3D printed chitosan-pectin hydrogels: from rheological characterization to scaffold development and assessment

gels Article 3D Printed Chitosan-Pectin Hydrogels: From Rheological Characterization to Scaffold Development and Assessment Iratxe Zarandona 1, Carlos Bengoechea 2, EstefaníaÁlvarez-Castillo 2, Koro de la Caba 1,3,* , Antonio Guerrero 2and Pedro Guerrero 1,3,4,*   Citation: Zarandona, I.; Bengoechea, C.; Álvarez-Castillo, E.; de la Caba, K.; Guerrero, A.; Guerrero, P. 3D Printed Chitosan-Pectin Hydrogels: From Rheological Characterization to Scaffold Development and Assessment. Gels 2021,7, 175. https://doi.org/10.3390/gels7040175 Academic Editor: Michael T. Cook Received: 3 October 2021 Accepted: 18 October 2021 Published: 21 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 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/). 1 BIOMAT Research Group, University of the Basque Country (UPV/EHU), Escuela de Ingeniería de Gipuzkoa, Plaza de Europa 1, 20018 Donostia-San Sebastián, Spain; [email protected] 2Departamento de Ingeniería Química, Universidad de Sevilla, Escuela Politécnica Superior, Calle Virgen de África, 7, 41011 Sevilla, Spain; [email protected] (C.B.); malvar[email protected] (E.Á.-C.); aguerrer[email protected] (A.G.) 3BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain 4Proteinmat Materials SL, Avenida de Tolosa 72, 20018 Donostia-San Sebastián, Spain *Correspondence: [email protected] (K.d.l.C.); pedr[email protected] (P.G.) Abstract: Chitosan-pectin hydrogels were prepared, and their rheological properties were assessed in order to select the best system to develop scaffolds by 3D printing. Hydrogels showed a weak gel behavior with shear thinning flow properties, caused by the physical interactions formed between both polysaccharides, as observed by FTIR analysis. Since systems with high concentration of pectin showed aggregations, the system composed of 2 wt% chitosan and 2 wt% pectin (CHI2PEC2) was selected for 3D printing. 3D printed scaffolds showed good shape accuracy, and SEM and XRD analyses revealed a homogeneous and amorphous structure. Moreover, scaffolds were stable and kept their shape and size after a cycle of compression sweeps. Their integrity was also maintained after immersion in PBS at 37 ◦ C, showing a high swelling capacity, suitable for exudate absorption in wound healing applications. Keywords: chitosan; pectin; 3D printing; rheology; scaffolds 1. Introduction Natural polymers are commonly used for biomedical purposes due to their high biocompatibility, biodegradability, and bioactivity [ 1 ]. In this sense, hydrogels from natural polymers for 3D bioprinting are gaining notoriety to produce scaffolds for applications such as wound dressing [ 2 ]. In particular, polysaccharides and proteins are applied as bioinks for 3D printing [ 3 – 5 ]. Among them, chitosan has caught attention due to its antimicrobial activity [ 6 , 7 ]. Chitosan is a linear polysaccharide derived from chitin, found in the exoskeleton of crustaceans and in fungi [ 8 ]. Furthermore, due to its positive charge in acidic media, it is the only natural cationic polysaccharide. Thus, it has the ability to interact with negatively charged biomolecules by electrostatic interactions that affect mucoadhesion, hemostatic activity, antimicrobial activity, cell permeation capacity, and cytocompatibility [9]. Chitosan has been studied for different biomedical applications, including drug delivery and tissue engineering [ 10 – 13 ]. However, chitosan presents challenges in 3D printing, since it is too soft to self-support its structure and would collapse or deform due to its own weight [ 14 , 15 ]. Consequently, in order to overcome these drawbacks, chitosan can be combined with other polymers to promote physical and/or chemical crosslinking [ 8 , 16 ]. Although physically crosslinked materials show lower mechanical strength compared to chemically crosslinked ones, physically crosslinked hydrogels exhibit shear thinning behavior and facilitate flow through the needle of the 3D printer [ 17 ]. In Gels 2021,7, 175. https://doi.org/10.3390/gels7040175 https://www.mdpi.com/journal/gels Gels 2021,7, 175 2 of 17 this regard, chitosan bioinks reinforced with other biopolymer such as gelatin have been successfully printed, controlling the stability of the scaffolds as a function of the gelatin content [18]. In this work, pectin has been selected to reinforce chitosan hydrogels. Pectin is an anionic heteropolysaccharide with gelling properties, extracted from citrus fruits [ 19 , 20 ]. Depending on the esterification degree, pectin can be classified as high or low methoxy pectin [ 21 ]. Pectin is highly available, biodegradable, and non-toxic, which makes it a potential material for biomedical applications [ 22 , 23 ]. Moreover, the mixture of chitosan and pectin forms a polyelectrolyte complex mainly conformed by electrostatic interactions between the amino groups of chitosan and the carboxylic groups of pectin [24]. The aim of this work was to prepare chitosan-pectin hydrogels with different concentrations in order to select the optimal composition for 3D printing and analyze the properties of the printed scaffold. To accomplish this challenge, chitosan-pectin hydrogels were assessed from a rheological perspective. The hydrogel that fitted 3D printing requirements was selected, 3D printed, and the resulting scaffolds were physicochemically and mechanically characterized. 