2.5.3. Cell Assays and Staining

In order to evaluate cell viability within the hydrogel scaffold over a 14-day period, staining with fluorescein diacetate (FDA) and propidium iodide (PI) was carried out to detect live and dead cells, respectively. In brief, scaffolds were washed with PBS at different time points (1, 4, 7 and 14 days) and incubated with 20 µg/mL FDA and 6 µg/mL PI for 10 min at r.t. Then, samples were washed with

PBS 3× and fluorescence images were taken using Nikon Ti-Ecllipse microscope (Nikon Instruments Europe B.V., Amsterdam, The Netherlands). Quantification was performed by image analysis using the Image-J 1.52p software counting both green and red cells with the function "find maxima." The cell viability percentage was calculated by quantifying the live cells between the total amount of cells in at least five images for three independent samples. As a control, cell viability studies of cells encapsulated in bulk hydrogels without using the static mixer were performed on the first day. Analysis of variance (ANOVA) using the Tukey grouping method of the results for the printed samples was performed at each time with respect to the first day at a significance level of \*\*\* *p* < 0.05 with respect to non-printed samples at a significance level of ### *p* < 0.05.

Fluorescence staining of nuclei was carried out to quantify cell proliferation within the 3D constructs over a 14-day period. Cells were fixed with PFA (paraformaldehyde) 3.7% *w*/*v* for 15 min at different time points (1, 4, 7, and 14 days), which is followed by permeabilization with 0.5% Triton -X 100 in PBS for 15 min and incubated with 1:1000 DAPI (4',6-diamidino-2-fenilindol) dillution in PBS for 20 min. Lastly, samples were imaged using a LSM 880 confocal microscope (Zeiss, Jena, Germany). Image analysis was performed using the Image-J 1.52p software using the functions "Z project" and "find maxima" to count the number of nuclei observed in all the z levels analyzed by confocal. Cell quantification with ImageJ was performed in three images per sample in a 425.1 × 425.1 µm tested area, and quantification obtained on day 1 was normalized to 100%. Analysis of variance (ANOVA) using the Tukey grouping method of the results for the printed samples was performed at each time point at a significance level of \* *p* < 0.05.

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

### *3.1. 3D Printing with a Static Mixing Tool*

The printing conditions to obtain stable threads from the two-component hydrogel system using the static mixer tool were evaluated. Once the two components enter the static mixer, diffusion and a covalent reaction between the amine groups of CMCh and aldehyde groups of HAox is started. Non-covalent interactions such as hydrogen bonds or ionic interactions might further stabilize the gel [69–71] and can be beneficial for the extrusion process. For high fidelity printing without clogging the nozzle, the crosslinking kinetics must be adjusted to ensure adequate mixing and good printing quality. The polymer concentration and components ratio, the gelation kinetics, and the viscosity of the inks are relevant parameters to adjust.

The matching between hydrogel crosslinking kinetics and extrusion speed is essential to obtain an adequate crosslinking degree in the static mixer to allow flow while providing smooth and stable strands [39,41]. The crosslinking kinetics for different CMCh and HAox weight concentrations (CMCh1/HAox1, CMCh1/HAox2, CMCh2/HAox2, CMCh2/HAox4, CMCh3/HAox3, and CMCh3/HAox6) were studied in rheological experiments. Figure 2A presents the variation of the shear modulus G<sup>0</sup> and the loss modulus G" as a function of time for the CMCh/HAox formulations that gave a measurable gelation point. As the system began to crosslink through the formation of Schiff base linkages, G<sup>0</sup> increased at a faster speed than G", which indicates a change in the viscoelastic behavior of the system to a more solid-like state. These differential growth speeds led to crossover point of G<sup>0</sup> and G", defined as a gelation point, which indicates that the 3D hydrogel network was formed [66,72]. The corresponding gelation time ranged from 0.90 ± 0.06 to 4.68 ± 0.10 min for the formulations studied (Figure 2B). Regarding ink composition, gelation time decreased with a dropping CMCh/HAox ratio and with an increasing CMCh concentration. The printability of the ink formulations was evaluated by image analysis of printed threads (Figure 2C). Printed threads with 1 wt % CMCh were liquid, which are in agreement with the rheology data that did not show gel formation (undergelation). Broken lines with small gel blocks were visible for 3 wt % CMCh formulations. In these cases, gel formation was faster than the residence time of the solution in the static mixer (over-gelation), and the shear force needed to extrude the ink caused gel fracture. For the intermediate CMCh compositions

