The following materials were used: i) CS powder (95% deacetylation degree; cat. no. C105799; Aladdin Scientific Corp.), ii) β-GP pentahydrate (cat. no. D106347; Aladdin Scientific Corp.), iii) sodium fluorescein (NaF; cat. no. F105615; Aladdin), iv) TiO₂ NPs (20-40 nm; cat. no. NM000800; Beijing Solarbio Science & Technology Co., Ltd.), v) L929 murine fibroblast cells (cat. no. KGG1306-1), vi) RPMI-1640 medium (containing newborn calf serum, double antibiotics; cat. no. KGL1509-500), vii) Cell Counting Kit-8 (CCK-8) cell proliferation assay kit (cat. no. KGA9305-500), viii) LIVE/DEAD cell viability assay kit (cat. no. KGA9501-1000), ix) bovine serum albumin (BSA) (standard grade, heat-treated) (cat. no. KGL2314-10), x) bicinchoninic acid (BCA) protein quantification assay kit (cat. no. KGB2101-250), all from Jiangsu KeyGen Biotech Co., Ltd., xi) mouse anti-proliferating cell nuclear antigen (PCNA) antibody (1:1,000; cat. no. BM0104) and xii) goat anti-mouse IgG/HRP antibody (1:10,000; cat. no. BA1056) both from Boster Biological Technology Co. Ltd.

The primary apparatus were the following: i) CO₂ cell culture incubator, ii) laboratory heating magnetic stirrer, iii) Fourier-transform infrared spectrometer (Nicolet 8700; Thermo Fisher Scientific, Inc.), iv) rheometer (TA Instruments), v) inverted fluorescence microscope (Zeiss GmbH); vi) electrophoresis system, vii) blotting apparatus (Bio-Rad Laboratories, Inc.), viii) automated digital gel imaging system (Tanon GIS 2010; Tanon Science and Technology Co., Ltd.), ix) LAS X4.4.0 (Leica Microsystems GmbH), x) microplate reader (Anthos ht2; Anthos Labtec), xi) mechanical testing machine (ZwickRoell; Zwick GmbH & Co.) and xii) Vibra-Cell^TM ultrasonic disruptor (Sonics & Materials, Inc.).

Preparation of CS solution. CS powder (95% deacetylation degree) was dissolved in an ice acetic acid solution (2% w/v concentration in 0.1 mol/l solution). The mixture was stirred at 25˚C overnight until complete dissolution. After filtration through a 150-mesh silk screen, the solution was allowed to stand and underwent high-pressure steam sterilization (151˚C for 15 min), followed by storage at 4˚C.

Preparation of β-GP solution. β-GP powder (5.6 g) was dissolved at a concentration of 56% w/v in deionized water, stirred at room temperature for 10 min until complete dissolution, and then sterilized by filtration through a 0.2-µm filter. The solution was stored at 4˚C.

Preparation of TiO₂ NP solution. After 6 h of ultraviolet sterilization, the TiO₂ NP powder was added to deionized water at a concentration of 1% w/v. The mixture was sonicated (the ultrasonic power was 240W) to obtain a uniform dispersion and was immediately used.

CS/β-GP/TiO₂ NP hydrogel preparation. The CS solution was then stirred in an ice bath using a magnetic stirrer. The β-GP solution was added dropwise in a 9:1 ratio, followed by continuous stirring at room temperature for 10 min. Subsequently, different proportions (0, 0.5, 1, and 1.5%) of TiO₂ NP solution were added dropwise to the prepared CS/β-GP solution, followed by 20 min of stirring at room temperature. The mixture was sealed and placed in a 37˚C water bath incubator to solidify for 24 h.

Fourier-transform infrared spectroscopy (FTIR) was used to analyze the formation of copolymers in the hydrogels. Hydrogels with varying contents of TiO₂ NPs were prepared, and FTIR was conducted in the wavelength range of 400-4,000 cm^-1 with four scans obtained per sample. The scanning resolution was set at 4 cm^-1, and the scanning speed was 0.2 mm/sec.

