*3.1. Oxidation of Alginate via NaIO<sup>4</sup>*

Since there are a large number of hydrophilic groups such as hydroxyl and carboxyl groups on the backbone of alginate molecules, they can easily form intramolecular hydrogen bonds, resulting in the strong and rigid molecular structure of alginate, which may restrict its scope of application in drug delivery [36,37]. However, the inert dihydroxy groups on the alginate backbone can be oxidized to generate the reactive dialdehyde groups by exploiting the oxidative properties of NaIO4, which significantly improved its biodegradability [23,38]. The cleavage of the C2–C3 bond of alginate uronic acid monomer could occur in the oxidation reaction of NaIO4, which strictly destroyed the rigid structure of the alginate molecular backbone, thus enhancing its molecular flexibility [24]. In this work, the SA was partially oxidized to a theoretical extent (5%, 10%, 15%, and 20%) and the oxidation degree (OD)—defined as the percentage of oxidized uronic acid groups in the alginate—was determined by the consumption of NaIO<sup>4</sup> with the results shown in Table 1. It was obvious that the OD of OSA increased with the increase in the amount of NaIO4, and the actual OD was close to the theoretical OD. This result directly indicated that most of the added NaIO<sup>4</sup> participated in the oxidation reaction, and the oxidation reaction of alginate with NaIO<sup>4</sup> was feasible and active.

### *3.2. Molecular Structure and Thermal Stability of OSA*

The molecular structure and specific functional groups of the resultant OSA could be confirmed by FT-IR and <sup>1</sup>H NMR spectroscopy. As shown in Figure 4a, both SA and OSA revealed the broad hydroxyl stretching vibration absorption peaks in the range of 4000~3000 cm−<sup>1</sup> [39]. In detail, SA exhibited main characteristic peaks at 2926, 1616, and 1417 cm−<sup>1</sup> owing to the C-H stretching vibration of the polysaccharide structure and the asymmetric and symmetric stretching vibration of -COO− [40]. Additionally, the absorption peaks at 1030 cm−<sup>1</sup> were attributed to the C-O stretching vibration on the polysaccharide skeleton [41]. In comparison with SA, OSA had a new characteristic peak at 1734 cm−<sup>1</sup> , which was assigned to the vibration absorption peak of the C=O bond on the aldehyde group. This peak was too weak to be detected for the hemiacetal formation of the free aldehyde groups [42]. In addition, the hydroxyl stretching vibration absorption peak at 3437 cm−<sup>1</sup> in the spectrum of SA became blue-shifted to 3443 cm−<sup>1</sup> in the spectrum of OSA, implying a decline in the amount of -OH groups of alginate. These results indicated that the adjacent hydroxyl groups at the C-2 and C-3 positions on alginate uronic acid monomer were oxidized to aldehyde groups by NaIO<sup>4</sup> [25,41]. Moreover, OSA displayed similar FT-IR spectroscopy results to the raw SA, which demonstrated the periodate ion only cleaved the C2–C3 linkage by the oxidation reaction, leading to the formation of a dialdehyde [24].

Figure 4b shows the <sup>1</sup>H NMR spectra of SA and OSA. The proton peaks of SA and OSA ranging from 5.0 to 3.5 ppm were assigned to the hydrogen atoms of native alginate backbone [40]. Compared with SA, the two new proton peaks were discovered at 5.3 and 5.6 ppm in the spectrum of OSA, which were attributed to a hemiacetalic proton generated from the hydroxyl groups of aldehyde and its neighbors, confirming the achievement of the oxidation with NaIO<sup>4</sup> [23]. Moreover, the proton peaks of SA appearing at 3.7~3.55 ppm that were assigned to the H2 of α-L-guluronic acid decreased and moved to the high field for cleavage of the C2–C3 bond of the uronic acid monomer [24]. The appearance of these new proton signal peaks and the change of the proton signal peaks also directly proved the successful oxidation of alginate with NaIO4.