2. Results and Discussion 2.1. Rheological Characterization 2.1.1. Linear Viscoelastic Properties Rheological properties of chitosan-pectin systems were characterized to determine the most suitable hydrogel composition for 3D printing. Frequency sweep tests were performed at 4 and 25 ◦C to confirm chitosan-pectin gel formation and observe the time dependence of the systems (Figure 1). Regardless of the temperature used during the test (25 or 4 ◦ C), the single system containing only chitosan (CHI2PEC0) showed a predominantly viscous response with the loss modulus (G 00 ) greater than storage modulus (G 0 ) in the low frequency regime (Figure 1a,c) and a crossover point at high frequency (i.e., at 8.60 Hz and 6.45 Hz for 25 ◦ C and 4 ◦ C, respectively). This difference in the crossover frequency suggests that the CHI2PEC0 system needed longer relaxation times at lower temperatures, as a consequence of a slightly more elastic behavior [ 1 ]. This behavior is qualitatively similar to that one previously found for single CHI solutions [ 25 ]. However, the values of G 0 and G 00 were higher in the present study since the molecular weight was almost twice the value used in the previous study. In contrast, CHI0PEC3 systems presented a predominantly viscous behavior (G 00 > G 0 ) in the whole frequency range (Figure 1b,d), indicating a liquidlike behavior at both temperatures. On the other hand, binary systems containing both biopolymers, chitosan, and pectin (Figure 1a–d), showed a weak gel-like behavior, with G 0 values higher than G 00 , regardless of the frequency and temperature analyzed. The similar and moderate frequency dependence shown by both moduli, for every chitosan/pectin system is typical of weak gels with non-covalent bonding among the components of the formulation [ 26 ]. It is worth noting that a gel-like behavior typically results in proper stability to maintain dimensional firmness during deposition on the printing bed [27]. In this sense, parameters aand bfrom the power law model for G 0 (Equation (1)) were used to predict gel firmness. The power law coefficient, a, represents the magnitude of G 0 at a frequency of 1 Hz, and the power law exponent, b, indicates the dependence of the elastic modulus on frequency, where an exponent value equal to zero means that G 0 does not depend on frequency [ 28 ]. Power law parameters are shown in Table 1as a function of the hydrogel composition and temperature. As may be observed, an increase in pectin content led to an increase in parameter agiving rise to an enhancement of the gel strength, either at 25 or 4 ◦ C. Moreover, a high level of gel strength was maintained at the highest pectin content even if the chitosan content was reduced from 2 to 1%. As for parameter b, results revealed that when pectin concentration increased and chitosan concentration decreased, bparameter decreased, resulting in less frequency-dependent hydrogels. Therefore, the increase of pectin led to a stronger hydrogel [ 29 ]. Moreover, comparing the same hydrogels at different temperatures, 4 and 25 ◦ C, no significant differences (p> 0.05) were generally Gels 2021,7, 175 3 of 17 observed for parameters aand b. The exceptions were found at the lowest concentrations of chitosan (CHI1PEC3 and CHI0PEC3) that showed lower values for parameter bat low temperature. In any case, parameter branged between 0.18 and 0.36, indicating a relatively marked frequency dependence. This response was reported to correspond to a weak gel behavior [30]. Gels 2021, 7, x FOR PEER REVIEW 3 of 18 0.01 0.1 1 10 10-5 10-4 10-3 10-2 10-1 100 101 102 103 104 G', G'' / (Pa) Frequency / (Hz) CHI2PEC3 G' 25C CHI2PEC3 G'' 25C CHI2PEC2 G' 25C CHI2PEC2 G'' 25C CHI2PEC0 G' 25C CHI2PEC0 G'' 25C 0.01 0.1 1 10 10-5 10-4 10-3 10-2 10-1 100 101 102 103 104 G', G'' / (Pa) Frequency / (Hz) CHI2PEC3 G' 25C CHI2PEC3 G'' 25C CHI1.5PEC3 G' 25C CHI1.5PEC3 G'' 25C CHI1PEC3 G' 25C CHI1PEC3 G'' 25C CHI0PEC3 G' 25C CHI0PEC3 G'' 25C (a) (b) 0.01 0.1 1 10 10-3 10-2 10-1 100 101 102 103 104 G', G'' / (Pa) Frequency / (Hz) CHI2PEC3 G' 4C CHI2PEC3 G'' 4C CHI2PEC2 G' 4C CHI2PEC2 G'' 4C CHI2PEC0 G' 4C CHI2PEC0 G'' 4C 0.01 0.1 1 10 10-3 10-2 10-1 100 101 102 103 104 G', G'' / (Pa) Frequency / (Hz) CHI2PEC3 G' 4C CHI2PEC3 G'' 4C CHI1.5PEC3 G' 4C CHI1.5PEC3 G'' 4C CHI1PEC3 G' 4C CHI1PEC3 G'' 4C CHI0PEC3 G' 4C CHI0PEC3 G'' 4C (c) (d) Figure 1. Elastic (G’) and viscous (G”) moduli as a function of frequency from 0.01 to 10 Hz: (a,c) for chitosan-pectin hydrogels with 2% chitosan at 25 and 4 °C, respectively; (b,d) for chitosan-pectin hydrogels with 3% pectin at 25 and 4 °C, respectively. In this sense, parameters a and b from the power law model for G’ (Equation (1)) were used to predict gel firmness. The power law coefficient, a, represents the magnitude of G’ at a frequency of 1 Hz, and the power law exponent, b, indicates the dependence of the elastic modulus on frequency, where an exponent value equal to zero means that G’ does not depend on frequency [28]. Power law parameters are shown in Table 1 as a function of the hydrogel composition and temperature. As may be observed, an increase in pectin content led to an increase in parameter a giving rise to an enhancement of the gel strength, either at 25 or 4 °C. Moreover, a high level of gel strength was maintained at the highest pectin content even if the chitosan content was reduced from 2 to 1%. As for parameter b, results revealed that when pectin concentration increased and chitosan concentration decreased, b parameter decreased, resulting in less frequency-dependent hydrogels. Therefore, the increase of pectin led to a stronger hydrogel [29]. Moreover, comparing the same hydrogels at different temperatures, 4 and 25 °C, no significant differences (p > 0.05) were generally observed for parameters a and b. The exceptions were found at the lowest concentrations of chitosan (CHI1PEC3 and CHI0PEC3) that showed lower values for parameter b at low temperature. In any case, parameter b ranged between 0.18 and 0.36, indicating a relatively marked frequency dependence. This response was reported to correspond to a weak gel behavior [30]. Figure 1. Elastic (G 0 ) and viscous (G 00 ) moduli as a function of frequency from 0.01 to 10 Hz: ( a , c ) for chitosan-pectin hydrogels with 2% chitosan at 25 and 4 ◦C, respectively; (b,d) for chitosan-pectin hydrogels with 3% pectin at 25 and 4 ◦C, respectively. In order to determine the linear viscoelastic range (LVR), stress sweep tests at 25 ◦ C and 4 ◦ C were carried out, which provide information on the unperturbed structure of the system. To delimit the LVR, the critical strain ( γc ) was determined, and values are shown in Table 2. Results revealed significant differences (p< 0.05) between single and binary systems, since a decrease of critical strain was observed for the latter, suggesting that the hydrogel network had lower deformation capacity due to the interactions between chitosan and pectin [ 31 ]. Chitosan may act as an efficient cross-linker in pectin systems at acidic pH, promoting an upward evolution in the viscoelastic