Hydrogels with varying TiO₂ NP content were analyzed using a rheometer with a cone and plate geometry featuring a cone angle of 2˚, a diameter of 20 mm, and a gap of 0.054 mm. The plate was equilibrated to the initial temperature (25˚C), and a temperature ramp test was performed in the range of 25-50˚C, for 250 min. The ramp rate was 0.1˚C/min, the frequency was 1.5 Hz, the shear strain was controlled at 1%, and the angular frequency was set to 10 rad/sec. Data were collected every second to determine solid-state transition temperatures. Cold traps were employed in all the rheometers to minimize solvent evaporation.

The injectability of the hydrogel was evaluated using a mechanical testing machine equipped with a 5 kN load cell. During the test, 10 ml of each hydrogel were loaded into a custom injection mold. The test was conducted at room temperature with a displacement of 5 mm and a crosshead speed of 30 mm/min, simulating the speed of manually injecting the hydrogel from a syringe. Each experiment was repeated three times to ensure accuracy and reliability. The load required as function of piston displacement was measured, and the average maximum force value for each composition was determined.

Cell proliferation toxicity test (CCK-8 assay). L929 murine fibroblast cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibiotics, according to the manufacturer's instructions. Upon reaching 90% confluence, cells were trypsinized and passaged during the logarithmic growth phase. L929 cells were seeded at a density of 4x10³ cells/well in 96-well plates. After 24 h of culture under 37˚C and 5% CO₂, the original culture medium was aspirated. The control group received 100 µl RPMI-1640 medium, while the other groups were treated with 90 µl RPMI-1640 medium (containing fetal bovine serum and double antibiotics) along with 10 µl of different concentrations of CS/β-GP/TiO₂ NP hydrogel (0, 0.5, 1 and 1.5%). On days 1, 3, 5 and 7, the CCK-8 cell proliferation toxicity assay was performed (concentration, 10%). The supernatants were collected, and the optical density (OD) at a wavelength of 450 nm was measured using an ELISA reader.

Cell viability and cytotoxicity staining. L929 cells were seeded at a density of 8x10³ cells/well in a 96-well plate. After 24 h of culture under 37˚C and 5% CO₂, the original culture medium was aspirated. The control group received 100 µl RPMI-1640 medium, while the other groups were treated with 90 µl RPMI-1640 medium (containing fetal bovine serum and double antibiotics) along with 10 µl of different concentrations of CS/β-GP/TiO₂ NP hydrogel (0, 0.5, 1 and 1.5%). After 24 h of incubation at 37˚C, the cells were stained using Calcein-AM and PI dyes (included in the LIVE/DEAD cell viability assay kit). The cells were incubated with the staining solution for 2 h at 37˚C and observed under an inverted fluorescence microscope at an excitation wavelength of 490 nm for live cells and 545 nm for dead cells.

Western blot analysis. L929 cells in good growth condition were seeded at a density of 5x10⁵ cells/well in 6-well plates. When cell growth reached 80-90% confluence, the intervention groups were treated with 200 µl of CS/β-GP/TiO₂ NP hydrogel (0, 0.5, 1 and 1.5%) along with 1.8 ml of RPMI-1640 medium. The control group received 2 ml of the medium solution. After 24 h of incubation, the total cellular protein was extracted using Cell Lysis Buffer for Western or IP (cat. no. P0013; Beyotime Insitute of Biotechnology), and the protein concentration was determined using a BCA protein quantification assay kit. Samples were boiled in 5X loading buffer for 10 min. For western blot analysis, 20 µg of protein was loaded per lane to ensure consistent sample loading. Electrophoresed at 80 V at a constant voltage until they entered the separating gel (10%), and then transferred to a PVDF membrane. The membrane was blocked with TBST buffer containing 5% skim milk powder at room temperature for 1 h. Mouse anti-PCNA antibody (1:1,000) was added and incubated overnight at 4˚C. After washing the membrane with TBST (including 0.1% Tween-20), goat anti-mouse IgG/HRP antibody (1:10,000) was added and the membrane was incubated for 1 h at 4˚C. Protein expression was detected using a highly sensitive ECL luminescence reagent (Epizyme Biomedical Technology). β-actin (cat. no. BA2305; Boster Biological Technology Co., Ltd.) was used as the reference protein. ImageJ 1.45 (National Institutes of Health) was used for densitometric analysis. PCNA is a sensitive and specific marker of cell proliferation, capable of accurately reflecting the proliferative activity of cells. It has been widely used in biocompatibility studies of various biomaterials, especially in the fields of tissue engineering and regenerative medicine (11-14).

