*2.1. Materials*

Sodium alginate (SA, M<sup>W</sup> = 1,106,340, G/M = 2:1), sodium periodate (NaIO4), D-Glucono-δ-lactone (GDL), nanosized hydroxyapatite (HAP), and sodium dihydrogen phosphate (NaH2PO4) were bought from Aladdin Chemical Reagent Co., Ltd., Shanghai, China. Anhydrous ethanol, hydrochloric acid, potassium chloride, sodium chloride (NaCl), sodium hydroxide (NaOH), and pH 7.4 phosphate buffer solution (PBS) were obtained from Zhongyao Chemical Reagent Co., Ltd., Beijing, China. Starch and ethylene glycol were bought from Macklin Co. Ltd., Shanghai, China. All chemicals were analytical grade and were used without further purification.

### *2.2. Methods*

### 2.2.1. Synthesis of Oxidized Sodium Alginate

The oxidation of SA was carried out in aqueous solution using NaIO<sup>4</sup> as the oxidant to prepare oxidized sodium alginate (OSA) with various theoretical oxidation degrees based on the reported methods [23,24]. Briefly, 5 g of SA was fully dissolved in 200 mL of distilled water in a dark bottle; then, 50 mL of anhydrous ethanol was added under vigorous stirring to obtain 2.0% (*w*/*v*) SA solution. Afterwards, a certain amount of NaIO<sup>4</sup> was added to initiate the oxidation reaction at 25 ◦C in N<sup>2</sup> atmosphere to obtain OSA. Various ratios of NaIO<sup>4</sup> to the number of repetitive uronic acid of alginate (5, 10, 15, or 20 mol %) were used, as shown in Table 1. Each reaction was performed for a period of 24 h until about 10 mL of ethylene glycol was incorporated to reduce the unreacted NaIO4. Subsequently, 4 times the volume of anhydrous ethanol and 4 g of NaCl were added to precipitate OSA; then, they were placed in the dialysis bag with a molecular weight cutoff of 3500 to dialyze against deionized water for 5 days. Finally, the dried OSA was obtained by freeze-drying the dialyzed OSA solution.


**Table 1.** Oxidation reaction parameters with NaIO<sup>4</sup> for OSA with various OD.

### 2.2.2. Determination of Oxidation Degree of OSA by UV–Vis Absorption Spectroscopy

The oxidation degree (OD) of OSA could be determined by UV–Vis absorption spectroscopy based on the difference between the initial and final amount of NaIO<sup>4</sup> during the oxidation reaction. Before adding the ethylene glycol to quench the oxidation reaction, 1 mL of reaction solution was transferred and diluted to 250 mL with distilled water. Then, 3.5 mL of this diluted solution was mixed with 1.50 mL of indicator solution that was prepared by mixing equal volumes of 20% (*w*/*v*) KI and 1% (*w*/*v*) soluble starch solutions, using pH 7.0 PBS as the solvent. The absorbance of diluted mixed solution was rapidly measured with a Shimadzu UV-1800 (Shimadzu, Kyoto, Japan) UV–visible spectrophotometer at 290 nm. The concentration of NaIO<sup>4</sup> in the solution was determined with a calibration curve whose linear regression equation was fitted as *<sup>y</sup>* = 0.2188 <sup>×</sup> *<sup>x</sup>*(10−<sup>5</sup> mol/L) + 0.0146 (R<sup>2</sup> = 0.9990), which was prepared by standard concentration of NaIO<sup>4</sup> in PBS in the range of 0.5~2.5 <sup>×</sup> <sup>10</sup>−<sup>5</sup> mol/L, as presented in Figure 3. Therefore, the actual OD of OSA was calculated by the amount of NaIO<sup>4</sup> according to the following equation:

$$OD = \frac{N\_{\bar{l}} - N\_r}{N\_o} \times 100\% \tag{1}$$

where *N<sup>i</sup>* , *N<sup>r</sup>* , and *No*, respectively, represent the initial moles of NaIO4, the residual moles of NaIO4, and initial moles of uronic acid of alginate.

of NaIO4, and initial moles of uronic acid of alginate.