moduli of samples [ 32 ]. Additionally, electrostatically stabilized complexes are formed in the presence of both biopolymers, where also hydrogen bonding and hydrophobic interactions may play a role [33]. Gels 2021,7, 175 4 of 17 Table 1. The effect of chitosan and pectin concentration on acoefficient and bexponent of the power law model for the storage modulus in the frequency sweep of the hydrogels at 25 and 4 ◦C. T (◦C) Sample a(Pa·Hz b)bR2 25 CHI2PEC0 20.1 ±1.1 a,A 0.677 ±0.015 a,A 0.9922 CHI2PEC2 340 ±19 b,B 0.360 ±0.008 b,B 0.9992 CHI2PEC3 759 ±3c,C 0.328 ±0.001 b,BC 0.9980 CHI1.5PEC3 485 ±18 b,B 0.300 ±0.011 bc,C 0.9992 CHI1PEC3 725 ±130 c,C 0.236 ±0.013 c,D 0.9977 CHI0PEC3 0.0938 ±0.0002 a,A 1.580 ±0.059 d,E 0.9858 4 CHI2PEC0 26.9 ±0.4 A0.676 ±0.001 A0.9959 CHI2PEC2 381 ±4B0.346 ±0.003 BC 0.9991 CHI2PEC3 1233 ±37 D0.303 ±0.011 BC 0.9998 CHI1.5PEC3 753 ±81 C0.295 ±0.011 C0.9996 CHI1PEC3 1209 ±68 D0.176 ±0.005 F0.9965 CHI0PEC3 8.90 ±0.49 A1.125 ±0.022 G0.9835 a–c, A–G Two means followed by the same letter in the same column are not significantly (p> 0.05) different according to the Tukey’s multiple range test. Table 2. Critical strain ( γc ), and values for G 0 at 1 Hz (G 01 ), and tan δ at 1 Hz (tan δ1 ) within the linear viscoelastic range for the hydrogels tested at 25 ◦C and 4 ◦C. T (◦C) Sample γcG01(Pa) tan δ1 25 CHI2PEC0 0.392 ±0.016 a,A 23.0 ±1.4 a,A 1.20 ±0.03 a,A CHI2PEC2 0.034 ±0.002 b,B 257 ±18 b,B 0.64 ±0.03 a,AB CHI2PEC3 0.037 ±0.004 b,B 763 ±41 c,F 0.54 ±0.01 a,AB CHI1.5PEC3 0.033 ±0.001 b,B 486 ±21 d,CD 0.53 ±0.04 a,AB CHI1PEC3 0.025 ±0.001 b,B 599 ±31 e,DE 0.39 ±0.04 a,B CHI0PEC3 0.752 ±0.137 c,C 0.114 ±0.010 a,A 21.52 ±0.92 b,C 4 CHI2PEC0 0.333 ±0.006 A29.4 ±0.3 A1.201 ±0.016A CHI2PEC2 0.0243 ±0.001 B417 ±62 BC 0.590 ±0.003 AB CHI2PEC3 0.0380 ±0.0008 B1023 ±52 G0.505 ±0.009 AB CHI1.5PEC3 0.0218 ±0.0008 B660 ±21 EF 0.498 ±0.008 AB CHI1PEC3 0.0231 ±0.0004 B1023 ±93 G0.301 ±0.006 B CHI0PEC3 0.295 ±0.013 A11.3 ±1.1 A2.103 ±0.085 D a–e, A–G Two means followed by the same letter in the same column are not significantly (p> 0.05) different according to the Tukey’s multiple range test. In addition, regarding G 0 values at 1 Hz (G 01 ) shown in Table 3for the LVR, significant differences (p< 0.05) were observed for the hydrogels tested at 25 ◦ C. Thus, the single systems (CHI0PEC3 and CHI2PEC0) showed the lowest G 0 values, between 0.11 and 22.96 Pa, respectively. No significant (p> 0.05) differences were observed between G 01 values at 25 ◦ C and 4 ◦ C for CHI0PEC3, CHI2PEC0, and CHI2PEC2 hydrogels. However, G 01 for CHI1PEC3, CHI1.5PEC3, and CHI2PEC3 samples showed a significant increase ( p< 0.05 ) with increasing temperature from 4 ◦ C to 25 ◦ C, especially for CHI1PEC3 hydrogels. Therefore, an increase in hydrogel stiffness was driven by a higher proportion of pectin [34]. The predominantly viscous response observed for CHI0PEC3 and CHI2PEC0 systems can be confirmed with the values of tan δ at 1 Hz (tan δ1 ), which are higher than unity for these two systems at 25 ◦ C and 4 ◦ C. In contrast, all the binary chitosan-pectin hydrogels presented tan δ1 values lower than 1, indicative of a predominantly elastic behavior. Among chitosan-pectin hydrogels, no significant difference (p> 0.05) was observed for tan δ1 , displaying values around 0.5. Therefore, considering that hydrogels exhibited small critical strain values ( γc < 0.05), moderately low loss tangent values (tan δ1 > 0.1), chitosan-pectin hydrogels can be considered “weak gels” [ 30 ]. It is worth mentioning that these gels have the capacity to flow without fracture, recover their structure, and achieve the properties required for 3D printing [35]. Gels 2021,7, 175 5 of 17 Table 3. Zero-shear rate viscosity ( η0 ), characteristic time ( λ ), flow index (n), and correlation coefficient (R 2 ) of Williamson model for chitosan-pectin hydrogels at 25 and 4 ◦C. T (◦C) Sample η0(Pa·s) λ(s) n R2 25 CHI2PEC0 113.84 ±15.43 a,A 14.2 ±1.0 a,A 0.448 ±0.016 a,A 0.999 CHI2PEC2 19,658 ±2287 a,AB 431 ±116 b,B 0.266 ±0.008 b,BCD 0.999 CHI2PEC3 29,432 ±7419 a,ABC 198 ±74 a,AB 0.191 ±0.008 b,CD 0.999 CHI1.5PEC3 36,336 ±9625 a,ABC 1040 ±135 c,C 0.265 ±0.022 b,BCD 0.999 CHI1PEC3 152,729 ±31,136 b,D 2621 ±57 d,F 0.204 ±0.018 b,CD 0.999 CHI0PEC3 0.501 ±0.137 a,A 0.0009 ±0.0004 a,A 0.674 ±0.133 c,E 0.970 4 CHI2PEC0 115.71 ±8.58 A8.75 ±0.91 A0.438 ±0.013 A0.999 CHI2PEC2 37,051 ±1973 ABC 2632 ±222 F0.305 ±0.007 BC 0.999 CHI2PEC3 48,159 ±6715 BC 570 ±124 B0.249 ±0.010 CD 0.999 CHI1.5PEC3 64,136 ±7242 C2131 ±198 E0.288 ±0.007 BC 0.999 CHI1PEC3 207,319 ±28281 E1544 ±282 D0.168 ±0.001 D0.999 CHI0PEC3 8.050 ±1.237 A0.119 ±0.028 A0.374 ±0.023 AB 0.999 a–d, A–F Two means followed by the same letter in the same column are not significantly (p> 0.05) different according to the Tukey’s multiple range test. Temperature sweep tests from 25 to 80 ◦ C were carried out at 1 Hz to analyze the effect of temperature on the rheological behavior of the hydrogels. As shown in Figure 2a, G 00 values were higher than G 0 for chitosan hydrogels without pectin (CHI2PEC0) at low temperatures, indicating a fluid-like viscoelastic behavior until the crossover point was reached around 58 ◦ C. From that temperature on, hydrogels exhibited a predominantly elastic response (i.e., G 0 became higher than G 00 ), due to the formation of chitosan clusters by hydrophobic interactions [ 36 ]. In contrast, in absence of chitosan, pectin systems (CHI0PEC3) exhibited a viscous behavior, with G 00 values clearly higher than G 0 in the whole range of temperatures, as shown in Figure 2B. In any case, these two single systems showed mechanical spectra corresponding to polymer solutions below the threshold for the critical gel behavior. Gels 2021, 7, x FOR PEER REVIEW 6 of 18 chitosan-pectin hydrogels, except CHI2PEC2, presented an increase of both moduli until 80 °C. 30 40 50 60 70 80 100 101 102 103 104 105 G', G'' / (Pa) T (ºC) CHI2PEC3 G' CHI2PEC3 G'' CHI2PEC2 G' CHI2PEC2 G'' CHI2PEC0 G' CHI2PEC0 G'' 40 60 80 100 101 102 103 104 105 G', G'' / (Pa) T (ºC) CHI2PEC3 G' CHI2PEC3 G'' CHI1.5PEC3 G' CHI1.5PEC3 G'' CHI1PEC3 G' CHI1PEC3 G'' CHI0PEC3 G' CHI0PEC3 G'' (a) (b) Figure 2. Elastic (G’) and viscous (G”) moduli as a function of temperature from 25 to 80 °C for (a) chitosan-pectin hydrogels with 2% chitosan and (b) chitosan-pectin hydrogels with 3% pectin. 