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**Figure 4.** (**a**) FT-IR spectra and (**b**) 1H NMR spectra of SA and OSA. **Figure 4.** (**a**) FT-IR spectra and (**b**) <sup>1</sup>H NMR spectra of SA and OSA. confirmed the presence of the anticipated peaks for Na (1069.02 eV), O (530.23 eV), N

Figure 4b shows the 1H NMR spectra of SA and OSA. The proton peaks of SA and OSA ranging from 5.0 to 3.5 ppm were assigned to the hydrogen atoms of native alginate backbone [40]. Compared with SA, the two new proton peaks were discovered at 5.3 and 5.6 ppm in the spectrum of OSA, which were attributed to a hemiacetalic proton generated from the hydroxyl groups of aldehyde and its neighbors, confirming the achievement of the oxidation with NaIO4 [23]. Moreover, the proton peaks of SA appearing at 3.7~3.55 ppm that were assigned to the H2 of α-L-guluronic acid decreased and moved to the high field for cleavage of the C2–C3 bond of the uronic acid monomer [24]. The appearance of these new proton signal peaks and the change of the proton signal peaks also directly proved the successful oxidation of alginate with NaIO4. As a complementary technique, XPS was performed to further verify the surface As a complementary technique, XPS was performed to further verify the surface composition of resultant OSA. As shown in Figure 5a, XPS spectra of SA and OSA confirmed the presence of the anticipated peaks for Na (1069.02 eV), O (530.23 eV), N (397.54 eV), and C (284.68 eV), with an O/Na ratio close to the theoretical value of 6 [43], indicating that partial oxidation of alginate by NaIO<sup>4</sup> preserved the main structure and functional groups of alginate. In addition, the peak assignment of XPS C1s narrow scans of SA and OSA that could elucidate the bonding atmosphere has been marked in the spectra in Figure 5b according to the previous report [44]. It can be observed that the C1s narrow scan revealed three peaks at 285.46 eV (O–C–O), 283.87 eV (C–O), and 282.20 eV (C–C), respectively. It is found that the peak ratio of the O–C–O species to the C–O species in the C1s narrow scan of OSA was significantly higher than that of SA, due to the partial oxidation of alginate by NaIO4, which was consistent with previous results reported by Jejurikar et al. [24]. Thus, these results further verified the successful partial oxidation of alginate by NaIO4. (397.54 eV), and C (284.68 eV), with an O/Na ratio close to the theoretical value of 6 [43], indicating that partial oxidation of alginate by NaIO4 preserved the main structure and functional groups of alginate. In addition, the peak assignment of XPS C1s narrow scans of SA and OSA that could elucidate the bonding atmosphere has been marked in the spectra in Figure 5b according to the previous report [44]. It can be observed that the C1s narrow scan revealed three peaks at 285.46 eV (O–C–O), 283.87 eV (C–O), and 282.20 eV (C–C), respectively. It is found that the peak ratio of the O–C–O species to the C–O species in the C1s narrow scan of OSA was significantly higher than that of SA, due to the partial oxidation of alginate by NaIO4, which was consistent with previous results reported by Jejurikar et al. [24]. Thus, these results further verified the successful partial oxidation of alginate by NaIO4.

**Figure 5.** (**a**) XPS spectra and (**b**) XPS C1s narrow scans of SA and OSA.

The crystal structure of OSA was evaluated by XRD. As shown in Figure 6, the XRD patterns of SA and OSA were proved to be amorphous structures, consistent with previous reports [45]. The typical characteristic diffraction peaks of SA at 2θ = 14.5◦ and 22.0◦ represented the hydrated crystalline structure generated from the intramolecular hydrogen bonds of SA [46]. Compared with SA, the diffraction peaks of OSA were shifted to

2 θ = 14.9◦ and 23.0◦ , and the peak at 2 θ = 23.0◦ was a broader peak. These results implied that the structure of SA was changed during the oxidation process and the intermolecular hydrogen bonds were weakened with the decrease in molecular weight. intermolecular hydrogen bonds were weakened with the decrease in molecular weight.

