Chitosan–Type-A-Gelatin Hydrogels Used as Potential Platforms in Tissue Engineering for Drug Delivery

Citation: Mehdi-Sefiani, H.; Granados-Carrera, C.M.; Romero, A.; Chicardi, E.; Domínguez-Robles, J.; Perez-Puyana, V.M. Chitosan–Type-A- Gelatin Hydrogels Used as Potential Platforms in Tissue Engineering for Drug Delivery. Gels 2024,10, 419. https://doi.org/10.3390/ gels10070419 Academic Editor: Michael Arkas Received: 7 May 2024 Revised: 17 June 2024 Accepted: 23 June 2024 Published: 26 June 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). gels Article Chitosan–Type-A-Gelatin Hydrogels Used as Potential Platforms in Tissue Engineering for Drug Delivery Hanaa Mehdi-Sefiani 1,2, Carmen Mª Granados-Carrera 2,*, Alberto Romero 2,* , Ernesto Chicardi 1, Juan Domínguez-Robles 3and Víctor Manuel Perez-Puyana 2 1Department of Engineering and Materials Science and Transportation, University of Seville, 41012 Seville, Spain 2Department of Chemical Engineering, Faculty of Chemistry, University of Seville, 41012 Seville, Spain 3Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain *Correspondence: [email protected] (C.M.G.-C.); alromer[email protected] (A.R.) Abstract: Hydrogels are materials made of crosslinked 3D networks of hydrophilic polymer chains that can absorb and retain significant amounts of water due to their hydrophilic structure without being dissolved. In relation to alternative biomaterials, hydrogels offer increased biocompatibility and biodegradability, giving them distinct advantages. Thus, hydrogel platforms are considered to have the potential for the development of biomedical applications. In this study, the main objective was the development of hybrid hydrogels to act as a drug delivery platform. These hydrogels were made from chitosan (CH) and type A gelatin (G), two natural polymers that provide a supportive environment for cellular attachment, viability, and growth, thanks to their unique properties. Particularly, the use of gelatins for drug delivery systems provides biodegradability, biocompatibility, and non-toxicity, which are excellent properties to be used in the human body. However, gelatins have some limitations, such as thermal instability and poor mechanical properties. In order to improve those properties, the aim of this work was the development and characterization of hybrid hydrogels with different ratios of CH–G (100–0, 75–25, 50–50, 25–75, 0–100). Hydrogels were characterized through multiple techniques, including Fourier transform infrared (FTIR) spectroscopy, rheological and microstructural studies, among others. Moreover, a model hydrophilic drug molecule (tetracycline) was incorporated to evaluate the feasibility of this platform to sustain the release of hydrophilic drugs, by being tested in a solution of Phosphate Buffer Solution at a pH of 7.2 and at 37 ◦ C. The results revealed that the synergy between chitosan and type A gelatin improved the mechanical properties as well as the thermal stability of it, revealing that the best ratios of the biopolymers are 50–50 CH–G and 75–25 CH–G. Thereby, these systems were evaluated in a controlled release of tetracycline, showing a controlled drug delivery of 6 h and highlighting their promising application as a platform for controlled drug release. Keywords: chitosan; type A gelatin; hydrogels; characterization; drug-delivery 1. Introduction A drug delivery system (DDS) is defined as the technology that introduces an adequate dosage of therapeutic substance into the body to achieve effectively and safely its appropriate therapeutic effect [ 1 ]. Thus, the main objective of a DDS is to ensure that drugs are administered in the required place in the body and at the correct time, avoiding adverse effects [ 2 ]. However, conventional DDSs (tablets, capsules, syrups, etc.) have limitations that are mainly attributable to their poor bioavailability, fluctuations of the drug concentration in plasma, limited solubility, poor tissue distribution of drug particles, and uncontrollable drug release characteristics [ 1 , 3 , 4 ]. Consequently, to establish new methods for achieving controlled and targeted drug release, the design and development of new Gels 2024,10, 419. https://doi.org/10.3390/gels10070419 https://www.mdpi.com/journal/gels Gels 2024,10, 419 2 of 17 DDSs have been considered [ 5 ]. In fact, chemists and chemical engineers are contributing to improve the development of controlled drug delivery technologies, which