(2 wt %), semi-solid printed strands were observed, so 2 wt % CMCh was considered a minimum concentration threshold for gel formation. The CMCh2/HAox2 formulation yielded broad lines with low shape fidelity. A feasible region was found for CMCh2/HAox4 formulation, which rendered smooth lines with shape fidelity. In this formulation, the crosslinking degree achieved in the mixing head provided adequate viscosity for extrusion with enough mechanical stability for high fidelity printing. CMCh2/HAox4, with a gelation time of 3.64 ± 0.43 min, was selected as the most appropriate ink for the subsequent experiments. *Polymers* **2020**, *12*, x FOR PEER REVIEW 7 of 17 CMCh2/HAox4 formulation, which rendered smooth lines with shape fidelity. In this formulation, the crosslinking degree achieved in the mixing head provided adequate viscosity for extrusion with enough mechanical stability for high fidelity printing. CMCh2/HAox4, with a gelation time of 3.64 ± 0.43 min, was selected as the most appropriate ink for the subsequent experiments.

**Figure 2.** (**A**) Storage (G′) and loss (G″) moduli obtained in time sweep rheological experiments, (**B**) gelation times, defined as G′ and G″ crossover points, and (**C**) light microscope pictures of printed samples of CMCh/HAox formulations with different weight concentrations of CMCh and HAox **Figure 2.** (**A**) Storage (G<sup>0</sup> ) and loss (G") moduli obtained in time sweep rheological experiments, (**B**) gelation times, defined as G<sup>0</sup> and G" crossover points, and (**C**) light microscope pictures of printed samples of CMCh/HAox formulations with different weight concentrations of CMCh and HAox solutions. Scale bars in a white color correspond to 500 µm.

solutions. Scale bars in a white color correspond to 500 µm.

printing quality, and the resolution.

While printing the CMCh2/HAox4 mixture, bubbles were observed in the needle (Figure 3A, black arrows). In addition, the printed lines had an irregular shape (Figure 3A). We hypothesized that the different viscosities of the precursor solutions due to the different molecular weights of the polymers [64,73] would be the reason for these features. Figure 3B shows that the viscosity of 2 wt % CMCh is 2 orders of magnitude higher than the viscosity of 4 wt % HAox. A different viscosity of the precursor's solutions is reported to lead to non-homogeneous mixtures due to their different flow through the mixer during extrusion [39,41]. Different strategies have been used in order to adjust viscosities of precursor solutions when using static mixers. For example, Hozumi et al. [41] used carboxymethyl cellulose as a thickening agent, and Bootsma et al. [39] distributed the solution with the largest impact on viscosity in the two syringes. In order to increase the viscosity of the HAox solution, we supplemented it with non-oxidized HA. The viscosity of different HAox-HA blends is also plotted in Figure 3B. All tested solutions presented a shear thinning behavior that facilitates extrusion and shape fidelity [5]. The addition of increasing amounts of HA to the HAox solution lead to a higher viscosity of the mixture. Based on the obtained results, the addition of 0.4 wt % of HA to the 4 wt % HAox solution resulted in a similar viscosity to the 2 wt % CMCh solution. This addition did not influence crosslinking kinetics of the formulation (Figure S1). The printing test with While printing the CMCh2/HAox4 mixture, bubbles were observed in the needle (Figure 3A, black arrows). In addition, the printed lines had an irregular shape (Figure 3A). We hypothesized that the different viscosities of the precursor solutions due to the different molecular weights of the polymers [64,73] would be the reason for these features. Figure 3B shows that the viscosity of 2 wt % CMCh is 2 orders of magnitude higher than the viscosity of 4 wt % HAox. A different viscosity of the precursor's solutions is reported to lead to non-homogeneous mixtures due to their different flow through the mixer during extrusion [39,41]. Different strategies have been used in order to adjust viscosities of precursor solutions when using static mixers. For example, Hozumi et al. [41] used carboxymethyl cellulose as a thickening agent, and Bootsma et al. [39] distributed the solution with the largest impact on viscosity in the two syringes. In order to increase the viscosity of the HAox solution, we supplemented it with non-oxidized HA. The viscosity of different HAox-HA blends is also plotted in Figure 3B. All tested solutions presented a shear thinning behavior that facilitates extrusion and shape fidelity [5]. The addition of increasing amounts of HA to the HAox solution lead to a higher viscosity of the mixture. Based on the obtained results, the addition of 0.4 wt % of HA to the 4 wt % HAox solution resulted in a similar viscosity to the 2 wt % CMCh solution. This addition did not influence crosslinking kinetics of the formulation (Figure S1). The printing test with CMCh2/HAox4-HA0.4 formulation (Figure 3C) showed regular and smooth lines without broken parts and no bubbles were