The crystal structure of OSA was evaluated by XRD. As shown in Figure 6, the XRD patterns of SA and OSA were proved to be amorphous structures, consistent with previous reports [45]. The typical characteristic diffraction peaks of SA at 2θ = 14.5° and 22.0° represented the hydrated crystalline structure generated from the intramolecular hydrogen bonds of SA [46]. Compared with SA, the diffraction peaks of OSA were shifted to 2 θ = 14.9° and 23.0°, and the peak at 2 θ = 23.0° was a broader peak. These results implied that the structure of SA was changed during the oxidation process and the

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**Figure 5.** (**a**) XPS spectra and (**b**) XPS C1s narrow scans of SA and OSA.

**Figure 6.** XRD patterns of SA and OSA.

**Figure 6.** XRD patterns of SA and OSA. Thermogravimetric analysis was the most effective method to characterize the different thermal stability of SA and OSA, which could indirectly reflect the change of molecular structures of alginate during the oxidation reaction. During the test, since the mass of the samples will change with the increase in temperature, the thermal stability of SA and OSA can be judged by comparing the initiating decomposition temperature and the final weight loss rate [47]. As shown in Figure 7a, the weight loss of the samples at the temperature below 100 °C corresponds to the evaporation of water in the samples. Major weight loss occurred at the temperature ranging from 200 °C to 300 °C, which resulted from dehydration of hydroxyl group along the alginate backbone and its thermal decomposition of hexuronic segments [48,49]. The total weight loss rates of SA and OSA at 800 °C were 74% and 77%, respectively. Compared with SA, OSA possessed lower residual weight, which may be ascribed to the oxidation of hydroxyl groups of SA. From the DTG curves of SA and OSA in Figure 7b, it can be observed that both SA and OSA displayed two main weight loss stages. The maximum rate of the first weight loss stage occurred at 100~150 °C, which was ascribed to the removal of physically adsorbed water and bound water from the polymer materials [27,28], while the second weight loss stage occurred at 200~300 °C and was attributed to the thermal decomposition of the polymer materials, which was gradually thermally cracked into CO, CO2, and H2O, resulting in a rapid decline in their weight [33]. The temperatures corresponding to the maximum rates Thermogravimetric analysis was the most effective method to characterize the different thermal stability of SA and OSA, which could indirectly reflect the change of molecular structures of alginate during the oxidation reaction. During the test, since the mass of the samples will change with the increase in temperature, the thermal stability of SA and OSA can be judged by comparing the initiating decomposition temperature and the final weight loss rate [47]. As shown in Figure 7a, the weight loss of the samples at the temperature below 100 ◦C corresponds to the evaporation of water in the samples. Major weight loss occurred at the temperature ranging from 200 ◦C to 300 ◦C, which resulted from dehydration of hydroxyl group along the alginate backbone and its thermal decomposition of hexuronic segments [48,49]. The total weight loss rates of SA and OSA at 800 ◦C were 74% and 77%, respectively. Compared with SA, OSA possessed lower residual weight, which may be ascribed to the oxidation of hydroxyl groups of SA. From the DTG curves of SA and OSA in Figure 7b, it can be observed that both SA and OSA displayed two main weight loss stages. The maximum rate of the first weight loss stage occurred at 100~150 ◦C, which was ascribed to the removal of physically adsorbed water and bound water from the polymer materials [27,28], while the second weight loss stage occurred at 200~300 ◦C and was attributed to the thermal decomposition of the polymer materials, which was gradually thermally cracked into CO, CO2, and H2O, resulting in a rapid decline in their weight [33]. The temperatures corresponding to the maximum rates of the thermal decomposition weight loss of SA and OSA were 249.6 and 232.2 ◦C, respectively. Obviously, the initiating thermal decomposition temperature of OSA was lower than that of raw SA, indicating that the thermal stability of OSA decreased after the oxidation reaction. This result was ascribed to the destruction of the intramolecular hydrogen bond for the cleavage of the C2–C3 bond of alginate uronic acid monomer during the oxidation process, thereby improving the molecular flexibility of alginate.

of the thermal decomposition weight loss of SA and OSA were 249.6 and 232.2 °C, respectively. Obviously, the initiating thermal decomposition temperature of OSA was lower than that of raw SA, indicating that the thermal stability of OSA decreased after the oxidation reaction. This result was ascribed to the destruction of the intramolecular hydrogen bond for the cleavage of the C2–C3 bond of alginate uronic acid monomer during the oxidation process, thereby improving the molecular flexibility of alginate.