have numerous advantages such as efficacy, less toxicity, and the improvement of patient compliance and convenience [6]. A DDS includes a wide variety of methods, including liposomes, micelles, and nanoparticles [ 2 ]. One way to achieve a controlled DDS is the development of an adequate encapsulation system that provides protection for the drug, preventing the surrounding environment of the body from carrying its uncontrolled release or out-of-place activation [ 7 ]. Over the last few years, biomaterials have attracted interest in the development of controlled DDSs. Particularly, hydrogels have been demonstrated as the ideal pharmaceutical carrier in biomedical areas due to their unique physical properties, such as porosity, biocompatibility, biodegradability, and nontoxicity, along with their controlled release properties and ability to protect drugs [8–11]. Furthermore, hydrogels are one of the outstanding classes of polymer-based drugcontrolled release systems for their unique pharmacokinetic properties among various DDSs [ 12 ]. Thus, hydrogels have been widely useful due to their ability to manufacture and combine with natural and synthetic components, as well as the properties mentioned above [ 13 ]. Polymer-based hydrogels are three-dimensional (3D) networks of hydrophilic polymer that possess the capacity to maintain their structural integrity while absorbing and retaining large amounts of water and biological fluids [ 8 , 14 ]. Moreover, as the DDS’s release mechanism would be affected by the properties of the hydrogel, such as porosity or swelling degree, polymer-based hydrogels have the capability to release the drugs gradually following a specific release kinetic achieved by modulating their compositions and structures [ 14 , 15 ] Furthermore, it can be highlighted that currently, the existence of “smart hydrogels” permit their use as sensors, thanks to their responsiveness towards stimuli such as temperature, solvents, pH, light, molecules, and mechanical force [13]. Recently, attention has been focused on finding materials that are non-toxic and biocompatible [ 16 ]. Particularly, natural polymers are materials obtained from human beings, plants, or animals, which have poor mechanical characteristics; but, due to their high affinity with water, gelling capacity, mucoadhesiveness, nontoxicity, and negative antigenicity in live organisms, they are widely used in tissue engineering (in biomedical applications, among others) [ 17 , 18 ]. Some examples of natural polymers, such as collagen, gelatin, alginate, and chitosan, can be mentioned [ 19 ]. Numerous strategies have been used to develop natural polymeric-based hydrogels with improved mechanical properties; among them, we can distinguish double networking [ 20 ], physical, enzymatic, or chemical crosslinking [18], and electrochemistry functionalization [21]. On the one hand, chitosan (CH) is a natural polymer derived from the deacetylation of chitin, which is present in the exoskeletons of crustaceans. Chitosan and its derivatives have become promising biological materials due to their distinctive molecular structure, excellent biological activities, and their properties such as biocompatibility, biodegradability, cytocompatibility, non-allergenicity, non-toxicity, and mucoadhesiveness, as well as their antitumor, antioxidant, antibacterial, and antifungal activities [ 22 – 27 ]. The reactive functional groups found in chitosan play an important role in aiding the synthesis of three-dimensional hydrogel. Chitosan-based hydrogels have been widely used in medical and pharmaceutical fields for years. One of their advantages is their complex network architecture, which offers a versatile platform that allows the incorporation of various bioactive compounds [ 23 ]. Additionally, chitosan has the capability to improve the bioavailability and stability of multiple small molecule drugs, peptides, and proteins, as well as sustain drug release due to its mucoadhesive properties. Moreover, chitosan-based drug delivery systems can aim at specific cells or tissues by adding to them specific ligands or antibodies [ 24 , 27 ]. Furthermore, the properties of chitosan-based hydrogels can be further tuned by altering their composition or combining them with other natural or synthetic polymers [ 24 ]. For example, Vasile et al. [ 28 ] developed a chitosan composite incorporating ZnO nanoparticles. In this way, potential material for wound care was obtained