CMCh2/HAox4-HA0.4 formulation (Figure 3C) showed regular and smooth lines without broken parts and no bubbles were formed during the printing process. The CMCh2/HAox4-HA0.4 formed during the printing process. The CMCh2/HAox4-HA0.4 formulation provided stable filaments with low deviance from the needle geometry and minimized collapsing between the superposed layers visible in the cross-points (Figure 3C). These observations indicate that static mixing of solutions with comparable viscosities improves mixing performance, printing quality, and the resolution. The printing protocol described in this case allows high fidelity printing of hydrogel structures with low-viscosity ink solutions, which is favorable for cell laden scaffolds [5,27,32]. The hydrogel viscosity, flow rate, and gelation kinetics of the components as they pass through the static mixer affect the mixing performance, homogeneity, and self-support capacity of the bio-ink.

*Polymers* **2020**, *12*, x FOR PEER REVIEW 8 of 17

**Figure 3.** (**A**) Image of the needle during printing. Black arrows highlight bubbles inside the bio-ink. Light microscopy pictures of printed strands and 3D printed scaffolds using CMCh2/HAox4 formulation. (**B**) Viscosity measurements of 2 wt % CMCh, 4 wt % HAox, and different HAox/HA blends. (**C**) Image of the needle during printing and light microscopy pictures of printed strands and **Figure 3.** (**A**) Image of the needle during printing. Black arrows highlight bubbles inside the bio-ink. Light microscopy pictures of printed strands and 3D printed scaffolds using CMCh2/HAox4 formulation. (**B**) Viscosity measurements of 2 wt % CMCh, 4 wt % HAox, and different HAox/HA blends. (**C**) Image of the needle during printing and light microscopy pictures of printed strands and 3D printed scaffolds using CMCh2/HAox4-HA0.4 formulation.

3D printed scaffolds using CMCh2/HAox4-HA0.4 formulation. *3.2. Characterization of the Optimized Bio-Ink* In order to confirm the formation of covalent crosslinks between the CMCh and HAox components of the printing mixture, the CMCh2/HAox4-HA0.4 formulation was characterized by The printing protocol described in this case allows high fidelity printing of hydrogel structures with low-viscosity ink solutions, which is favorable for cell laden scaffolds [5,27,32]. The hydrogel viscosity, flow rate, and gelation kinetics of the components as they pass through the static mixer affect the mixing performance, homogeneity, and self-support capacity of the bio-ink.

#### FTIR spectroscopy (see Figure S2). The characteristic peaks corresponding to the functional groups *3.2. Characterization of the Optimized Bio-Ink*