2.1.2. Flow Properties Additionally, the flow behavior of chitosan-pectin hydrogels was determined, and flow curves are displayed in Figure 3. All chitosan-pectin hydrogels with 2% chitosan presented a similar shear-thinning behavior at 25 °C (Figure 3a) and 4 °C (Figure 3c), with a marked decrease of viscosity as shear rate increased, clearly describing a power law decay region and showing a tendency towards a zero-shear limiting viscosity at low shear rate. Once again, the single CHI solution (CHI2PEC0) displayed a similar shear thinning response, although showing higher viscosity values than those found previously (due to the highest molecular weight) [25]. Moreover, flow properties were dependent on pectin concentration; when pectin concentration increased, viscosity also increased. However, binary systems containing 3% pectin did not reflect any apparent influence of chitosan concentration on the flow curves, neither at 25 °C (Figure 3b) nor at 4 °C (Figure 3d). In contrast, pectin hydrogels without chitosan were found to be independent on shear rate at 25 °C, indicating a Newtonian fluid behavior, whereas at 4 °C, a decrease of viscosity was observed at high shear rates, indicating a non-Newtonian shear-thinning behavior. Moreover, the viscosity at 4 °C increased 10 times with respect to the viscosity at 25 °C, as also reported by other authors [37,38]. It is worth mentioning that the shear thinning behavior of the hydrogels facilitates their flow during 3D printing. Upon hydrogel ejection from the nozzle of the 3D printer (25 °C) and its deposition on the printer bed (4 °C), the shear rate undergoes a sudden decrease which entails a remarkable increase in the viscosity of the shear-thinning hydrogel, leading to an enhancement of the dimensional stability of the 3D printed scaffold [39]. Figure 2. Elastic (G 0 ) and viscous (G 00 ) moduli as a function of temperature from 25 to 80 ◦ C for ( a ) chitosan-pectin hydrogels with 2% chitosan and (b) chitosan-pectin hydrogels with 3% pectin. Regarding chitosan-pectin systems (Figure 2a,b), all samples showed higher values of G 0 than G 00 , reflecting the elastic behavior of the samples in the range between 25 and 80 ◦ C. Moreover, all chitosan-pectin systems presented nearly constant values of both moduli between 25 and 58 ◦ C, suggesting that no difference was expected when printing at room or physiological temperature. However, for temperatures above 58 ◦ C, all chitosan-pectin hydrogels, except CHI2PEC2, presented an increase of both moduli until 80 ◦C. Gels 2021,7, 175 6 of 17 2.1.2. Flow Properties Additionally, the flow behavior of chitosan-pectin hydrogels was determined, and flow curves are displayed in Figure 3. All chitosan-pectin hydrogels with 2% chitosan presented a similar shear-thinning behavior at 25 ◦ C (Figure 3a) and 4 ◦ C (Figure 3c), with a marked decrease of viscosity as shear rate increased, clearly describing a power law decay region and showing a tendency towards a zero-shear limiting viscosity at low shear rate. Once again, the single CHI solution (CHI2PEC0) displayed a similar shear thinning response, although showing higher viscosity values than those found previously (due to the highest molecular weight) [ 25 ]. Moreover, flow properties were dependent on pectin concentration; when pectin concentration increased, viscosity also increased. However, binary systems containing 3% pectin did not reflect any apparent influence of chitosan concentration on the flow curves, neither at 25 ◦ C (Figure 3b) nor at 4 ◦ C (Figure 3d). In contrast, pectin hydrogels without chitosan were found to be independent on shear rate at 25 ◦ C, indicating a Newtonian fluid behavior, whereas at 4 ◦ C, a decrease of viscosity was observed at high shear rates, indicating a non-Newtonian shear-thinning behavior. Moreover, the viscosity at 4 ◦ C increased 10 times with respect to the viscosity at 25 ◦ C, as also reported by other authors [ 37 , 38 ]. It is worth mentioning that the shear thinning behavior of the hydrogels facilitates their flow during 3D printing. Upon hydrogel ejection from the nozzle of the 3D printer (25 ◦ C) and its deposition on the printer bed (4 ◦ C), the shear rate undergoes a sudden decrease which entails a remarkable increase in the viscosity of the shear-thinning hydrogel, leading to an enhancement of the dimensional stability of the 3D printed scaffold [39]. Gels 2021, 7, x FOR PEER REVIEW 7 of 18 0.01 0.1 1 10 100 100 101 102 103 104 105 h / (Pas) ɣ / (1/s) CHI2PEC3 25C CHI2PEC2 25C CHI2PEC0 25C 0.01 0.1 1 10 100 100 101 102 103 104 105 h / (Pas) ɣ / (1/s) CHI2PEC3 25C CHI1.5PEC3 25C CHI1PEC3 25C CHI0PEC3 25C (a) (b) 0.01 0.1 1 10 100 100 101 102 103 104 105 h / (Pas) ɣ / (1/s) CHI2PEC3 4C CHI2PEC2 4C CHI2PEC0 4C 0.01 0.1 1 10 100 100 101 102 103 104 105 h / (Pas) ɣ / (1/s) CHI2PEC3 4C CHI1.5PEC3 4C CHI1PEC3 4C CHI0PEC3 4C (c) (d) Figure 3. Steady state flow curves of chitosan-pectin hydrogels: (a,c) for chitosan-pectin hydrogels with 2% chitosan at 25 and 4 °C, respectively; (b,d) for chitosan-pectin hydrogels with 3% pectin. Lines reproduce the fitting to the Williamson model. Flow curves were fitted to the Williamson model (Equation (2)) and fitting parameters are presented in Table 4. Concerning the zero-shear rate viscosities for the hydrogels at 25 °C, an increase from 19.7 to 1523 kPa·s was observed following this increasing sequence: CHI2PEC2 < CHI2PEC3 < CHI1.5PEC3 < CHI1PEC3, indicating that when the amount of pectin increased and that of chitosan decreased, the formation of polymer aggregates was promoted [40,41]. Regarding the effect of temperature, no significant difference (p < 0.05) was observed between the values of the zero-shear rate viscosity at 25 and 4 °C. Regarding the viscosity in the 3D printing process, taking into consideration that the nozzle diameter used was 0.84 mm, with a printing velocity of 2.8 mm/s, the shear rate can be calculated using Equation (3). The estimated value for the shear rate at the nozzle was between 42 and 60 s−1, corresponding to a viscosity between 12 and 23 Pa·s for all binary systems. Bearing in mind these results, viscosity during 3D printing decreased 103 times compared to the