that Gels 2024,10, 419 3 of 17 combines the antibacterial properties of their components with a controlled release of the antibiotic (gentamicin). In other research completed by Xu et al. [ 29 ], chemical chitosan and catechol hydrogels were crosslinked with genipin in order to study the release of lidocaine. The results showed that catechol increased the hydrogel density, and the drug was more effective. Moreover, Ren et al. [ 12 ] developed crosslinked injectable hydrogels by oxidizing the blend of chitosan, gelatin, and dopamine as the drug of the system. The results showed stable mechanical strength and good degradability. Gelatin is a natural polymer obtained through hydrolytic degradation of type I collagen. This biopolymer has been widely used in biomedical applications due to its high biodegradability, biocompatibility, non-toxicity, non-immunogenicity, and highwater absorption [ 18 , 30 – 32 ]. Notably, these properties render gelatin an excellent candidate for drug-delivery vehicles [ 31 ]. Gelatin has recently become a significant factor in tailoring drug release in pharmaceutical formulations [18]. In fact, gelatin is the main ingredient in hard and soft capsules in pharmaceutical applications since it melts above 30 ◦ C and easily releases drugs. Additionally, the hydrophilic nature of gelatin facilitates the permeation of body fluids into the particles, which leads to an increase in the diffusion-mediated release of drugs [ 30 ]. There are two types of gelatins, type A and type B gelatin, which are obtained by an acid-treated and alkali-treated process [ 31 ]. In particular, type A gelatin (G) has demonstrated great biocompatibility and the ability to form hydrogels [ 32 ]. According to the literature, gelatin hydrogels are a promising choice for delivering therapeutic or pharmaceutical compounds, thanks to their distinct properties. They can be designed to react to changes in temperature or pH levels, ensuring accurate drug release in response to specific bodily conditions. Furthermore, they are highly effective in delivering drugs to specific locations, which enhances their efficacy while minimizing side effects [18]. In particular, several investigations have exhibited the potential of chitosan and gelatin carriers in drug delivery systems [ 25 ]. First, Mahdizadeh et al. [ 33 ] developed a chitosan– gelatin carrier, which proved through drug release, release kinetics, and physical and mechanical studies its capability to be used as an insulin nanoparticle carrier. In another study, Mathew et al. [ 34 ] developed dopamine-loaded chitosan/gelatin nanocomposites that demonstrated to be excellent drug delivery carriers with optimal antioxidant and antibacterial properties because of their sustained release in a neutral medium, as well as their capability to inhibit the DPPH radical and Staphylococcus aureus. Finally, Wu et al. synthesized microspheres combining gelatin with an anti-cancer medication (pacific yew tree bark) and chitosan in order to achieve a high drug entrapment efficiency [26]. In this context, the purpose of this work was the development and characterization of chitosan–type-A-gelatin hydrogels by studying different biopolymeric ratios, as well as evaluating their rheological and microstructural properties. Moreover, the incorporation of a model hydrophilic drug molecule (tetracycline) to evaluate the feasibility of this platform to sustain the release of hydrophilic drugs. This research sets a strong foundation for further exploration into the efficacy and practicality of this novel approach to drug delivery and its potential application in tissue engineering. 2. Results and Discussion 2.1. Chemical Characterization: Fourier Transform Infrared Spectroscopy (FTIR) FTIR characterization is necessary to ensure that the interactions between chitosan and type A gelatin occurred. Particularly, the peaks of the FTIR informed about the stretching and bending of the bounds. For this purpose, FTIR measurements of the different hydrogels were carried out and the results are shown in Figure 1. Figure 1A shows the spectrum of the type A gelatin-based hydrogel. First, the presence of C-N and C=O bands occur in a frequency range between 1750 and 1600 cm −1 . On the other hand, in a frequency range between 1600 and 1500 cm −1 , a signal called Amide I is indicated, which is associated with the stretching of C=O. Then, Amide II signal appears in the range 1500–1375 cm −1 for N-H bending. N-H