of the CMCh and HAox/HA precursors were observed in the mixture [56,57,62], together with a band at 1653 cm−<sup>1</sup> , which can be attributed to the stretching vibration of the C=N bond of the Schiff base formed by a reaction of amine and aldehyde groups. This indicates that covalent crosslinking was successfully achieved [63,64,74]. Furthermore, a peak was observed at 885 cm−<sup>1</sup> , corresponding to the hemiacetal structure obtained due to the unreacted aldehyde groups of HAox after crosslinking [62]. Intensity of this peak is lower than in the HAox spectrum, which indicates that the rest of the aldehyde groups had participated in the crosslinking reaction. The viscoelastic properties of the crosslinked CMCh2/HAox4-HA0.4 hydrogel were studied by rheology in frequency sweep experiments. Hydrogel formation was corroborated since storage modulus was always higher than loss modulus. Additionally, a slight frequency-dependent viscoelastic behavior was observed. Presumably, the shear modulus values were mainly due to the In order to confirm the formation of covalent crosslinks between the CMCh and HAox components of the printing mixture, the CMCh2/HAox4-HA0.4 formulation was characterized by FTIR spectroscopy (see Figure S2). The characteristic peaks corresponding to the functional groups of the CMCh and HAox/HA precursors were observed in the mixture [56,57,62], together with a band at 1653 cm−<sup>1</sup> , which can be attributed to the stretching vibration of the C=N bond of the Schiff base formed by a reaction of amine and aldehyde groups. This indicates that covalent crosslinking was successfully achieved [63,64,74]. Furthermore, a peak was observed at 885 cm−<sup>1</sup> , corresponding to the hemiacetal structure obtained due to the unreacted aldehyde groups of HAox after crosslinking [62]. Intensity of this peak is lower than in the HAox spectrum, which indicates that the rest of the aldehyde groups had participated in the crosslinking reaction.

covalent crosslinking of the CMCh and HAox functional groups, and HA did not influence crosslinking kinetics or final modulus (Figure S1). Gels were soft with a shear modulus in the range of 50–100 Pa (Figure 4A). This value indicates that these hydrogel scaffolds are promising candidates for regeneration of soft tissues [39], and is comparable to reported chitosan/hyaluronic acid injectable hydrogels with encapsulated cells for abdominal reparation and adhesion prevention [39,63,74,75]. The viscosity of the crosslinked CMCh2/HAox4-HA0.4 bio-ink vs. shear rate is plotted in Figure 4B. The ink viscosity found was relatively low, especially when compared to air pressure-based extruded inks (in the range of 30–6 × 10<sup>7</sup> mPa) [1,2], which is a desirable feature since low-viscosity bio-inks usually allow higher cell viability [5,27,32]. Solution behaved as a non-Newtonian fluid, where viscosity decreased linearly with an increasing shear rate. This shear thinning behavior is a favorable property for printing. It implies a decrease in the viscosity when the shear stress increases inside the The viscoelastic properties of the crosslinked CMCh2/HAox4-HA0.4 hydrogel were studied by rheology in frequency sweep experiments. Hydrogel formation was corroborated since storage modulus was always higher than loss modulus. Additionally, a slight frequency-dependent viscoelastic behavior was observed. Presumably, the shear modulus values were mainly due to the covalent crosslinking of the CMCh and HAox functional groups, and HA did not influence crosslinking kinetics or final modulus (Figure S1). Gels were soft with a shear modulus in the range of 50–100 Pa (Figure 4A). This value indicates that these hydrogel scaffolds are promising candidates for regeneration of soft tissues [39], and is comparable to reported chitosan/hyaluronic acid injectable hydrogels with encapsulated cells for abdominal reparation and adhesion prevention [39,63,74,75]. The viscosity of the crosslinked CMCh2/HAox4-HA0.4 bio-ink vs. shear rate is plotted in Figure 4B. The ink viscosity found was relatively low, especially when compared to air pressure-based extruded inks (in the range of

30–6 × 10<sup>7</sup> mPa) [1,2], which is a desirable feature since low-viscosity bio-inks usually allow higher cell viability [5,27,32]. Solution behaved as a non-Newtonian fluid, where viscosity decreased linearly with an increasing shear rate. This shear thinning behavior is a favorable property for printing. It implies a decrease in the viscosity when the shear stress increases inside the needle under applied pressure, which is followed by a sharp increase of viscosity after extrusion. This facilitates both extrusion and shape fidelity [5]. *Polymers* **2020**, *12*, x FOR PEER REVIEW 9 of 17 needle under applied pressure, which is followed by a sharp increase of viscosity after extrusion. This facilitates both extrusion and shape fidelity [5].