**Figure 3.** Calibration curve of NaIO<sup>4</sup> as a function of its concentration.

### **Figure 3.** Calibration curve of NaIO4 as a function of its concentration. 2.2.3. Characterization of Oxidized Sodium Alginate

2.2.3. Characterization of Oxidized Sodium Alginate The successful synthesis of OSA was confirmed by Fourier transform infrared spectrophotometer (FT-IR), 1H nuclear magnetic resonance (1H NMR), and X-ray Photoelectron Spectroscopy (XPS). The FT-IR measurement was performed on disks that were prepared by compressing the mixture of KBr and a slight amount of sample using a Nicolet-6700 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA). The FT-IR spectra of the sample were recorded in the range of wavenumbers between 4000 and 400 cm−1 for 64 scans with a spectral resolution of 4.0 cm−1. The 1H NMR spectroscopy of the sample that was dissolved in D2O (99%) with a concentration of 10 mg/mL was recorded on an AV 400 NMR nuclear magnetic resonance spectrometer (Bruker, Ettlingen, Germany) at 25 °C. The surface elemental composition of the sample was examined by XPS using an axis ultra DLD apparatus (Kratos, Manchester, UK), which was equipped with a monochromatic Al KR X-ray source operating at 15 kV, 10 mA (150 W). The spectra were collected in fixed analyzer transmission mode (FAT): survey scans at 1200~0 eV with 1.0 eV steps at an analyzer pass energy of 160 eV; narrow scans at 0.1 eV steps at an analyzer pass energy of 20 eV. The crystalline structure and thermal stability of OSA were examined by X-ray Diffraction (XRD) and thermogravimetric analysis (TGA). The XRD pattern was performed on an AXS/D8 X-ray diffractometer (Bruker, Cambridge, UK) with The successful synthesis of OSA was confirmed by Fourier transform infrared spectrophotometer (FT-IR), <sup>1</sup>H nuclear magnetic resonance (1H NMR), and X-ray Photoelectron Spectroscopy (XPS). The FT-IR measurement was performed on disks that were prepared by compressing the mixture of KBr and a slight amount of sample using a Nicolet-6700 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA). The FT-IR spectra of the sample were recorded in the range of wavenumbers between 4000 and 400 cm−<sup>1</sup> for 64 scans with a spectral resolution of 4.0 cm−<sup>1</sup> . The <sup>1</sup>H NMR spectroscopy of the sample that was dissolved in D2O (99%) with a concentration of 10 mg/mL was recorded on an AV 400 NMR nuclear magnetic resonance spectrometer (Bruker, Ettlingen, Germany) at 25 ◦C. The surface elemental composition of the sample was examined by XPS using an axis ultra DLD apparatus (Kratos, Manchester, UK), which was equipped with a monochromatic Al KR X-ray source operating at 15 kV, 10 mA (150 W). The spectra were collected in fixed analyzer transmission mode (FAT): survey scans at 1200~0 eV with 1.0 eV steps at an analyzer pass energy of 160 eV; narrow scans at 0.1 eV steps at an analyzer pass energy of 20 eV. The crystalline structure and thermal stability of OSA were examined by X-ray Diffraction (XRD) and thermogravimetric analysis (TGA). The XRD pattern was performed on an AXS/D8 X-ray diffractometer (Bruker, Cambridge, UK) with Cu-Kα radiation (λ = 0.154 nm). The measurement was conducted in a step scan mode at a scanning speed of 0.025◦/s over a 2θ range of 5◦~60◦ . The thermal property of OSA was determined by a Q600 thermogravimetric analyzer (TA Instrument, New Castle, DE, USA). The TGA test was carried out under a N<sup>2</sup> atmosphere at a heating rate of 10 K/min with the range of 30~800 ◦C.