bending is also present in the range 1250–1125 cm −1 for the Amide III signal [ 35 ]. On the other hand, Figure 1E shows the Gels 2024,10, 419 4 of 17 profile of the chitosan-based hydrogel. In this case, in a frequency range between 1750 and 1600 cm −1 , the presence of a CH=N band is indicated. Subsequently, the signal for C=O stretch (Amide I) occurs in the range 1600–1500 cm −1 and the signal for the N-H bending (Amide II) occurs in the range of 1500 and 1375 cm −1 . Finally, in a range of 1125–1000 cm −1 the presence of a C-O band is indicated [36–39]. Gels 2024, 10, x FOR PEER REVIEW 4 of 17 Figure 1. Fourier Transform Infrared Spectroscopy (FTIR) of the different hydrogels synthesized: (A) 0–100 CH–G; (B) 25–75 CH–G; (C) 50–50 CH–G; (D) 75–25 CH–G; (E) 100–0 CH–G. Figure 1A shows the spectrum of the type A gelatin-based hydrogel. First, the presence of C-N and C=O bands occur in a frequency range between 1750 and 1600 cm −1 . On Figure 1. Fourier Transform Infrared Spectroscopy (FTIR) of the different hydrogels synthesized: (A) 0–100 CH–G; (B) 25–75 CH–G; (C) 50–50 CH–G; (D) 75–25 CH–G; (E) 100–0 CH–G. Gels 2024,10, 419 5 of 17 Figure 1B–D show the profiles of the binary systems. In the case of 25–75 CH–G (Figure 1B), it can be highlighted that the signal for C=O stretch (Amide I) occurs in the range 1600–1500 cm −1 and the signal for the N-H bending occurs in the range of 1500 and 1375 cm −1 (Amide II) and in the range of 1250–1125 cm −1 (Amide III) as it occurred in Figure 1A associated with the spectrum of the type A gelatin hydrogel. What is different is that the CH=N band appears in the range of 1750 and 1600 cm −1 , which is characteristic of the chitosan-based hydrogel’s spectrum. On the other hand, the 75–25 CH–G spectrum (Figure 1D) indicates the presence of a CH=N band in a frequency range between 1750 and 1600 cm −1 . Furthermore, the stretching of C=O (Amide I) in the range 1600–1500 cm −1 and N-H bending (Amide II) in the range 1500–1375 cm −1 are also indicated. Finally, a band of C-O occurs in the range 1125–1000 cm −1 . In the 50–50 CH–G spectrum (Figure 1C), the spectrum of both biopolymers shows synergy, resulting in previously significant peaks being attenuated. The peak at 1012 cm −1 is similar to that of the type A based-hydrogel spectrum. The presence of a CH=N band in a frequency range between 1750 and 1600 cm −1 is also observed. Finally, the stretching of C=O (Amide I) in the range of 1600–1500 cm −1 and N-H bending (Amide II) in the range of 1500–1375 cm−1can be seen [36–38]. 2.2. Rheological Characterization of Hydrogels Apart from evaluating the physicochemical properties of the hydrogels, rheological characterization was also carried out. Firstly, strain sweep tests were performed in order to determine the linear viscoelastic interval, which is the interval where elastic and viscous moduli are independent of deformation as well as the critical deformation, which refers to the maximum deformation supported by the sample within the linear viscoelastic interval (Table 1) [ 40 ]. Attending to the critical deformation shown in Table 1, it can be noted that type A gelatin-based hydrogel has critical deformation values higher than the critical deformation values of chitosan-based hydrogel. This difference may be due to the fact that chitosan-based hydrogel is a rigid material that offers a specific structure, while the type A gelatin system is more deformable and allows its chains to be deformed without breaking them [ 41 ]. However, when the hydrogel has a higher proportion of chitosan (75–25 system), the system becomes rigid but improves the values obtained by the unitary chitosan system. Conversely, the systems with a higher proportion of type A gelatin show a deformable characteristic, which is better than values obtained by the unitary type A gelatin system studied at 5 ◦ C. On the other hand, it can be highlighted that the temperature of measurements could affect the deformable capabilities of the unitary systems. In fact, the critical strain obtained at 5 ◦ C does not follow a defined trend. Particularly, the unitary type A gelatin system evaluated at 5 ◦ C shows a significant decrease in its deformable capability. In contrast, when the unitary chitosan system and the systems containing chitosan were assessed at low temperatures, they showed a slight increase in their deformable capability. Moreover, the critical strain of binary hydrogels increased in comparison