer solution was prepared by dissolving the respective buffer salts in distilled water, followed by adjustment of the target pH using NaOH or HCl. The final pH was determined using pH test papers (Duotest^®; Macherey-Nagel Co., Ltd., Düren, Germany).

3.4. Rheological Characterization (Dynamic Viscoelastic Analysis)

Dynamic viscoelasticity measurements were performed at a controlled temperature of 25 °C on pregel solutions subjected to varying durations of UV treatment (0, 40, and 120 min) to investigate the effect of UV exposure on their viscoelastic properties. The storage and loss moduli were determined using an MCR-101 rotary rheometer (Anton Paar, Graz, Austria) with a plate-plate geometry (25 mm in diameter and a 0.7 mm gap). Measurements were performed at a frequency (ω) of 0.1 rad/s with a strain value of 3%, which was chosen to ensure that the deformations remained within the linear viscoelastic region. The storage modulus (G′) and loss modulus (G″) were recorded as a function of the UV exposure period.

3.5. Characterization of pH-Responsive Swelling and Its Reversibility

Hydrogel discs (20 mm in diameter and 2 mm thick) prepared as described in Section 3.2 were subjected to pH-responsive swelling studies. The procedure began with an initial swelling step, in which the hydrogel discs were immersed in ultrapure water for 1 h. After this period, excess surface water was carefully removed using a Kimwipe, and the swollen hydrogels were weighed (W₁). Following the initial swelling, the pre-swollen hydrogels were immersed in pH buffer solutions ranging from 4 to 10 prepared as described in Section 3.3 for 24 h. Excess surface water was removed using a Kimwipe before weighing the hydrogels (W₂). The swelling ratio was then calculated using the following equation: swelling ratio = W₂/W₁. Here, W₁ represents the weight after initial swelling in ultrapure water, and W₂ is the weight after swelling in the pH buffer solution.

The reversibility of the pH response was evaluated using the following procedure. The prepared hydrogels were first immersed in ion-exchanged water for 1 h, after which their weights were measured. Next, the hydrogels were immersed in a pH 4 buffer solution for 3 days and reweighed. Before changing to the next buffer solution, the hydrogels were immersed in distilled water for 3 days. This was followed by immersing them in a pH 10 buffer solution for 3 days and weighing them again after 3 days of immersion in ion-exchanged water. Finally, after another 3 days of immersion in distilled water, the hydrogels were immersed in the pH 4 buffer solution for a second 3 day period and weighed once more. Thus, for the reversibility evaluation, the hydrogels were subjected to solvent exchanges in the order of pH 4, pH 10, and pH 4, with a 3 day distilled water immersion period between each buffer solution change. Similarly, the reversibility was also assessed by changing the solvent in the order of pH 10, pH 4, and pH 10, following the same procedure of distilled water immersion between buffer changes. To evaluate the stability and reversibility of the swelling behavior, measurements were performed twice on similarly adjusted gels and averaged. All of the measurements were performed at room temperature (25 ± 1 °C) using an analytical balance with an accuracy of 0.1.

3.6. Mechanical Properties at Different pH Values

The elastic modulus of the hydrogels at various pH values was determined using a rheometer (Creep Meter RE2-33005B, Yamaden, Japan) at room temperature (20 °C). For elasticity measurements, hydrogel samples were prepared by injecting 0.125 g of riboflavin-collagen solution into each well of a silicone mold with a flat bottom (well dimensions: 1 cm × 1 cm). UV irradiation was applied using the same equipment described in Section 3.2 to obtain collagen gels within the mold. The gels were prepared with UV irradiation times of 40 min and 120 min. Following gel formation, 0.125 g of the pH solutions prepared in Section 3.3 was added to each mold well containing the gel. The samples were then left to equilibrate for 1 d to ensure complete swelling. Push-in tests were performed on the gels while they remained in the mold cells. The measurement conditions were as follows: strain rate of 30%; measurement speed of 1 mm/s; contact area diameter of 3 mm; fixture diameter of 3 mm; and applied temperature of 24 °C. Stress–strain curves were obtained for 12 collagen gel samples. Young’s modulus was calculated from the slope of the linear region in each stress–strain curve.

The elasticity moduli of the hydrogels at various pH values were determined at the Analytical Research Center for Experimental Sciences of Saga University. This research was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) through the Advanced Research Infrastructure Sharing Promotion Program (Support Program for Introduction of New Shared Systems) under grant number JPMXS0422400020.