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**Figure 7.** (**a**) TGA curves and (**b**) DTG curves of SA and OSA. **Figure 7.** (**a**) TGA curves and (**b**) DTG curves of SA and OSA. The weight-average molecular weights (MW) of the water-soluble OSA were measured via GPC, which used 0.05% sodium azide as the mobile phase. Figure 8a depicts

### *3.3. In Vitro Biodegradation Analysis 3.3. In Vitro Biodegradation Analysis* GPC chromatographs of SA and OSA with various ODs. It can be observed that the

The weight-average molecular weights (MW) of the water-soluble OSA were measured via GPC, which used 0.05% sodium azide as the mobile phase. Figure 8a depicts GPC chromatographs of SA and OSA with various ODs. It can be observed that the molecular weight of OSA decreased with an increase in the OD of OSA, similar to the previous results [50]. The SA and OSA with various ODs were immersed in the PBS buffer at 37 °C for in vitro biodegradation, and the biodegradation performance of the samples was studied by measuring their molecular weight at a specific time. As shown in Figure 8b, SA underwent less biodegradation because SA was hardly biodegradable in the body [9,10]. In contrast, OSA showed significant biodegradability, especially within 10 days of the onset of biodegradation, which could exhibit great potentials as the degradable hydrogel scaffolds or controlled release drug delivery system in biomedical field. Additionally, the MW of OSA decreased very rapidly with the increase in their OD, and all of the OSA with various ODs revealed similar biodegradation trends. These behaviors were attributed to the cleavage of the C2–C3 bond of alginate uronic acid, as oxidation occurred because this was a free-radical-independent biodegradation process, which The weight-average molecular weights (MW) of the water-soluble OSA were measured via GPC, which used 0.05% sodium azide as the mobile phase. Figure 8a depicts GPC chromatographs of SA and OSA with various ODs. It can be observed that the molecular weight of OSA decreased with an increase in the OD of OSA, similar to the previous results [50]. The SA and OSA with various ODs were immersed in the PBS buffer at 37 ◦C for in vitro biodegradation, and the biodegradation performance of the samples was studied by measuring their molecular weight at a specific time. As shown in Figure 8b, SA underwent less biodegradation because SA was hardly biodegradable in the body [9,10]. In contrast, OSA showed significant biodegradability, especially within 10 days of the onset of biodegradation, which could exhibit great potentials as the degradable hydrogel scaffolds or controlled release drug delivery system in biomedical field. Additionally, the M<sup>W</sup> of OSA decreased very rapidly with the increase in their OD, and all of the OSA with various ODs revealed similar biodegradation trends. These behaviors were attributed to the cleavage of the C2–C3 bond of alginate uronic acid, as oxidation occurred because this was a free-radical-independent biodegradation process, which showed that the biodegradation depended directly on the concentration of NaIO<sup>4</sup> in the reaction solution [23]. Consequently, the biodegradation rate of alginate could be controlled by regulating the OD of OSA to meet different application requirements. molecular weight of OSA decreased with an increase in the OD of OSA, similar to the previous results [50]. The SA and OSA with various ODs were immersed in the PBS buffer at 37 °C for in vitro biodegradation, and the biodegradation performance of the samples was studied by measuring their molecular weight at a specific time. As shown in Figure 8b, SA underwent less biodegradation because SA was hardly biodegradable in the body [9,10]. In contrast, OSA showed significant biodegradability, especially within 10 days of the onset of biodegradation, which could exhibit great potentials as the degradable hydrogel scaffolds or controlled release drug delivery system in biomedical field. Additionally, the MW of OSA decreased very rapidly with the increase in their OD, and all of the OSA with various ODs revealed similar biodegradation trends. These behaviors were attributed to the cleavage of the C2–C3 bond of alginate uronic acid, as oxidation occurred because this was a free-radical-independent biodegradation process, which showed that the biodegradation depended directly on the concentration of NaIO4 in the reaction solution [23]. Consequently, the biodegradation rate of alginate could be controlled by regulating the OD of OSA to meet different application requirements.