to unitary hydrogels (100–0 CH–G and 0–100 CH–G) due to the synergistic effect produced by the combination of both biopolymers. So, it can be verified that the presence of gelatin in the structure increases the critical strain of the system. On the other hand, the critical strain obtained at 40 ◦ C follows a defined trend. However, it can be noted that the increase in the proportion of type A gelatin induces a higher value of critical deformation. Finally, these values can be compared with those obtained in another work by Sánchez-Cid et al. [ 41 ]. Specifically, the critical deformation values of unitary chitosan-based hydrogel obtained at 5 ◦ C are too similar to those obtained in this work. After the critical deformation of each system was obtained, a time sweep test was carried out before gelation to analyze the evolution of both elastic (G ′ ) and viscous (G ′′ ) moduli during the gelation process. Figure 2shows how the gelation process increases the elastic modulus (G ′ ) of the different hydrogels evaluated. Moreover, the distance between G ′ and G ′′ increases with time, which indicates that the systems are gaining a more solid Gels 2024,10, 419 6 of 17 character. However, after one hour, the values of the elastic modulus (G ′ ) and the viscous modulus (G′′) became constant. Table 1. Critical strain, G ′ and tan δ at 1 Hz (indicated as G ′1 and tan ( δ ) 1 ) and critical temperature obtained for CH–G hydrogels with different biopolymers’ proportions at two temperatures, 5 ◦ C and 40 ◦C. System CH–G Temperature (◦C) Critical Strain (%) G′1 (Pa) tan (δ)1 (-) Critical Temperature (◦C) 0–100 5 25.0 ±0.6 1160 ±253 0.029 ±0.007 32.5–35.0 40 100.2 ±0.1 0.40 ±0.01 8.85 ±1.49 25–75 5 100.8 ±0.1 877 ±145 0.027 ±0.004 31.0–36.0 40 100.6 ±0.1 182 ±56 0.032 ±0.006 50–50 5 65.3 ±0.5 3880 ±528 0.021 ±0.001 35.0–40.0 40 64.0 ±0.5 710 ±26 0.032 ±0.003 75–25 5 66.1 ±0.4 1646 ±56 0.072 ±0.001 >40.0 40 41.3 ±0.4 1574 ±38 0.090 ±0.014 100–0 5 10.4 ±0.2 3907 ±1106 0.117 ±0.006 >40.0 40 6.8 ±0.5 10,262 ± 5810 0.088 ±0.008 Gels 2024, 10, x FOR PEER REVIEW 6 of 17 values can be compared with those obtained in another work by Sánchez-Cid et al. [41]. Specifically, the critical deformation values of unitary chitosan-based hydrogel obtained at 5 °C are too similar to those obtained in this work. Table 1. Critical strain, G′ and tan δ at 1 Hz (indicated as G′1 and tan (δ)1) and critical temperature obtained for CH–G hydrogels with different biopolymers’ proportions at two temperatures, 5 °C and 40 °C. System CH–G Temperature (°C) Critical Strain (%) G′1 (Pa) tan (δ)1 (-) Critical Temperature (°C) 0–100 5 25.0 ± 0.6 1160 ± 253 0.029 ± 0.007 32.5–35.0 40 100.2 ± 0.1 0.40 ± 0.01 8.85 ± 1.49 25–75 5 100.8 ± 0.1 877 ± 145 0.027 ± 0.004 31.0–36.0 40 100.6 ± 0.1 182 ± 56 0.032 ± 0.006 50–50 5 65.3 ± 0.5 3880 ± 528 0.021 ± 0.001 35.0–40.0 40 64.0 ± 0.5 710 ± 26 0.032 ± 0.003 75–25 5 66.1 ± 0.4 1646 ± 56 0.072 ± 0.001 >40.0 40 41.3 ± 0.4 1574 ± 38 0.090 ± 0.014 100–0 5 10.4 ± 0.2 3907 ± 1106 0.117 ± 0.006 >40.0 40 6.8 ± 0.5 10,262 ± 5810 0.088 ± 0.008 After the critical deformation of each system was obtained, a time sweep test was carried out before gelation to analyze the evolution of both elastic (G′) and viscous (G″) moduli during the gelation process. Figure 2 shows how the gelation process increases the elastic modulus (G′) of the different hydrogels evaluated. Moreover, the distance between G′ and G″ increases with time, which indicates that the systems are gaining a more solid character. However, after one hour, the values of the elastic modulus (G′) and the viscous modulus (G″) became constant. 0 500 2500 3000 3500 10 0 10 1 10 2 10 3 CH − G 0 − 100 25 − 75 50 − 50 75 − 25 100 − 0 G' G'' G', G'' / (Pa) Time (s) Figure 2. Time sweep test of chitosan–type-A-gelatin hydrogels during the gelation process. Figure 2. Time sweep test of chitosan–type-A-gelatin hydrogels during the gelation process. Subsequently, frequency sweep tests were also carried out at different temperatures, and the results are shown in Figure 3. Firstly, it is important to note that Figure 3A,B present the profile of elastic (G ′ ) and viscous (G ′′ ) moduli in the frequency range studied of the different systems at 5 ◦ C and 40 ◦ C, respectively. According to the profiles obtained for the five systems, G ′ values are always higher than G‘’ values, thus showing a predominantly solid behavior, which also indicates the elastic response of the hydrogel