**Figure 4.** (**A**) Frequency sweep experiment and (**B**) viscosity analysis of CMCh2/HAox4-HA0.4 bio-**Figure 4.** (**A**) Frequency sweep experiment and (**B**) viscosity analysis of CMCh2/HAox4-HA0.4 bio-ink formulation.

### ink formulation. *3.3. Characterization of the 3D Printed Hydrogel Sca*ff*olds*

*3.3. Characterization of the 3D Printed Hydrogel Scaffolds* 2- and 4-layered grid square scaffolds (12 × 12 mm<sup>2</sup> printed area) were printed using the 2- and 4-layered grid square scaffolds (12 × 12 mm<sup>2</sup> printed area) were printed using the CMCh2/HAox4-HA0.4 formulation (Figure 5). Good printing accuracy and resolution was observed and stable scaffolds with filaments of uniform dimensions (diameter 357 ± 58 µm) were obtained.

CMCh2/HAox4-HA0.4 formulation (Figure 5). Good printing accuracy and resolution was observed and stable scaffolds with filaments of uniform dimensions (diameter 357 ± 58 μm) were obtained. Swelling and degradation rates are relevant parameters when using hydrogels' bio-inks since they affect the fidelity and stability of the bio-printed scaffolds, as well as allow cellular ingrowth and tissue regeneration [27,76,77]. In this study, swelling and degradability of the printed scaffolds were analyzed by imaging the scaffolds after incubation in PBS for given times and by quantification of the width of the strands (Figure 6A–C). Figure 6A shows the microscopy images of 2-layer printed scaffolds after 4 h of immersion in PBS. A 49 ± 9% swelling was observed under these conditions (Figure 6A). A progressive and notable decrease in scaffold volume was observed with incubation time, up to nearly complete degradation after 7 days (Figure 6B). This degradation rate is slightly faster than previously reported for CMCh/HAox injectable hydrogels (10–14 days) [63,74,75], which can be assigned to the higher surface area and open structure of the grid scaffold that makes them more sensitive for degradation. Schiff's base crosslinked hydrogels have low stability due to the dynamic nature of the bond [57,78,79]. Thus, to increase the long-term stability of the scaffolds, a new crosslinking approach was proposed since stability is directly related to the crosslinking degree of the hydrogel [76,80,81]. A post-printing crosslinking step was adopted by immersing the scaffold in 20 mM FeCl<sup>3</sup> for 7 min. Fe (III) forms coordination complexes with hyaluronic acid units at a physiological pH [82], which are expected to act as additional crosslinking points in the printed scaffold. Figure 6A shows lower swelling (19 ± 8%) of the printed scaffold after the second crosslinking step and 4 h after swelling. Additionally, slower degradation of the scaffold was observed after post-printing stabilization (Figure 6C). The scaffold maintained its structural integrity up to 28 days of incubation, although signs of erosion were appreciated in the last stage. In conclusion, the treatment with iron

scaffolds printed using CMCh2/HAox4-HA0.4 formulation.

ink formulation.

facilitates both extrusion and shape fidelity [5].

(III) leads to 3D scaffolds with higher structural integrity and long-term stability. This reinforces the stability of Schiff's base crosslinked hydrogels, which has remained a challenging issue [45,78]. 2- and 4-layered grid square scaffolds (12 × 12 mm<sup>2</sup> printed area) were printed using the CMCh2/HAox4-HA0.4 formulation (Figure 5). Good printing accuracy and resolution was observed and stable scaffolds with filaments of uniform dimensions (diameter 357 ± 58 μm) were obtained.

**Figure 4.** (**A**) Frequency sweep experiment and (**B**) viscosity analysis of CMCh2/HAox4-HA0.4 bio-

*Polymers* **2020**, *12*, x FOR PEER REVIEW 9 of 17

needle under applied pressure, which is followed by a sharp increase of viscosity after extrusion. This

**Figure 5.** Camera pictures (**left**) and light microscopy pictures (**right**) of 2-layer and 4-layer square **Figure 5.** Camera pictures (**left**) and light microscopy pictures (**right**) of 2-layer and 4-layer square scaffolds printed using CMCh2/HAox4-HA0.4 formulation.