**Figure 8.** (**a**) GPC chromatographs of SA and OSA with various OD; (**b**) biodegradation curves of SA and OSA with various OD in PBS solution. **Figure 8.** (**a**) GPC chromatographs of SA and OSA with various OD; (**b**) biodegradation curves of SA and OSA with various OD in PBS solution.

**Figure 8.** (**a**) GPC chromatographs of SA and OSA with various OD; (**b**) biodegradation curves of

SA and OSA with various OD in PBS solution.

### *3.4. Gelation Study of OSA* preliminarily determined by observing the results of the viscosity variation with the shear rate, as shown in Figure 9a. Since the formation time of hydrogel was related to the

*3.4. Gelation Study of OSA* 

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OSA with oxidation degrees ranging from 5% to 20% was formulated into the corresponding concentration of homogeneous hydrogel, and their gelation ability was preliminarily determined by observing the results of the viscosity variation with the shear rate, as shown in Figure 9a. Since the formation time of hydrogel was related to the molecular weight of the oxidized alginate derivatives, high molecular weight of OSA could greatly reduce the formation time of hydrogel. It could be found that the viscosity (η) of the SA hydrogel exhibited a slight variation with the shear rate, which displayed typical Newtonian liquid characteristics. This result was mainly attributed to the highly stretched rigid molecular structure of SA [37]. On the contrary, the OSA hydrogel with various ODs displayed apparent shear-thinning behavior, which was ascribed to the destruction of the intramolecular hydrogen bond for cleavage of the C2–C3 bond of the alginate uronic acid monomer during the oxidation process. In comparison with SA hydrogel, the OSA hydrogel exhibited distinctly lower viscosity (η) at the same shear rate, indicating that the gelation ability of OSA significantly decreased due to the oxidation of the adjacent hydroxyl groups at C-2 and C-3 positions on alginate uronic acid monomer [24]. Simultaneously, the viscosity of OSA hydrogel decreased with the increase in OD at the same shear rate, which indicated that the hydrogel could be prepared by controlling the OD. molecular weight of the oxidized alginate derivatives, high molecular weight of OSA could greatly reduce the formation time of hydrogel. It could be found that the viscosity (η) of the SA hydrogel exhibited a slight variation with the shear rate, which displayed typical Newtonian liquid characteristics. This result was mainly attributed to the highly stretched rigid molecular structure of SA [37]. On the contrary, the OSA hydrogel with various ODs displayed apparent shear-thinning behavior, which was ascribed to the destruction of the intramolecular hydrogen bond for cleavage of the C2–C3 bond of the alginate uronic acid monomer during the oxidation process. In comparison with SA hydrogel, the OSA hydrogel exhibited distinctly lower viscosity (η) at the same shear rate, indicating that the gelation ability of OSA significantly decreased due to the oxidation of the adjacent hydroxyl groups at C-2 and C-3 positions on alginate uronic acid monomer [24]. Simultaneously, the viscosity of OSA hydrogel decreased with the increase in OD at the same shear rate, which indicated that the hydrogel could be prepared by controlling the OD.

OSA with oxidation degrees ranging from 5% to 20% was formulated into the corresponding concentration of homogeneous hydrogel, and their gelation ability was

**Figure 9.** (**a**) Viscosity of SA hydrogel and OSA hydrogel with various ODs as a function of shear rate at 25 °C; (**b**) viscosity of SA hydrogel and OSA hydrogel with various ODs as a function of shear rate; (**c**) storage (G') and loss (G'') moduli of SA hydrogel and OSA hydrogel with various ODs as functions of angular frequency (ω). **Figure 9.** (**a**) Viscosity of SA hydrogel and OSA hydrogel with various ODs as a function of shear rate at 25 ◦C; (**b**) viscosity of SA hydrogel and OSA hydrogel with various ODs as a function of shear rate; (**c**) storage (G0 ) and loss (G00) moduli of SA hydrogel and OSA hydrogel with various ODs as functions of angular frequency (ω).