system. Furthermore, a constant profile in the frequency range corroborates the stability of the systems. On the other hand, related to the temperature of measurements, it can be noted that at Gels 2024,10, 419 7 of 17 high temperatures (Figure 3B), the presence of a higher proportion of gelatin leads to an increase in the values of G ′ and G ′′ . However, the evolution of the 0–100 CH–G system at 40 ◦ C is not the same as the other ones, showing the low stability of type A gelatin at high frequencies. Thus, it is important to note that an increase in the proportion of chitosan leads to an increase in viscoelastic parameters due to its structural characteristics [ 42 ]. Comparing the elastic modulus and loss tangent at 1 Hz included in Table 1, it can be observed that the G ′ values of the systems generally increased with a lower temperature of measurement and the presence of a high proportion of chitosan. Moreover, the tan ( δ ) 1 values shown in Table 1indicate that using a higher temperature of measurement, the systems showed a decrease in their solid character since the tan δ value increase suggests that the value of G ′′ is greater than the G ′ . Furthermore, the tan δ values decrease with the presence of chitosan, indicating that with a higher proportion of chitosan, the systems obtained had higher solid character. These differences highlight the better mechanical resistance of hydrogels that have a small amount of chitosan due to the necessity of a material that can withstand mechanical stress during the cell growth and proliferation stage. Finally, highlighted are the similarities between the profile of the frequency curve of the unitary type A gelatin-based hydrogel obtained at 5 ◦ C and the one obtained in research done by Mehdi-Sefiani, H. et al. [ 32 ]. On the other hand, we found similarities between the frequency curves obtained for binary hydrogels made by chitosan and the collagen studied by Sánchez-Cid et al. [40]. Gels 2024, 10, x FOR PEER REVIEW 7 of 17 Subsequently, frequency sweep tests were also carried out at different temperatures, and the results are shown in Figure 3. Firstly, it is important to note that Figure 3A,B present the profile of elastic (G′) and viscous (G″) moduli in the frequency range studied of the different systems at 5 °C and 40 °C, respectively. According to the profiles obtained for the five systems, G′ values are always higher than G‘’ values, thus showing a predominantly solid behavior, which also indicates the elastic response of the hydrogel system. Furthermore, a constant profile in the frequency range corroborates the stability of the systems. On the other hand, related to the temperature of measurements, it can be noted that at high temperatures (Figure 3B), the presence of a higher proportion of gelatin leads to an increase in the values of G′ and G″. However, the evolution of the 0–100 CH–G system at 40 °C is not the same as the other ones, showing the low stability of type A gelatin at high frequencies. Thus, it is important to note that an increase in the proportion of chitosan leads to an increase in viscoelastic parameters due to its structural characteristics [42]. Comparing the elastic modulus and loss tangent at 1 Hz included in Table 1, it can be observed that the G′ values of the systems generally increased with a lower temperature of measurement and the presence of a high proportion of chitosan. Moreover, the tan (δ) 1 values shown in Table 1 indicate that using a higher temperature of measurement, the systems showed a decrease in their solid character since the tan δ value increase suggests that the value of G″ is greater than the G′. Furthermore, the tan δ values decrease with the presence of chitosan, indicating that with a higher proportion of chitosan, the systems obtained had higher solid character. These differences highlight the better mechanical resistance of hydrogels that have a small amount of chitosan due to the necessity of a material that can withstand mechanical stress during the cell growth and proliferation stage. Finally, highlighted are the similarities between the profile of the frequency curve of the unitary type A gelatin-based hydrogel obtained at 5 °C and the one obtained in research done by Mehdi-Sefiani, H. et al. [32]. On the other hand, we found similarities between the frequency curves obtained for binary hydrogels made by chitosan and the collagen studied by Sánchez-Cid et al. [40]. Figure 3. Frequency sweep test of