### scaffolds printed using CMCh2/HAox4-HA0.4 formulation. *3.4. CMCh2*/*HAox4-HA0.4 Based Bio-Printing*

In general, 3D printed hydrogels have been demonstrated to protect cells from mechanical damage induced during the extrusion process, while providing an appropriate environment for the encapsulated cells after printing (by mimicking the ECM) [27,30]. Nevertheless, it is a critical aspect in bio-fabrication to evaluate whether viscosity, in situ crosslinking, and printing process are compatible with encapsulated living cells [4,10]. Thus, the ability of CMCh2/HAox4-HA0.4 formulation to be used as a bio-ink was tested by printing scaffolds with encapsulated L929 fibroblasts. Cell viability in the bio-printed scaffolds was studied during a 14-day period. Live/dead staining allowed imaging of the cells in 2-layer printed scaffolds (Figure 7A). Abundance of cells homogeneously distributed in the scaffold were observed, which reflects the good mixing performance during the printing process. Additionally, cells were released from the hydrogel after 7 and 14 days of culture, which can be due to the degradation rate of the scaffolds. This is a desirable characteristic for potential regenerative approaches for wound healing, where delivered cells would migrate out of the scaffold to heal the injured site [4]. Quantification of cell viability is displayed in Figure 7B, together with data from 3D cultures in non-printed hydrogels of the same formulation as the control. Cells in the printed or non-printed materials showed viability around 60–65% after 1 day of incubation. There are no significant differences between printed and non-printed formulation, which indicates that the printing process did not affect the cells short-term viability. Cell viability in the printed scaffold increased at longer culture times and reached 96% and 95% 7 and 14 days of culture, respectively, which are both significantly different from printed and non-printed formulations after 1 day of incubation. These data suggest that neither the covalent reaction responsible of gelation nor the shear stress produced by the printing process or the stabilization process with iron (III) cause adverse long-term effects on the cells. The proliferation rate of the cells in the scaffolds was analyzed after DAPI staining. Cell proliferation increased over the 14-day period in the printed scaffolds (Figure 7C), and values reached after 14 days

of incubation were significantly different from those found after 1 and 4 days of culturing. Lastly, the scaffolds maintained their structural integrity during the whole culture processes (Figure 7D), which indicates that the optimized printed formula and the subsequent stabilization step with iron produced mechanically robust scaffolds with good biocompatibility. observed after post-printing stabilization (Figure 6C). The scaffold maintained its structural integrity up to 28 days of incubation, although signs of erosion were appreciated in the last stage. In conclusion, the treatment with iron (III) leads to 3D scaffolds with higher structural integrity and long-term stability. This reinforces the stability of Schiff's base crosslinked hydrogels, which has remained a challenging issue [45,78].

crosslinking step and 4 h after swelling. Additionally, slower degradation of the scaffold was

*Polymers* **2020**, *12*, x FOR PEER REVIEW 10 of 17

Swelling and degradation rates are relevant parameters when using hydrogels' bio-inks since they affect the fidelity and stability of the bio-printed scaffolds, as well as allow cellular ingrowth and tissue regeneration [27,76,77]. In this study, swelling and degradability of the printed scaffolds were analyzed by imaging the scaffolds after incubation in PBS for given times and by quantification of the width of the strands (Figure 6A–C). Figure 6A shows the microscopy images of 2-layer printed scaffolds after 4 h of immersion in PBS. A 49 ± 9% swelling was observed under these conditions (Figure 6A). A progressive and notable decrease in scaffold volume was observed with incubation time, up to nearly complete degradation after 7 days (Figure 6B). This degradation rate is slightly faster than previously reported for CMCh/HAox injectable hydrogels (10–14 days) [63,74,75], which can be assigned to the higher surface area and open structure of the grid scaffold that makes them more sensitive for degradation. Schiff's base crosslinked hydrogels have low stability due to the dynamic nature of the bond [57,78,79]. Thus, to increase the long-term stability of the scaffolds, a new crosslinking approach was proposed since stability is directly related to the crosslinking degree of the hydrogel [76,80,81]. A post-printing crosslinking step was adopted by immersing the scaffold in 20 mM FeCl<sup>3</sup> for 7 min. Fe (III) forms coordination complexes with hyaluronic acid units at a physiological pH [82], which are expected to act as additional crosslinking points in the printed