zero-shear rate viscosity. These results are in accordance with the empirical ink viscosities reported during 3D printing, which are in a range from 0.5 to 20 Pa·s at high shear rates (102–103 s−1), while the range moves from 102 to 103 Pa·s at lower shear rates (≤0.1 s−1) [42–44]. While all binary systems follow the criteria for high shear rates, only CHI2PEC2 and CHI1.5PEC3 binary systems comply with the requirement at low shear rates (i.e., viscosity lower than 103 Pa·s). In order to select the most suitable system for 3D printing applications, the following features can be considered: (i) Systems containing 3% pectin showed apparent viscosities higher than 1000 Pa·s at low shear rates (<0.1 s−1); (ii) Higher concentrations of pectin with lower concentrations of chitosan showed pectin aggregation and clustering and, thus, heterogeneity; (iii) There was no significant difference in the rheological behavior among Figure 3. Steady state flow curves of chitosan-pectin hydrogels: ( a , c ) for chitosan-pectin hydrogels with 2% chitosan at 25 and 4◦C, respectively; (b,d) for chitosan-pectin hydrogels with 3% pectin. Lines reproduce the fitting to the Williamson model. Gels 2021,7, 175 7 of 17 Flow curves were fitted to the Williamson model (Equation (2)) and fitting parameters are presented in Table 4. Concerning the zero-shear rate viscosities for the hydrogels at 25 ◦ C, an increase from 19.7 to 1523 kPa · s was observed following this increasing sequence: CHI2PEC2 < CHI2PEC3 < CHI1.5PEC3 < CHI1PEC3, indicating that when the amount of pectin increased and that of chitosan decreased, the formation of polymer aggregates was promoted [ 40 , 41 ]. Regarding the effect of temperature, no significant difference (p< 0.05) was observed between the values of the zero-shear rate viscosity at 25 and 4 ◦ C. Regarding the viscosity in the 3D printing process, taking into consideration that the nozzle diameter used was 0.84 mm, with a printing velocity of 2.8 mm/s, the shear rate can be calculated using Equation (3). The estimated value for the shear rate at the nozzle was between 42 and 60 s −1 , corresponding to a viscosity between 12 and 23 Pa · s for all binary systems. Bearing in mind these results, viscosity during 3D printing decreased 10 3 times compared to the zero-shear rate viscosity. These results are in accordance with the empirical ink viscosities reported during 3D printing, which are in a range from 0.5 to 20 Pa · s at high shear rates (10 2 –10 3 s −1 ), while the range moves from 10 2 to 10 3 Pa · s at lower shear rates ( ≤ 0.1 s −1 ) [ 42 – 44 ]. While all binary systems follow the criteria for high shear rates, only CHI2PEC2 and CHI1.5PEC3 binary systems comply with the requirement at low shear rates (i.e., viscosity lower than 10 3 Pa · s). In order to select the most suitable system for 3D printing applications, the following features can be considered: (i) Systems containing 3% pectin showed apparent viscosities higher than 1000 Pa·s at low shear rates (<0.1 s−1); (ii) Higher concentrations of pectin with lower concentrations of chitosan showed pectin aggregation and clustering and, thus, heterogeneity; (iii) There was no significant difference in the rheological behavior among the rest of chitosan-pectin hydrogels. Therefore, taking all these factors into account, CHI2PEC2 hydrogels were selected for 3D printing. Table 4. Denotation and composition of chitosan-pectin systems. System Designation Chitosan Concentration (w/v%) Pectin Concentration (w/v%) Single CHI2PEC0 2.0 0.0 systems CHI0PEC3 0.0 3.0 CHI2PEC2 2.0 2.0 Binary CHI2PEC3 2.0 3.0 systems CHI1.5PEC3 1.5 3.0 CHI1PEC3 1.0 3.0 2.2. CHI2PEC2 Hydrogel Characterization The FTIR spectra of chitosan, pectin, and CHI2PEC2 are shown in Figure 4a. Both chitosan and pectin showed bands at 3000–3600 cm −1 , associated with O-H bonds in both polymers and to N-H bonds in chitosan, and bands between 1150 and 890 cm −1 associated with the C-O-C of the saccharide ring [ 33 ]. The differences were observed in the range between 1300 and 1750 cm −1 . Regarding chitosan spectrum, the band at 1644 cm −1 , attributed to C=O stretching; a band at 1558 cm −1 , associated to NH2 bending; and two bands at 1418 and 1376 cm −1 , corresponding to CH3 deformation, were observed [ 45 , 46 ]. For pectin spectrum, a band at 1738 cm −1 , assigned to C = O of the ester bonds, and the band at 1604 cm −1 , associated with the asymmetric stretching vibration of COO- were observed [ 47 ]. Some displacements of these bands were observed for CHI2PEC2. In particular, the band related to the ester group in pectin shifted from 1738 cm −1 to 1732 cm −1 in CHI2PEC2. Moreover, the absorption band corresponding to C=O stretching at 1644 cm −1 for chitosan and at 1604 cm −1 for pectin shifted to 1628 cm −1 for CHI2PEC2. All these band displacements indicate physical interactions by hydrogen bonding between both biopolymers (Figure 4b). Gels 2021,7, 175 8 of 17 Gels 2021, 7, x FOR PEER REVIEW 8 of 18 the rest of chitosan-pectin hydrogels. Therefore, taking all these factors into account, CHI2PEC2 hydrogels were selected for 3D printing. Table 4. Denotation and composition of chitosan-pectin systems. System Designation Chitosan Concentration (w/v%) Pectin Concentration (w/v%) Single CHI2PEC0 2.0 0.0 systems CHI0PEC3 0.0 3.0 CHI2PEC2 2.0 2.0 Binary CHI2PEC3 2.0 3.0 systems CHI1.5PEC3 1.5 3.0 CHI1PEC3 1.0 3.0 2.2. CHI2PEC2 Hydrogel Characterization The FTIR spectra of chitosan, pectin, and CHI2PEC2 are shown in Figure 4a. Both chitosan and pectin showed bands at 3000–3600 cm−1, associated with O-H bonds in both polymers and to N-H bonds in chitosan, and bands between 1150 and 890 cm−1 associated with the C-O-C of the saccharide ring [33]. The differences were observed in the range between 1300 and 1750 cm−1. Regarding chitosan spectrum, the band at 1644 cm−1, attributed to C=O stretching; a band at 1558 cm−1, associated to NH2 bending; and two bands at 1418 and 1376 cm−1, corresponding to CH3 deformation, were observed [45,46]. For pectin spectrum, a band at 1738 cm−1, assigned to C = O of the ester bonds, and the band at 1604 cm−1, associated with the asymmetric stretching vibration of COO- were observed [47]. Some displacements of these bands were observed for CHI2PEC2. In particular, the band related to the ester group in pectin shifted from 1738 cm−1 to 1732 cm−1 in CHI2PEC2. Moreover, the absorption band corresponding to C=O stretching at 1644 cm−1 for chitosan and at 1604 cm−1 for pectin shifted to 1628 cm−1 for CHI2PEC2. All these band displacements indicate physical interactions by hydrogen bonding between both biopolymers (Figure 4b). 