chitosan–type-A-gelatin hydrogels evaluated at 5 °C (A) and 40 °C (B). Subsequently, temperature ramps were evaluated, and the results are shown in Figure 4. Firstly, it can be noted that the chitosan-based hydrogels are thermal stable compared with hydrogels that were developed only with type A gelatin. To corroborate this information, the critical temperature of each system was obtained and included in Table 1. The value of the critical temperature also indicated that hydrogels with a higher proportion of chitosan or unitary chitosan hydrogel have greater thermal stability than Figure 3. Frequency sweep test of chitosan–type-A-gelatin hydrogels evaluated at 5 ◦ C (A) and 40 ◦C (B). Subsequently, temperature ramps were evaluated, and the results are shown in Figure 4. Firstly, it can be noted that the chitosan-based hydrogels are thermal stable compared with hydrogels that were developed only with type A gelatin. To corroborate this information, the critical temperature of each system was obtained and included in Table 1.The value of the critical temperature also indicated that hydrogels with a higher proportion of chitosan or unitary chitosan hydrogel have greater thermal stability than hydrogels elaborated with a higher proportion of type A gelatin. It can also be attributed to gelatin melting above 30 ◦C [30]. Furthermore, with the aim of studying if these hydrogels are capable of being used in medical applications, time sweep tests were further evaluated by maintaining the temperature at 40 ◦ C for a period of 1 h. As can be seen in Figure 5, all the systems remained stable at 40 ◦ C except the type A gelatin unitary hydrogel, which showed instability because of its degradation at temperatures above 30 ◦C, as noted in Figure 4. Gels 2024,10, 419 8 of 17 Gels 2024, 10, x FOR PEER REVIEW 8 of 17 hydrogels elaborated with a higher proportion of type A gelatin. It can also be attributed to gelatin melting above 30 °C [30]. 5 10152025303540 10 0 10 1 10 2 10 3 10 4 CH−G G' G'' 0−100 25−75 50−50 75−25 100−0 G',G'' / (Pa) Temperature (ºC) Figure 4. Temperature ramp test of chitosan–type-A-gelatin-based hydrogels. Furthermore, with the aim of studying if these hydrogels are capable of being used in medical applications, time sweep tests were further evaluated by maintaining the temperature at 40 °C for a period of 1 h. As can be seen in Figure 5, all the systems remained stable at 40 °C except the type A gelatin unitary hydrogel, which showed instability because of its degradation at temperatures above 30 °C, as noted in Figure 4. 0 50 100 150 200 250 300 10-3 10-2 10-1 100 101 102 103 104 CH − G 0 − 100 25 − 75 50 − 50 75 − 25 100 − 0 G' G'' G',G'' / (Pa) Time (s) Figure 5. Time sweep test of chitosan–type-A-gelatin-based hydrogels at 40 °C. Figure 4. Temperature ramp test of chitosan–type-A-gelatin-based hydrogels. Gels 2024, 10, x FOR PEER REVIEW 8 of 17 hydrogels elaborated with a higher proportion of type A gelatin. It can also be attributed to gelatin melting above 30 °C [30]. 5 10152025303540 10 0 10 1 10 2 10 3 10 4 CH−G G' G'' 0−100 25−75 50−50 75−25 100−0 G',G'' / (Pa) Temperature (ºC) Figure 4. Temperature ramp test of chitosan–type-A-gelatin-based hydrogels. Furthermore, with the aim of studying if these hydrogels are capable of being used in medical applications, time sweep tests were further evaluated by maintaining the temperature at 40 °C for a period of 1 h. As can be seen in Figure 5, all the systems remained stable at 40 °C except the type A gelatin unitary hydrogel, which showed instability because of its degradation at temperatures above 30 °C, as noted in Figure 4. 0 50 100 150 200 250 300 10-3 10-2 10-1 100 101 102 103 104 CH − G 0 − 100 25 − 75 50 − 50 75 − 25 100 − 0 G' G'' G',G'' / (Pa) Time (s) Figure 5. Time sweep test of chitosan–type-A-gelatin-based hydrogels at 40 °C. Figure 5. Time sweep test of chitosan–type-A-gelatin-based hydrogels at 40 ◦C. 2.3. Microstructural Characterization: Scanning Electron Microscopy (SEM) In order to study the microstructural characteristics of the different systems of hydrogels, a SEM imaging was obtained (Figure 6). Gels 2024,10, 419 9 of 17 Gels 2024, 10, x FOR PEER REVIEW 9 of 17 2.3. Microstructural Characterization: Scanning Electron Microscopy (SEM) In order to study the microstructural characteristics of the different systems of hydrogels, a SEM imaging was obtained (Figure 6). Figure 6. 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