**Figure 6.** (**A**) Microscopy images of CMCh2/HAox4-HA0.4 printed scaffolds just after printing, after swelling in PBS for 4 h, and after iron treatment and swelling in PBS for 4 h. (**B**) Degradation study of CMCh2/HAox4-HA0.4 scaffolds with no additional treatment after incubation in PBS and (**C**) after **Figure 6.** (**A**) Microscopy images of CMCh2/HAox4-HA0.4 printed scaffolds just after printing, after swelling in PBS for 4 h, and after iron treatment and swelling in PBS for 4 h. (**B**) Degradation study of CMCh2/HAox4-HA0.4 scaffolds with no additional treatment after incubation in PBS and (**C**) after iron treatment and incubation in PBS at different time points. Scale bars correspond to 500 µm.

iron treatment and incubation in PBS at different time points. Scale bars correspond to 500 µm.

These observations were consistent with other studies based on naturally derived hydrogels bioprinting [17,51,83]. For example, Akkineni et al. studied the encapsulation of endothelial cells in a low viscosity hydrogel core (1% gelatin and 3% alginate) by obtaining cell viability values around 65% one day after printing, which is comparable to our results. A high viscosity shell composed of 10% alginate and 1% gelatin and a secondary crosslinking with CaCl<sup>2</sup> provided the structural integrity to the scaffold [17]. In addition, Gu et al. presented the direct-write printing of stem cells within a polysaccharide-based bio-ink comprising alginate, carboxymethylchitosan, and agarose. The time course of dead cells content within the optimized bio-ink (containing 5% *w*/*v* of carboxymethylchitosan) demonstrated a relatively high (around 25%) cell death after printing. Subsequently, this decreased to around 10% by day 7, following a trend very similar to that of our work [83]. On the other hand, some reactive hydrogels have been reported for cell encapsulation such as: injectable hydrogels with proliferation trends similar to that found in our work [42], layered platforms with constant cell viability values around 70% until 5 days [37], or gradient formulations [36] where cell viability values slightly decrease with time until around 80% after 7 days. Based on the in-vitro studies, we conclude that the proposed printing technology and bio-ink formulation of this work are suitable as a 3D printing platform for potential biomedical applications as cell carriers in the tissue engineering field.

**Figure 7.** Biological results of CMCh2/HAox4-HA0.4 printed scaffolds loaded with L929 fibroblasts and treated with Fe over a 14-day period: (**A**) Fluorescence imaging of live/dead stained scaffolds at different culture days. (**B**) Quantification of live/dead results including bulk hydrogels at 1 day as a control. Analysis of variance (ANOVA) of the results for the printed samples was performed at each time point with respect to day 1 at a significance level of \*\*\* *p* < 0.05, and with respect to non-printed samples at a significance level of ### *p* < 0.05. (**C**) Proliferation assay by quantification of nuclei after DAPI staining. Analysis of variance (ANOVA) of the results for the printed samples was performed at each time point at a significance level of \*\* *p* < 0.01. (**D**) Picture of a stable printed scaffold after 14 **Figure 7.** Biological results of CMCh2/HAox4-HA0.4 printed scaffolds loaded with L929 fibroblasts and treated with Fe over a 14-day period: (**A**) Fluorescence imaging of live/dead stained scaffolds at different culture days. (**B**) Quantification of live/dead results including bulk hydrogels at 1 day as a control. Analysis of variance (ANOVA) of the results for the printed samples was performed at each time point with respect to day 1 at a significance level of \*\*\* *p* < 0.05, and with respect to non-printed samples at a significance level of ### *p* < 0.05. (**C**) Proliferation assay by quantification of nuclei after DAPI staining. Analysis of variance (ANOVA) of the results for the printed samples was performed at each time point at a significance level of \*\* *p* < 0.01. (**D**) Picture of a stable printed scaffold after 14 days of incubation. Scale bars correspond to 200 µm.