Ultrasound-triggered release of NaF from CS/β-GP/TiO₂ NP hydrogel. NaF, a water-soluble fluorescent dye, was used as a small-molecule drug analog to assess the ultrasound-controlled release of the hydrogel. Two hydrogel formulations were evaluated: i) CS/β-GP and ii) CS/β-GP/TiO₂ NPs containing 1.5% TiO₂ NPs. A total of 10 mg of NaF salt were added to 10 ml of double-distilled water, resulting in a NaF solution with a concentration of 1 mg/ml. During the preparation of composite hydrogels, the NaF solution was incorporated into the hydrogel mixture at a ratio of 100 µl/ml before mixing β-GP into the hydrogels, ensuring the uniform distribution of NaF. For release studies, a 1-ml sample of the hydrogel was immersed in 5 ml of a buffer medium consisting of 0.01 M calcium- and magnesium-free Dulbecco's phosphate-buffered saline (DPBS; cat. no. KGL2208-500; Jiangsu KeyGen Biotech Co., Ltd.) at pH 7.4 and maintained at 37˚C throughout the testing period. The diffusion-mediated release of NaF was evaluated at 0, 5, and 24 h after incubation without ultrasound treatment.

The ultrasound treatment was performed as revealed in Table I. The approach involved applying ultrasound for 5 h at time point 0 (ultrasound intensity=9.6 mW/cm²; 25% amplitude; 2.5 min/h; 37˚C; 5-h cycles). This scheme was based on previous studies conducted by Huebsch et al (15) and Levingstone et al (16). The amount of NaF released after ultrasound application was compared with the control group without ultrasound treatment at the 5-h time point. At each time point, 1 ml of the supernatant was collected, and the absorbance at 512 nm was measured using an ELISA reader to assess the NaF concentration.

Ultrasound-triggered release of BSA from CS/β-GP/TiO₂ NP hydrogel. Subsequently, the ultrasound-controlled release of BSA was investigated as a large-molecule drug/protein analog. For BSA release studies, two hydrogel compositions were tested: i) CS/β-GP and ii) CS/β-GP/TiO₂ NPs containing 1.5% TiO₂ NPs, prepared as aforementioned. BSA was added to double-distilled water to achieve a concentration of 10 mg/ml and then added to the hydrogel to create a 1 mg/ml BSA solution. Subsequently, 1 ml of the hydrogel sample was suspended in a buffer medium containing 5 ml of 0.01 M calcium-magnesium-free DPBS at pH 7.4 and 37˚C.

Release studies were conducted at 0, 5, 24, 29, 72 and 77 h without ultrasound application. Ultrasound treatment was performed using three different approaches, as shown in Table I. For the first ultrasound treatment, ultrasound was applied for 5 h at time point 0 (ultrasound intensity=9.6 mW/cm²; 25% amplitude; 2.5 min/h; 37˚C; 5-h cycles) (15,16). For the second ultrasound treatment, hydrogels were incubated for 24 h at 37˚C, followed by 5-h ultrasound treatment using the first ultrasound treatment protocol. For the third ultrasound treatment, hydrogels were incubated for 72 h at 37˚C, followed by 5-h ultrasound treatment using the first protocol. The results at the same time points were compared with those of the diffusion-based release control group without ultrasound treatment.

BSA was quantified using a BCA protein quantification assay kit. The assay was conducted according to the manufacturer's instructions, and the absorbance at 562 nm was measured using an ELISA reader to determine BSA concentration.

Unless otherwise specified, the experiments were conducted in triplicate. The data results were generally reported as the mean ± standard deviation (SD) and analyzed statistically based on this format. Statistical analyses were performed using GraphPad Prism 8 software (GraphPad; Dotmatics) and SPSS 25.0 (IBM Corp.). Statistical significance was determined using the unpaired t-test and one-way analysis of variance (ANOVA). Tukey's post hoc test was used after ANOVA. P<0.05 was considered to indicate a statistically significant dofference.