1000150020002500300035004000 Transmittance (a.u.) Wavenumber (cm-1) CHI PEC CHI2PEC2 (a) (b) Figure 4. (a) FTIR spectra of neat chitosan (CHI), neat pectin (PEC) and the hydrogel with 2 wt% chitosan and 2 wt% pectin (CHI2PEC2) and (b) representation of the interactions between chitosan and pectin. In order to evaluate the hydrogel adhesiveness to a biological surface, mucoadhesion analysis was carried out and the force displacement curve of one of the CHI2PEC2 replicates is shown in Figure 5. At the beginning of the test, when the sample is getting closer to the mucin disk, the force decreased due to the force applied to attach the hydrogel to the mucin disk. A significant increase of the force was observed as the hydrogel started Chitosan O O COO- O HO OH + O O COOCH3 HO OH O O OH O HO NH3 O OH HO NH2 O O OH O HO NH3 O OH HO NH2 Pectin Chitosan Figure 4. ( a ) FTIR spectra of neat chitosan (CHI), neat pectin (PEC) and the hydrogel with 2 wt% chitosan and 2 wt% pectin (CHI2PEC2) and (b) representation of the interactions between chitosan and pectin. In order to evaluate the hydrogel adhesiveness to a biological surface, mucoadhesion analysis was carried out and the force displacement curve of one of the CHI2PEC2 replicates is shown in Figure 5. At the beginning of the test, when the sample is getting closer to the mucin disk, the force decreased due to the force applied to attach the hydrogel to the mucin disk. A significant increase of the force was observed as the hydrogel started to detach from the mucin disk due to the exerted force done. From then on, the force started to decrease. The maximum detachment force (Fmax) and the work of adhesion (Wad) were calculated from the mucoadhesion analysis. The values found for Fmax and Wad were 0.21 ±0.02 N and 0.36 ± 0.03 N mm, respectively. These values are higher than those obtained for other polysaccharide systems (Fmax = 0.093 N and Wad = 0.029 N mm) for wound dressing applications [ 48 ]. Therefore, chitosan-pectin hydrogel showed excellent mucoadhesive properties, which are essential requirements for wound dressing materials in order to be adhered to the wound site to protect it from the external environment. This mucoadhesive capacity is associated with –OH and –NH2 groups in the biopolymers, which could be linked to mucins by hydrogen bonding; furthermore, as acidic pH was used for the hydrogel preparation, the amino groups of chitosan were protonated and, thus, strong electrostatic interactions could be formed with the anionic groups of mucin [ 49 ]. Since pectin and mucin are anionic compounds, hence, electrostatic repulsion charges might result in an uncoiling of polymer chains and facilitate chain entanglement and bond formation [50]. Gels 2021, 7, x FOR PEER REVIEW 9 of 18 to detach from the mucin disk due to the exerted force done. From then on, the force started to decrease. The maximum detachment force (Fmax) and the work of adhesion (Wad) were calculated from the mucoadhesion analysis. The values found for Fmax and Wad were 0.21 ± 0.02 N and 0.36 ± 0.03 N mm, respectively. These values are higher than those obtained for other polysaccharide systems (Fmax = 0.093 N and Wad = 0.029 N mm) for wound dressing applications [48]. Therefore, chitosan-pectin hydrogel showed excellent mucoadhesive properties, which are essential requirements for wound dressing materials in order to be adhered to the wound site to protect it from the external environment. This mucoadhesive capacity is associated with –OH and –NH2 groups in the biopolymers, which could be linked to mucins by hydrogen bonding; furthermore, as acidic pH was used for the hydrogel preparation, the amino groups of chitosan were protonated and, thus, strong electrostatic interactions could be formed with the anionic groups of mucin [49]. Since pectin and mucin are anionic compounds, hence, electrostatic repulsion charges might result in an uncoiling of polymer chains and facilitate chain entanglement and bond formation [50]. -12 -10 -8 -6 -4 -2 0 2 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 Force (N) Distance (mm) Fmax Wadh Figure 5. Force displacement curve of one replicate of CHI2PEC2 hydrogel in the mucoadhesion analysis. Additionally, texture profile analysis (TPA) can provide information related to the hardness, adhesiveness, and cohesiveness of the hydrogels, which are of great relevance to analyze their handleability for 3D printing [51]. In this regard, hardness, which is the maximum force required to produce the first deformation, showed a value of 5.9 ± 0.2 g. Adhesiveness is related to the capacity of a gel to adhere on a surface and, thus, higher adhesiveness values indicate that the gel needs shorter time to bond to the surface [52]. In the case of the CHI2PEC2 system, the adhesiveness value found was 8.7 ± 0.5 g·s. This adhesiveness, as well as the hardness observed for CHI2PEC2 hydrogel, was similar to values obtained for hyaluronic acid/carboxymethylcellulose systems [50]. Finally, cohesiveness is related to the strength capacity of a gel to maintain its own structure when subjected to a compressive stress [53]. CHI2PEC2 hydrogel exhibited a high cohesive value of 1.05 ± 0.04, indicative of a high capacity to maintain the three-dimensional structural integrity and, hence, to hold its structure after printing [54]. 2.3. 