### days of incubation. Scale bars correspond to 200 µm. **4. Conclusions**

**4. Conclusions** The present study describes the development of a reactive hydrogel bio-ink with an extrusion printing methodology based on a dual-syringe system with a static mixing tool. This method shows multiple advantages for 3D extrusion bio-printing. (1) Gelation during the extrusion process provides enough viscosity for printing with good shape fidelity while using low viscosity precursor solutions, (2) the crosslinking during extrusion provides enough structural integrity to retain the printed shape, and (3) the stability of the scaffold, if required for long-term culturing, can be increased in a simple incubation step. Bio-printed scaffolds obtained with our approach showed good biocompatibility, moderate swelling, and shape stability during 14 days of culturing. Since precursors' concentrations and printing conditions can be easily varied, this printing approach offers high versatility and we envision that it can be adaptable to a wide range of reactive systems with appropriate crosslinking The present study describes the development of a reactive hydrogel bio-ink with an extrusion printing methodology based on a dual-syringe system with a static mixing tool. This method shows multiple advantages for 3D extrusion bio-printing. (1) Gelation during the extrusion process provides enough viscosity for printing with good shape fidelity while using low viscosity precursor solutions, (2) the crosslinking during extrusion provides enough structural integrity to retain the printed shape, and (3) the stability of the scaffold, if required for long-term culturing, can be increased in a simple incubation step. Bio-printed scaffolds obtained with our approach showed good biocompatibility, moderate swelling, and shape stability during 14 days of culturing. Since precursors' concentrations and printing conditions can be easily varied, this printing approach offers high versatility and we envision that it can be adaptable to a wide range of reactive systems with appropriate crosslinking kinetics to be employed in the future for broad applications in regenerative medicine and tissue-engineering.

kinetics to be employed in the future for broad applications in regenerative medicine and tissueengineering. **Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1. Figure S1. (A) Storage **Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4360/12/9/1986/s1. Figure S1. (A) Storage (G<sup>0</sup> ) and loss (G") moduli obtained in time sweep rheological experiments and (B) gelation times, defined as G' and G" crossover points, obtained for CMCh2/HAox4 and CMCh2/HAox4-HA0.4 formulations. Figure S2. FTIR spectra of CMCh, HAox, and CMCh/HAox reactive hydrogel.

(G′) and loss (G″) moduli obtained in time sweep rheological experiments and (B) gelation times, defined as G' and G" crossover points, obtained for CMCh2/HAox4 and CMCh2/HAox4-HA0.4 formulations. Figure S2. FTIR spectra of CMCh, HAox, and CMCh/HAox reactive hydrogel. **Author Contributions:** M.P.-B. extensively contributed to the investigation and writing-original draft preparation. M.K.W.-B. contributed to methodology and draft preparation. B.V.-L., J.S.R., and A.d.C. contributed to supervision. All the authors contributed to analysis, discussion, and redaction of the paper. All authors have read and agreed to the published version of the manuscript.

**Author Contributions:** M.P.-B. extensively contributed to the investigation and writing-original draft preparation. M.K.W.-B. contributed to methodology and draft preparation. B.V.-L., J.S.R., and A.d.C. contributed to supervision. All the authors contributed to analysis, discussion, and redaction of the paper. All authors have read and agreed to the published version of the manuscript. **Funding:** This research was funded by CIBER-BBN, Spain and the Spanish Ministry of Economy and Competitivity (project MAT2017-84277-R and M. Puertas-Bartolomé scholarship BES-2015-075161). B. Vázquez-Lasa and J. San Román are members of the *SusPlast platform (Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy)* from the Spanish National Research Council (CSIC).

**Funding:** This research was funded by CIBER-BBN, Spain and the Spanish Ministry of Economy and Competitivity (project MAT2017-84277-R and M. Puertas-Bartolomé scholarship BES-2015-075161). B. Vázquez**Acknowledgments:** The authors acknowledge RegenHu Company, and, particularly, Sandro Figi, Dominic Ernst, Michael Kuster, and Andreas Scheidegger for the fruitful collaboration, development, and providing the mixing tool. The authors thank Emmanuel Terriac from INM, Germany, for assistance in the confocal imaging.

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