Composite hydrogels with TiO₂ NP contents of 0, 0.5, 1, and 1.5% in CS/β-GP/TiO₂ NPs were successfully synthesized.

Characterization analysis of pure CS/β-GP and CS/β-GP/TiO₂ NPs was conducted using FTIR, confirming the successful incorporation of TiO₂ NPs at different concentrations into the hydrogels (Fig. 1). The wide absorption peak at 3,600-3,200 cm^-1 corresponded to the stretching vibrations of hydroxyl groups (O-H) and amino groups (N-H). The peaks at 2,932 and 2,856 cm^-1 represented the antisymmetric and symmetric stretching vibrations of methylene groups (CH₂), respectively. The absorption peak at 1,648 cm^-1 corresponded to the amide I band stretching vibration of C=O. The peak at 1,557 cm^-1 corresponded to the bending vibration of C-N-H in the amide II band. The peak at 1,384 cm^-1 corresponded to the symmetric bending vibration of methyl groups (C-CH₃). The peaks at 1,122 and 1,079 cm^-1 corresponded to the stretching vibrations of C-O, while those at 1,079 and 974 cm^-1 corresponded to the antisymmetric and symmetric stretching vibrations of phosphate groups, respectively. The peak at 783 cm^-1 was possibly associated with the swinging vibration of C-H.

With increasing TiO₂ NP content, the absorption peak at 3,600-3,200 cm^-1 broadened, indicating the introduction of more hydroxyl groups (O-H), possibly owing to the residual water in TiO₂ NPs. The absorption peak of the amide I band C=O stretching vibration shifted from 1,628 cm^-1 to 1,648 cm^-1, indicating a blue shift. The peaks at 1,557 cm^-1, 1,384 cm^-1, and 1,300-700 cm^-1 intensified, which might be attributed to the vibrations of organic compounds present in TiO₂ NPs. New absorption peaks appeared at 500-700 cm^-1, possibly corresponding to the stretching and bending vibrations of Ti-O in the introduced TiO₂ NPs.

The thermal response characteristics of all the hydrogels were evaluated by rheological analysis. The solidification temperatures of pure CS/β-GP hydrogel and hydrogels with added TiO₂ NPs were found to be in the range of 36.5-37.3˚C (Fig. 2).

Injectability experiments were conducted to assess the feasibility of the prepared hydrogels as clinical injection materials. No significant differences in the injection forces between the four hydrogel groups were observed (Fig. 3). The injection force for the CS/β-GP group was 5.79±0.57 N, for the CS/β-GP + 0.5% TiO₂ NP group it was 5.63±0.41 N, for the CS/β-GP + 1.0% TiO₂ NP group it was 5.54±0.67 N, and for the CS/β-GP + 1.5% TiO₂ NP group it was 5.70±0.49 N. Additionally, all these values were lower than the maximum force (22.6 N) that a surgeon's hand can comfortably apply to an injector (17). This indicates that all the hydrogel formulations prepared were suitable for use as injection materials in a clinical setting.

The doubling time of L929 cells is generally 28-36 h. In the present experiment, the number of L929 cells increased by only ~2-fold over 7 days, which is indeed lower than the typically expected proliferation rate. It is considered that the possible reasons for this could be the use of a different culture medium or a lower initial cell density at the beginning of the experiment. The cell count increased over time, and the absorbance values at various time points after seeding the cells onto different composite hydrogels were not significantly different from those of the control group, which was cultured in conventional plastic 96-well plates (Fig. 4). There were no statistically significant differences between the groups (P>0.05).

Cell viability staining accurately distinguished live from dead cells on the material surface. The survival rates of cells co-cultured with various hydrogel formulations were not significantly different from those of the control group (Fig. 5), which were consistent with the CCK-8 results. Both the CCK-8 assay and cell viability staining indicated that the prepared composite hydrogels exhibited excellent biocompatibility. Due to the experimental results showing a very clear distinction between live and dead cells, with almost no dead cells detected across all groups and a live cell proportion close to 100%, further quantitative analysis was not conducted.