In addition, it can be also observed from Figure 9b,c that the SA hydrogel and OSA hydrogel with various ODs presented an upward trend for their storage modulus (G') and loss modulus (G'') as the angular frequency (ω) increased. In general, the storage modulus

In addition, it can be also observed from Figure 9b,c that the SA hydrogel and OSA hydrogel with various ODs presented an upward trend for their storage modulus (G0 ) and loss modulus (G00) as the angular frequency (ω) increased. In general, the storage modulus (G0 ) and loss modulus (G00) were, respectively, related to the strength of the internal structure and dynamic viscosity. By contrast, the SA hydrogel exhibited relatively better mechanical properties as the storage (G0 ) and loss (G00) moduli of SA hydrogel were apparently higher than that of OSA hydrogel at the same angular frequency (ω). Furthermore, a reduction in both of the storage (G0 ) and loss (G00) moduli was found when the OD increased, which could be owing to the decrease of the molecular weight. Thus, it could be concluded that the oxidation reaction would cause the decrease in the molecular weight of the alginate, further affecting the formation of hydrogel. As presented in Figure 10, only OSA with ODs of 5% and 10% could be cross-linked by Ca2+ to form hydrogel, while no hydrogels were formed when the OD exceeded 10%. Therefore, the critical OD of OSA to form a hydrogel was 10%, which was consistent with the previous report [23]. Meanwhile, OSA with low molecular weight was beneficial to biomedical applications, such as regenerative medicine, 3D-printed/composite scaffolds, and tissue engineering, because it was found that, for M<sup>W</sup> ≤ 50 kDa, alginate could be removed from the human body [11,12]. (G') and loss modulus (G'') were, respectively, related to the strength of the internal structure and dynamic viscosity. By contrast, the SA hydrogel exhibited relatively better mechanical properties as the storage (G') and loss (G'') moduli of SA hydrogel were apparently higher than that of OSA hydrogel at the same angular frequency (ω). Furthermore, a reduction in both of the storage (G') and loss (G'') moduli was found when the OD increased, which could be owing to the decrease of the molecular weight. Thus, it could be concluded that the oxidation reaction would cause the decrease in the molecular weight of the alginate, further affecting the formation of hydrogel. As presented in Figure 10, only OSA with ODs of 5% and 10% could be cross-linked by Ca2+ to form hydrogel, while no hydrogels were formed when the OD exceeded 10%. Therefore, the critical OD of OSA to form a hydrogel was 10%, which was consistent with the previous report [23]. Meanwhile, OSA with low molecular weight was beneficial to biomedical applications, such as regenerative medicine, 3D-printed/composite scaffolds, and tissue engineering, because it was found that, for MW ≤ 50 kDa, alginate could be removed from the human body [11,12].

**Figure 10.** The physical picture of OSA—5% and OSA—10% hydrogel. **Figure 10.** The physical picture of OSA—5% and OSA—10% hydrogel.

### *3.5. Cytocompatibility of OSA Hydrogel 3.5. Cytocompatibility of OSA Hydrogel*

After 2 days and 5 days incubation, the in vitro cytotoxicity of OSA—10% hydrogel was measured using the CCK—8 assay kit. As shown in Figure 11, the MC3T3-E1 cells displayed better proliferative activity on the OSA—10% hydrogel than that on the control group, indicating that the MC3T3-E1 cells could survive on the OSA—10% hydrogel and grow in their 3D pore structure. These results further indicated that the OSA hydrogel had no cytotoxicity, presenting excellent biocompatibility. Therefore, the OSA hydrogel with excellent cytocompatibility could realize their extended biomedical applications in After 2 days and 5 days incubation, the in vitro cytotoxicity of OSA—10% hydrogel was measured using the CCK—8 assay kit. As shown in Figure 11, the MC3T3-E1 cells displayed better proliferative activity on the OSA—10% hydrogel than that on the control group, indicating that the MC3T3-E1 cells could survive on the OSA—10% hydrogel and grow in their 3D pore structure. These results further indicated that the OSA hydrogel had no cytotoxicity, presenting excellent biocompatibility. Therefore, the OSA hydrogel with excellent cytocompatibility could realize their extended biomedical applications in regenerative medicine, 3D-printed/composite scaffolds, and tissue engineering.

regenerative medicine, 3D-printed/composite scaffolds, and tissue engineering.