3D Printed CHI2PEC2 Scaffold Characterization CHI2PEC2 scaffolds were successfully printed with shape fidelity as shown in Figure 6. After ink deposition, the material kept the set geometry and showed mechanical integrity. Figure 5. Force displacement curve of one replicate of CHI2PEC2 hydrogel in the mucoadhesion analysis. Gels 2021,7, 175 9 of 17 Additionally, texture profile analysis (TPA) can provide information related to the hardness, adhesiveness, and cohesiveness of the hydrogels, which are of great relevance to analyze their handleability for 3D printing [ 51 ]. In this regard, hardness, which is the maximum force required to produce the first deformation, showed a value of 5.9 ± 0.2 g. Adhesiveness is related to the capacity of a gel to adhere on a surface and, thus, higher adhesiveness values indicate that the gel needs shorter time to bond to the surface [ 52 ]. In the case of the CHI2PEC2 system, the adhesiveness value found was 8.7 ± 0.5 g · s. This adhesiveness, as well as the hardness observed for CHI2PEC2 hydrogel, was similar to values obtained for hyaluronic acid/carboxymethylcellulose systems [ 50 ]. Finally, cohesiveness is related to the strength capacity of a gel to maintain its own structure when subjected to a compressive stress [ 53 ]. CHI2PEC2 hydrogel exhibited a high cohesive value of 1.05 ± 0.04, indicative of a high capacity to maintain the three-dimensional structural integrity and, hence, to hold its structure after printing [54]. 2.3. 3D Printed CHI2PEC2 Scaffold Characterization CHI2PEC2 scaffolds were successfully printed with shape fidelity as shown in Figure 6. After ink deposition, the material kept the set geometry and showed mechanical integrity. Gels 2021, 7, x FOR PEER REVIEW 10 of 18 Figure 6. 3D printed CHI2PEC2 scaffold after drying at room temperature. 2.3.1. Physicochemical Properties Swelling of CHI2PEC2 scaffolds was evaluated in phosphate buffered saline (PBS) solution (pH = 7.4) at room temperature. As can be observed in Figure 7, swelling increased fast up to 1250% in the first 5 h, indicating that the samples increased more than 10 times their initial weights. Thereafter, swelling continued increasing more slowly up to 1830% at day 7. Since pectin moieties were negatively charged with -COO- groups at pH 7.4, electrostatic repulsions were promoted and, thus, the swelling ability increased [55,56]. In this context, having a high swelling capacity is desirable in order to absorb wound exudates that could slow the wound healing and macerate the nearby skin [57]. It is worth noting that samples kept their integrity after the 7 days of immersion. 0 2,000 4,000 6,000 8,000 10,000 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 Swelling (%) Immersion time (min) Equation y = A1*exp(-x/t1) + A2*exp(-x/t2) + y0 y0 1771.06695 ± 31.55655 A1 -842.51424 ± 23.03308 t1 33.87561 ± 2.18776 A2 -756.73002 ± 32.46403 t2 1619.85958 ± 216.65442 Adj. R-Square 0.99078 Figure 7. Swelling capacity of CHI2PEC2 scaffolds in PBS (pH = 7.4). The values of degradation degree (DD) after 1, 3, 7, 11, 15, 18, and 21 days of immersion into PBS at 37 °C are presented in Figure 8. No significant mass loss (p > 0.05) was observed up to day 7. After 7 days, a pronounced increase of DD up to 25% was observed, but samples kept their integrity, which indicates that the interactions between chitosan and pectin were strong enough. No significant differences (p > 0.05) were observed between days 7 and 18, at which an increase of DD and a mass loss of 43% were observed. Figure 6. 3D printed CHI2PEC2 scaffold after drying at room temperature. 2.3.1. Physicochemical Properties Swelling of CHI2PEC2 scaffolds was evaluated in phosphate buffered saline (PBS) solution (pH = 7.4) at room temperature. As can be observed in Figure 7, swelling increased fast up to 1250% in the first 5 h, indicating that the samples increased more than 10 times their initial weights. Thereafter, swelling continued increasing more slowly up to 1830% at day 7. Since pectin moieties were negatively charged with -COO- groups at pH 7.4, electrostatic repulsions were promoted and, thus, the swelling ability increased [ 55 , 56 ]. In this context, having a high swelling capacity is desirable in order to absorb wound exudates that could slow the wound healing and macerate the nearby skin [ 57 ]. It is worth noting that samples kept their integrity after the 7 days of immersion. The values of degradation degree (DD) after 1, 3, 7, 11, 15, 18, and 21 days of immersion into PBS at 37 ◦ C are presented in Figure 8. No significant mass loss (p> 0.05) was observed up to day 7. After 7 days, a pronounced increase of DD up to 25% was observed, but samples kept their integrity, which indicates that the interactions between chitosan and pectin were strong enough. No significant differences (p> 0.05) were observed between days 7 and 18, at which an increase of DD and a mass loss of 43% were observed. Gels 2021,7, 175 16 of 17 23. Ma, T.; Lv, L.; Ouyang, C.; Hu, X.; Liao, X.; Song, Y.; Hu, X. Rheological behavior and particle alignment of cellulose nanocrystal and its composite hydrogels during 3D printing. Carbohydr. Polym. 2021,253, 117217. [CrossRef] 24. de Souza, F.C.B.; de Souza, R.F.B.; Drouin, B.; Mantovani, D.; Moraes, Â.M. Phosphorylation of chitosan to improve osteoinduction of chitosan/xanthan-based scaffolds for periosteal tissue engineering. Int. J. Biol. Macromol. 2019,132, 178–189. [CrossRef] 25. Calero, N.; Muñoz, J.; Ramírez, P.; Guerrero, A. Flow behaviour, linear viscoelasticity and surface properties of chitosan aqueous solutions. Food Hydrocoll. 2010,24, 659–666. [CrossRef] 26. Liu, L.; Ciftci, O.N. Effects of high oil compositions and printing parameters on food paste properties and printability in a 3D printing food processing model. J. Food Eng. 2021,288, 110135. [CrossRef] 27. Montoya, J.; Medina, J.; Molina, A.; Gutiérrez, J.; Rodríguez, B.; Marín, R. Impact of viscoelastic and structural properties from starch-mango and starch-arabinoxylans hydrocolloids in 3D food printing. Addit. Manuf. 2021,39, 101891. [CrossRef] 28. Resch, J.J.; Daubert, C.R. Rheological and physicochemical properties of derivatized whey protein concentrate powders. Int. J. Food Prop. 2002,5, 419–434. [CrossRef] 29. Liu, Y.; Yu, Y.; Liu, C.; Regenstein, J.M.; Liu, X.; Zhou, P. Rheological and mechanical behavior of milk protein composite gel for extrusion-based 3D food printing. LWT 2019,102, 338–346. [CrossRef] 30. Manzoor, M.; Singh, J.; Bandral, J.D.; Gani, A.; Shams, R. Food hydrocolloids: Functional, nutraceutical and novel applications for delivery of bioactive compounds. Int. J. Biol. Macromol. 2020,165, 554–567. [CrossRef] 31. Perez-Puyana, V.; Rubio-Valle, J.F.; Jiménez-Rosado, M.; Guerrero, A.; Romero, A. Chitosan as a potential alternative to collagen for the development of genipin-crosslinked scaffolds. React. Funct. Polym. 