<sup>×</sup>100% <sup>−</sup> <sup>=</sup>

*<sup>N</sup> <sup>N</sup> OD* (1)

*o*

where *Ni*, *Nr*, and *No*, respectively, represent the initial moles of NaIO4, the residual moles

*i r N*

### Cu-Kα radiation (λ = 0.154 nm). The measurement was conducted in a step scan mode at 2.2.4. In Vitro Biodegradation of Oxidized Sodium Alginate

a scanning speed of 0.025°/s over a 2θ range of 5°~60°. The thermal property of OSA was determined by a Q600 thermogravimetric analyzer (TA Instrument, New Castle, DE, USA). The TGA test was carried out under a N2 atmosphere at a heating rate of 10 K/min with the range of 30~800 °C. 2.2.4. In Vitro Biodegradation of Oxidized Sodium Alginate The in vitro biodegradation of OSA was performed at 37 °C using PBS buffer containing 10,000 U/mL lysozyme to simulate the physiological conditions. A total 0.2 g The in vitro biodegradation of OSA was performed at 37 ◦C using PBS buffer containing 10,000 U/mL lysozyme to simulate the physiological conditions. A total 0.2 g of OSA with various ODs was immersed in the 100-mL PBS buffer for 60 days. At different time intervals (6 days, 12 days, 20 days, 28 days, 38 days, 48 days, and 60 days), 2 mL aliquot of samples were withdrawn and their molecular weights were determined by an e2695 Gel Permeation Chromatography equipped with an UltrahydrogelTM120 (7.8 <sup>×</sup> 300 mm<sup>2</sup> ) column (Waters, Milford, MA, USA). A total 0.05% sodium azide was used as the mobile phase with a flow rate of 0.6 mL/min at 40 ◦C. Similarly, each time interval for the sample was tested in parallel for 3 times to take the average value. Within the time interval, the biodegradation of OSA could be evaluated by its weight-average molecular weight (MW).

of OSA with various ODs was immersed in the 100-mL PBS buffer for 60 days. At different time intervals (6 days, 12 days, 20 days, 28 days, 38 days, 48 days, and 60 days), 2 mL

### 2.2.5. Gelation Ability of Oxidized Sodium Alginate homogeneous alginate hydrogel to be obtained by the control of the release of Ca2+ from

2.2.5. Gelation Ability of Oxidized Sodium Alginate

weight (MW).

*Polymers* **2022**, *14*, x FOR PEER REVIEW 6 of 15

aliquot of samples were withdrawn and their molecular weights were determined by an e2695 Gel Permeation Chromatography equipped with an UltrahydrogelTM120 (7.8 × 300 mm2) column (Waters, Milford, MA, USA). A total 0.05% sodium azide was used as the mobile phase with a flow rate of 0.6 mL/min at 40 °C. Similarly, each time interval for the sample was tested in parallel for 3 times to take the average value. Within the time interval, the biodegradation of OSA could be evaluated by its weight-average molecular

To examine the gelation ability of OSA, the internal gelation of OSA was conducted based on our previous work [33]. As shown in Scheme 1, this method allowed