**Figure 10.** The physical picture of OSA—5% and OSA—10% hydrogel.

*3.5. Cytocompatibility of OSA Hydrogel* 

**Figure 11.** Cell viability of MC3T3-E1 cells cultured on the OSA—10% hydrogel for 2 and 5 days, respectively.

After 2 days and 5 days incubation, the in vitro cytotoxicity of OSA—10% hydrogel was measured using the CCK—8 assay kit. As shown in Figure 11, the MC3T3-E1 cells displayed better proliferative activity on the OSA—10% hydrogel than that on the control group, indicating that the MC3T3-E1 cells could survive on the OSA—10% hydrogel and grow in their 3D pore structure. These results further indicated that the OSA hydrogel had no cytotoxicity, presenting excellent biocompatibility. Therefore, the OSA hydrogel with excellent cytocompatibility could realize their extended biomedical applications in

regenerative medicine, 3D-printed/composite scaffolds, and tissue engineering.

(G') and loss modulus (G'') were, respectively, related to the strength of the internal structure and dynamic viscosity. By contrast, the SA hydrogel exhibited relatively better mechanical properties as the storage (G') and loss (G'') moduli of SA hydrogel were apparently higher than that of OSA hydrogel at the same angular frequency (ω). Furthermore, a reduction in both of the storage (G') and loss (G'') moduli was found when the OD increased, which could be owing to the decrease of the molecular weight. Thus, it could be concluded that the oxidation reaction would cause the decrease in the molecular weight of the alginate, further affecting the formation of hydrogel. As presented in Figure 10, only OSA with ODs of 5% and 10% could be cross-linked by Ca2+ to form hydrogel, while no hydrogels were formed when the OD exceeded 10%. Therefore, the critical OD of OSA to form a hydrogel was 10%, which was consistent with the previous report [23]. Meanwhile, OSA with low molecular weight was beneficial to biomedical applications, such as regenerative medicine, 3D-printed/composite scaffolds, and tissue engineering, because it was found that, for MW ≤ 50 kDa, alginate could be removed from the human

### **4. Conclusions**

body [11,12].

In this work, the oxidation of SA with various ODs was achieved using NaIO<sup>4</sup> as the oxidant, and the structure and properties of the resultant OSA were characterized by multiple testing methods. Meanwhile, the effect of OD on the biodegradability and gelation ability of OSA was also investigated. The adjacent hydroxyl groups at C-2 and C-3 position on alginate uronic acid monomer were oxidized to aldehyde groups by NaIO4. On account of the cleavage of the C2–C3 bond of alginate uronic acid monomer during the oxidation process, the thermal stability of OSA was worse than that of SA. On the contrary, OSA possessed smaller molecular weight and better degradability in contrast to SA; the higher the OD of OSA, the better the degradability. Nevertheless, the gelation ability of OSA decreased with the increase in OD due to the decrease in molecular weight. It is worth noting that the critical OD of OSA to form a hydrogel was 10%. Thus, it could be concluded that both the formation and biodegradability of OSA hydrogel were controlled by the OD. This work aimed to improve the properties of alginate via the oxidation reaction to broaden its application in the biomedical field.

**Author Contributions:** Writing—original draft preparation, H.W.; methodology, X.C.; data curation, Y.W.; software, D.L.; conceptualization, X.S.; visualization, Z.L.; writing—review and editing, supervision, H.Y.; funding acquisition, Q.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, 51963009; the Natural Science Foundation of Hainan Province, 220MS035; the Key Research and Development Project of Hainan Province, ZDYF2019018; the Open Fund for Innovation and Entrepreneurship of College Students of Hainan Normal University, S202111658065X; the Scientific Research Fund of Jiangxi Provincial Education Department, GJJ211509. The APC was funded by the National Natural Science Foundation of China, 51963009.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. Samples of the compounds are available from the authors.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