2020,146, 104414. [CrossRef] 32. Marudova, M.; MacDougal, A.J.; Ring, S.G. Pectin–chitosan interactions and gel formation. Carbohydr. Res. 2004 ,339, 1933–1939. [CrossRef] [PubMed] 33. Rashidova, S.S.; Milusheva, R.Y.; Semenova, L.N.; Mukhamedjanova, M.Y.; Voropaeva, N.L.; Vasilyeva, S.; Faizieva, R.; Ruban, I.N. Characteristics of Interactions in the Pectin–Chitosan System. Chromatographia 2004,59, 779–782. [CrossRef] 34. Cernencu, A.I.; Lungu, A.; Stancu, I.C.; Serafim, A.; Heggset, E.; Syverud, K.; Iovu, H. Bioinspired 3D printable pectinnanocellulose ink formulations. Carbohydr. Polym. 2019,220, 12–21. [CrossRef] [PubMed] 35. Pieczywek, P.M.; Cie´sla, J.; Płazi´nski, W.; Zdunek, A. Aggregation and weak gel formation by pectic polysaccharide homogalacturonan. Carbohydr. Polym. 2021,256, 117566. [CrossRef] 36. Tang, Y.F.; Du, Y.M.; Hu, X.W.; Shi, X.W.; Kennedy, J.F. Rheological characterisation of a novel thermosensitive chitosan/poly(vinyl alcohol) blend hydrogel. Carbohydr. Polym. 2007,67, 491–499. [CrossRef] 37. Chen, Y.; Zhang, J.G.; Sun, H.J.; Wei, Z.J. Pectin from Abelmoschus esculentus: Optimization of extraction and rheological properties. Int. J. Biol. Macromol. 2014,70, 498–505. [CrossRef] 38. Birch, N.P.; Barney, L.E.; Pandres, E.; Peyton, S.R.; Schiffman, J.D. Thermal-Responsive Behavior of a Cell Compatible Chitosan/Pectin Hydrogel. Biomacromolecules 2015,16, 1837–1843. [CrossRef] 39. Schwab, A.; Levato, R.; D’Este, M.; Piluso, S.; Eglin, D.; Malda, J. Printability and Shape Fidelity of Bioinks in 3D Bioprinting. Chem. Rev. 2020,120, 10850–10877. [CrossRef] [PubMed] 40. Norcino, L.B.; de Oliveira, J.E.; Moreira, F.K.V.; Marconcini, J.M.; Mattoso, L.H.C. Rheological and thermo-mechanical evaluation of bio-based chitosan/pectin blends with tunable ionic cross-linking. Int. J. Biol. Macromol. 2018 ,118, 1817–1823. [CrossRef] [PubMed] 41. Tsianou, M.; Kjøniksen, A.L.; Thuresson, K.; Nyström, B. Light Scattering and Viscoelasticity in Aqueous Mixtures of Oppositely Charged and Hydrophobically Modified Polyelectrolytes. Macromolecules 1999,32, 2974–2982. [CrossRef] 42. Chen, X.; Yue, Z.; Winberg, P.C.; Dinoro, J.N.; Hayes, P.; Beirne, S.; Wallace, G.G. Development of rhamnose-rich hydrogels based on sulfated xylorhamno-uronic acid toward wound healing applications. Biomater. Sci. 2019,7, 3497. [CrossRef] [PubMed] 43. Robinson, S.S.; O’Brien, K.W.; Zhao, H.; Peele, B.N.; Larson, C.M.; MacMurray, B.C.; Van Meerbeek, I.M.; Dunham, S.N.; Shepherd, R.F. Integrated soft sensors and elastomeric actuators for tactile machines with kinesthetic sense. Extrem. Mech. Lett. 2015 ,5, 47–53. [CrossRef] 44. Tian, K.; Bae, J.; Bakarich, S.E.; Yang, C.; Gately, R.D.; Spinks, G.M.; in het Panhuis, M.; Suo, Z.; Vlassak, J.J. 3D Printing of Transparent and Conductive Heterogeneous Hydrogel–Elastomer Systems. Adv. Mater. 2017,29, 1604827. [CrossRef] 45. Barbosa, H.F.G.; Francisco, D.S.; Ferreira, A.P.G.; Cavalheiro, É.T.G. A new look towards the thermal decomposition of chitins and chitosans with different degrees of deacetylation by coupled TG-FTIR. Carbohydr. Polym. 2019 ,225, 115232. [CrossRef] [PubMed] 46. Mauricio-Sánchez, R.A.; Salazar, R.; Luna-Bárcenas, J.G.; Mendoza-Galván, A. FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning. Vib. Spectrosc. 2018,94, 1–6. [CrossRef] 47. Priyadarshi, R.; Kim, S.M.; Rhim, J.W. Pectin/pullulan blend films for food packaging: Effect of blending ratio. Food Chem. 2021 , 347, 129022. [CrossRef] 48. Singh, B.; Sharma, S.; Dhiman, A. Design of antibiotic containing hydrogel wound dressings: Biomedical properties and histological study of wound healing. Int. J. Pharm. 2013,457, 82–91. [CrossRef] [PubMed] 49. Sahatsapan, N.; Rojanarata, T.; Ngawhirunpat, T.; Opanasopit, P.; Tonglairoum, P. 6-Maleimidohexanoic acid-grafted chitosan: A new generation mucoadhesive polymer. Carbohydr. Polym. 2018,202, 258–264. [CrossRef] 50. Russo, E.; Selmin, F.; Baldassari, S.; Gennari, C.G.M.; Caviglioli, G.; Cilurzo, F.; Minghetti, P.; Parodi, B. A focus on mucoadhesive polymers and their application in buccal dosage forms. J. Drug Deliv. Sci. Technol. 2016,32, 113–125. [CrossRef] Gels 2021,7, 175 17 of 17 51. Janarthanan, G.; Shin, H.S.; Kim, I.G.; Ji, P.; Chung, E.J.; Lee, C.; Noh, I. Self-crosslinking hyaluronic acid–carboxymethylcellulose hydrogel enhances multilayered 3D-printed construct shape integrity and mechanical stability for soft tissue engineering. Biofabrication 2020,12, 045026. [CrossRef] [PubMed] 52. Villanueva, J.G.V.; Huertas, P.A.S.; Galan, F.S.; Rueda, R.J.E.; Triana, J.C.B.; Rodriguez, J.P.C. Bio-adhesion evaluation of a chitosan-based bone bio-adhesive. Int. J. Adhes. Adhes. 2019,92, 80–88. [CrossRef] 53. Bhattacharyya, A.; Janarthanan, G.; Tran, H.N.; Ham, H.J.; Yoon, J.H.; Noh, I. Bioink homogeneity control during 3D bioprinting of multicomponent micro/nanocomposite hydrogel for even tissue regeneration using novel twin screw extrusion system. Chem. Eng. J. 2021,415, 128971. [CrossRef] 54. Xia, H.; Ren, M.; Zou, Y.; Qin, S.; Zeng, C. Novel Biocompatible Polysaccharide-Based Eutectogels with Tunable Rheological, Thermal, and Mechanical Properties: The Role of Water. Molecules 2020,25, 3314. [CrossRef] 55. Gerschenson, L.N.; Fissore, E.N.; Rojas, A.M.; Encalada, A.M.I.; Zukowski, E.F.; Coelho, R.A.H. Pectins obtained by ultrasound from agroindustrial by-products. Food Hydrocoll. 2021,118, 106799. [CrossRef] 56. Cesco, C.T.; Valente, A.J.M.; Paulino, A.T. Methylene Blue Release from Chitosan/Pectin and Chitosan/DNA Blend Hydrogels. Pharmaceutics 2021,13, 842. [CrossRef] [PubMed] 57. Long, J.; Etxeberria, A.E.; Nand, A.V.; Bunt, C.R.; Ray, S.; Seyfoddin, A. A 3D printed chitosan-pectin hydrogel wound dressing for lidocaine hydrochloride delivery. Mater. Sci. Eng. C 2019,104, 109873. [CrossRef] [PubMed] 58. Zarandona, I.; Estupiñán, M.; Pérez, C.; Alonso-Sáez, L.; Guerrero, P.; de la Caba, K. Chitosan Films Incorporated with Exopolysaccharides from Deep Seawater Alteromonas sp. Mar. Drugs 2020,18, 447. [CrossRef] 59. Soubhagya, A.S.; Moorthi, A.; Prabaharan, M. Preparation and characterization of chitosan/pectin/ZnO porous films for wound healing. Int. J. Biol. Macromol. 2020,157, 135–145. [CrossRef]