To examine the gelation ability of OSA, the internal gelation of OSA was conducted based on our previous work [33]. As shown in Scheme 1, this method allowed homogeneous alginate hydrogel to be obtained by the control of the release of Ca2+ from the insoluble hydroxyapatite (HAP) in the presence of D-glucono-δ-lactone (GDL), which avoided the unbalanced crosslinking density, thus enhancing the mechanical property and homogeneity of the hydrogel [34,35]. In detail, a certain amount of HAP was ultrasonically dispersed in 2% (*w*/*v*) OSA solution and stirred vigorously until a homogenous solution was formed. Then, the ion cross-linking of OSA was initiated by the addition of a certain amount of GDL under the magnetic stirring. In this study, HAP–GDL complex with the molar ratio of 1:10 was used as the cross-linking system, and the molar ratio of Ca2+ from HAP and carboxyl from OSA was fixed at 0.18. After stirring at high speed for 3 min, the mixture was quickly transferred into a 12-well tissue culture plate and physically cross-linked at 4 ◦C for 24 h to obtain homogeneous OSA hydrogel. The resultant OSA hydrogel was left for 30 min to eliminate any air bubbles for further rheological measurement. The rheological properties of the OSA hydrogel with various ODs were analyzed by steady shear test and dynamic sweep measurements using a rotational rheometer with parallel-plate geometry (DHR TA Instruments, New Castle, DE, USA) at 25 ◦C. Steady shear measurements were carried out to record the apparent viscosity (η) with the shear rate ranging from 0.1 to 1000 s−<sup>1</sup> , while the oscillation frequency sweep measurements were conducted to record storage modulus G0 and loss modulus G00 with the angular frequencies (ω) ranging from 0.1 to 100 rad/s and the strain amplitude was fixed at 1%. the insoluble hydroxyapatite (HAP) in the presence of D-glucono-δ-lactone (GDL), which avoided the unbalanced crosslinking density, thus enhancing the mechanical property and homogeneity of the hydrogel [34,35]. In detail, a certain amount of HAP was ultrasonically dispersed in 2% (*w*/*v*) OSA solution and stirred vigorously until a homogenous solution was formed. Then, the ion cross-linking of OSA was initiated by the addition of a certain amount of GDL under the magnetic stirring. In this study, HAP–GDL complex with the molar ratio of 1:10 was used as the cross-linking system, and the molar ratio of Ca2+ from HAP and carboxyl from OSA was fixed at 0.18. After stirring at high speed for 3 min, the mixture was quickly transferred into a 12-well tissue culture plate and physically cross-linked at 4 °C for 24 h to obtain homogeneous OSA hydrogel. The resultant OSA hydrogel was left for 30 min to eliminate any air bubbles for further rheological measurement. The rheological properties of the OSA hydrogel with various ODs were analyzed by steady shear test and dynamic sweep measurements using a rotational rheometer with parallel-plate geometry (DHR TA Instruments, New Castle, DE, USA) at 25 °C. Steady shear measurements were carried out to record the apparent viscosity (η) with the shear rate ranging from 0.1 to 1000 s−1, while the oscillation frequency sweep measurements were conducted to record storage modulus G′ and loss modulus G″ with the angular frequencies (ω) ranging from 0.1 to 100 rad/s and the strain amplitude was fixed at 1%.

**Scheme 1.** Schematic representation of the preparation of OSA and its ionic crosslinking by HAP– **Scheme 1.** Schematic representation of the preparation of OSA and its ionic crosslinking by HAP–GDL.

GDL. 2.2.6. In Vitro Cytotoxicity of Oxidized Sodium Alginate Hydrogel

2.2.6. In Vitro Cytotoxicity of Oxidized Sodium Alginate Hydrogel To verify the application potential of OSA hydrogels in the biomedical field, the osteoblastic MC3T3-E1 cells cultured with the culture medium containing 90% MEM-α, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin were applied to examine their cytocompatibility by using the Cell Counting Kit-8 (CCK-8) assay. The OSA—10% hydrogels were cut into circular disks with a diameter of 20 mm and a height To verify the application potential of OSA hydrogels in the biomedical field, the osteoblastic MC3T3-E1 cells cultured with the culture medium containing 90% MEM-α, 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin were applied to examine their cytocompatibility by using the Cell Counting Kit-8 (CCK-8) assay. The OSA—10% hydrogels were cut into circular disks with a diameter of 20 mm and a height of 10 mm; then, they were sterilized by cobalt 60 radiation with the irradiation intensity of 8 kGy. The MC3T3-E1 cells were seeded on the OSA—10% hydrogels in the 24-well tissue culture plates at a density of 5 <sup>×</sup> <sup>10</sup><sup>4</sup> per well, while the same cells were seeded on the tissue culture plates as a blank control. Afterwards, the culture medium was replenished to make the total amount of medium per well reach up to 500 µL. Subsequently, they were transferred to an incubator containing 5% CO2, 95% air, and 100% relative humidity at 37 ◦C and their culture media were replaced every 2 days. After 2 days and 5 days incubation, 50 µL of CCK-8 reagent was added to 500 µL of medium in each well, and they were placed in the incubator at 37 ◦C for 4 h. Finally, 100 µL of solution from each well was transferred

to a 96-well plate, whose absorbance value (OD) was determined by an X-mark microplate reader (Bio-rad, Hercules, CA, USA) at a wavelength of 450 nm. Since the OD of the cell medium on the OSA—10% hydrogel is proportional to the number of living cells, the cell viability of the MC3T3-E1 cells on the OSA—10% hydrogel could be judged by comparing the OD values.

### **3. Results and Discussion**