After co-culturing L929 cells with various hydrogel groups for 24 h, western blot analysis was performed to assess protein PCNA expression. No significant differences in cellular protein expression among the different groups were observed (P>0.05) (Fig. 6).

Considering the results from the injectability test, the CCK-8 cell proliferation assay, the cell viability staining experiment, and given that all CS/β-GP and TiO₂ NP polymer composites exhibited the required properties for injectable hydrogels and showed no cell toxicity, the CS/β-GP + 1.5% TiO₂ NP hydrogel was selected for further investigation into ultrasound-triggered drug release. This hydrogel was opted due to the high percentage of the sonosensitizer TiO₂ NPs.

The release of NaF from CS/β-GP and CS/β-GP + 1.5% TiO₂ NP thermosensitive hydrogels upon ultrasound stimulation was evaluated. When hydrogel samples were suspended in DPBS at 37˚C, the hydrogel remained in a gel state. After treatment with an ultrasonic disruptor, the main changes observed in the hydrogel samples were local structural damage or disintegration, rather than complete dissolution. The analysis of diffusion-related release for each hydrogel group revealed that NaF was gradually released over time, with up to 33.15±0.89% release observed within 24 h solely due to diffusion. At the 0 and 5-h time points, the incorporation of TiO₂ NPs into the thermo-responsive hydrogel had no significant impact on the NaF diffusion-related release. However, at the 24-h time point, the NaF release level from the CS/β-GP + 1.5% TiO₂ NP group was significantly lower than that from the CS/β-GP group (P<0.01) (Fig. 7A). This suggests that hydrogels containing TiO₂ NPs may reduce diffusion-related NaF release. Ultrasound increased the release of NaF from both hydrogel groups. For the CS/β-GP group, ultrasound treatment method 1 resulted in 24.16±1.32% NaF release, significantly increasing NaF release compared with the control group at the same time point (5 h; 16.05±0.07%; P<0.01). For the CS/β-GP + 1.5% TiO₂ NP group, the first ultrasound treatment method led to 27.77±0.98% NaF release, significantly increasing NaF release compared with the control group at the same time point (18.21±0.48%; P<0.01). Furthermore, using the first ultrasound treatment method, the release of NaF from the CS/β-GP + 1.5% TiO₂ NP group was significantly higher than that from the CS/β-GP group (P<0.01) (Fig. 7B).

The ultrasound-triggered release of BSA was evaluated for CS/β-GP and CS/β-GP + 1.5% TiO₂ NP hydrogels. The analysis of diffusion-related BSA release for each hydrogel group revealed relatively low BSA release levels over time, with 6.63±0.50% and 6.97±0.30% BSA released from CS/β-GP and CS/β-GP + 1.5% TiO₂ NP groups, respectively, at the 72-h time point (Fig. 8A). No significant differences in diffusion-related BSA release between CS/β-GP and CS/β-GP + 1.5% TiO₂ NP groups was observed. And under the experimental conditions, no significant degradation or disintegration of the hydrogel network was observed within 72 h. The physical integrity of the hydrogels remained intact. Ultrasound application increased the release of BSA from pure CS/β-GP and CS/β-GP + 1.5% TiO₂ NP hydrogels. For the CS/β-GP hydrogel, the BSA release recorded after ultrasound treatment method 1 was 24.94±1.82% (Fig. 8B), which was significantly higher than that for the control group at the same time point (5 h), which released only 3.46±0.34% BSA. Compared with the BSA level recorded for the first ultrasound treatment method, the levels recorded for the second and third ultrasound treatment methods were slightly lower, possibly due to BSA degradation over time. Notably, in the first ultrasound treatment method, the incorporation of TiO₂ NPs into the thermo-responsive hydrogel increased BSA release, with the CS/β-GP + 1.5% TiO₂ NP group releasing 49.42±0.55%, which was significantly higher than the release from the CS/β-GP group (24.94±1.82%). Similar increases were observed for the second and third ultrasound treatment method (Fig. 8C and D).