*2.16. Statistical analysis*

GraphPad Prism 9 software (San Diego, CA, USA) was used to perform the statistical analysis, using a significance value of *p* < 0.001. Data are presented as the mean ± standard error (SE). An ANOVA was performed to evaluate physical changes in scaffold fabrication and changes in percent swelling and permeability between scaffold fabrication methods and to analyze cell viability. To compare the four scaffold fabrication methods with respect to functionalization (without Np, with AuNp and with AuNp+Alg) a multivariate analysis was performed, with Tukey's post hoc test for multiple comparisons.

### **3. Results**

## *3.1. Characterization of Alginate–Chitosan Scaffolds*

Method 1 (without sonication) had the highest weight with respect to the other three fabrication methods (*p* < 0.001). Regarding thickness, the scaffolds generated by Method 2 (with sonication) were the thinnest, presenting significant changes with respect to Methods 1 and 3 (*p* < 0.001), see Supplementary Materials, Figure S2.

### *3.2. Swelling Degree*

In Figure 2A, the swelling percentages of the scaffolds according to the processing method can be observed. The swelling percentage of the scaffolds was higher than 2500% in three of the methods. The scaffolds elaborated with Method 3 (30 min crosslinking) were the ones with the highest swelling percentage. Figure 2B shows that the swelling percentage increases when functionalizing the scaffolds with AuNp and AuNp+Alg, particularly in Method 3, which had the highest values.

**Figure 2.** (**A**) Swelling percentages (%) for the four scaffold preparation methods. (**B**) Swelling percentages for the four preparation methods functionalized with AuNp and AuNp+Alg. (**C**) Permeability (m2) for the four scaffold preparation methods. (**D**) Permeability for the four preparation methods functionalized with AuNp and AuNp+Alg. (\*) *p* < 0.001 vs. Method 1, (\$) *p* < 0.001 vs. Method 2, (#) *p* < 0.001 vs. Method 3, (&) *p* < 0.001 vs. Method 4. (**E**) Porosity percentage (%) for the four scaffold preparation methods and the scaffolds functionalized with AuNp. (**F**) Degradation degree for the four scaffold preparation methods and (**G**) the functionalization of the four methods with AuNp. (**H**) Graphical representation of cell viability analysis for the four preparation methods functionalized with AuNp and AuNp+Alg. (\*) *p* < 0.001 vs. without Np. Data are presented as the mean ± SE. **Figure 2.** (**A**) Swelling percentages (%) for the four scaffold preparation methods. (**B**) Swelling percentages for the four preparation methods functionalized with AuNp and AuNp+Alg. (**C**) Permeability (m<sup>2</sup> ) for the four scaffold preparation methods. (**D**) Permeability for the four preparation methods functionalized with AuNp and AuNp+Alg. (\*) *p* < 0.001 vs. Method 1, (\$) *p* < 0.001 vs. Method 2, (#) *p* < 0.001 vs. Method 3, (&) *p* < 0.001 vs. Method 4. (**E**) Porosity percentage (%) for the four scaffold preparation methods and the scaffolds functionalized with AuNp. (**F**) Degradation degree for the four scaffold preparation methods and (**G**) the functionalization of the four methods with AuNp. (**H**) Graphical representation of cell viability analysis for the four preparation methods functionalized with AuNp and AuNp+Alg. (\*) *p* < 0.001 vs. without Np. Data are presented as the mean ± SE.

.

The scaffolds obtained with Method 3 were studied in their swelling in different aqueous media (pH dependence) over 7 days (Figure 3). Similar behavior can be observed in each medium over time, in which the swelling increases, reaches a maximum and does not decrease significantly at the total time studied. The Table 1 shows the percentage reached with respect to the maximum swelling (*S*max) of the scaffolds. At 5 min, the hydrogels reached between 53 and 94% of the *S*max, and at 60 min, most of the scaffolds reached between 90 and 100% of the *S*max. The swelling dependence of the alginate/chitosan scaffolds on pH showed a decrease in the swelling when going from acidic to basic pHs, from 2945 to 1880% (Figure 3C). The scaffolds obtained with Method 3 were studied in their swelling in different aqueous media (pH dependence) over 7 days (Figure 3). Similar behavior can be observed in each medium over time, in which the swelling increases, reaches a maximum and does not decrease significantly at the total time studied. The Table 1 shows the percentage reached with respect to the maximum swelling (*S*max) of the scaffolds. At 5 min, the hydrogels reached between 53 and 94% of the *S*max, and at 60 min, most of the scaffolds reached between 90 and 100% of the *S*max. The swelling dependence of the alginate/chitosan scaffolds on pH showed a decrease in the swelling when going from acidic to basic pHs, from 2945 to 1880% (Figure 3C).


**Table 1.** Maximum swelling (*S*max) as a function of pH.

## *3.3. Permeability*

Method 2 (sonication) has the highest permeability coefficient, being more than twice the one obtained with the other scaffold processing methods (Figure 2C). When functionalizing the scaffolds with AuNp and AuNp+Alg, a slight increase in permeability is observed in the four proposed scaffold elaboration methods (Figure 2D).

### *3.4. Porosity Polymers* **2022**, *14*, x FOR PEER REVIEW 9 of 21

It is observed that the scaffolds of Method 3 (30 min crosslinking) are the most porous, and this property increases to more than 95% when functionalized with AuNp (Figure 2E).

**Figure 3.** Swelling of alginate/chitosan scaffolds (Method 3) as a function of time in different aqueous media: (**A**) Milli-Q and PBS and (**B**) buffers prepared with Na2HPO4/citric acid for pH 3, 5, 8 and 9. In addition, their maximum swelling (*S*max) as a function of pH (**C**) and the percentages reached with respect to *S*max at 5, 40 and 60 min are shown (see Table 1). Maximum swelling trend lines are shown as an eye guide. (**D**) Percentage of degradation (%) of the scaffolds of Method 3 **Figure 3.** Swelling of alginate/chitosan scaffolds (Method 3) as a function of time in different aqueous media: (**A**) Milli-Q and PBS and (**B**) buffers prepared with Na2HPO4/citric acid for pH 3, 5, 8 and 9. In addition, their maximum swelling (*S*max) as a function of pH (**C**) and the percentages reached with respect to *S*max at 5, 40 and 60 min are shown (see Table 1). Maximum swelling trend lines are shown as an eye guide. (**D**) Percentage of degradation (%) of the scaffolds of Method 3 (more crosslinking time) as a function of the aqueous medium exposed for 7 days.

### (more crosslinking time) as a function of the aqueous medium exposed for 7 days. *3.5. Degradation*

**Table 1.** Maximum swelling (*S*max) as a function of pH.  **Swelling Referred to** *S***max (%) Medium 5 min 40 min 60 min**  pH 3 75 92 95 pH 5 76 97 100 Milli-Q 71 88 90 PBS 81 93 93 pH 8 53 57 58 Degradation analysis under cell culture conditions was performed on scaffolds made with the four proposed methods (Figure 2F) and on scaffolds of the four methods functionalized with AuNp (Figure 2G). It is observed that the scaffolds of Method 1 (without sonication) are the ones that degrade slower without AuNp, but when functionalized with AuNp, they degrade faster. In the case of Method 4, the opposite happens; they are the scaffolds that degrade faster without AuNp and with AuNp they degrade slower. In general, they degrade slightly faster with AuNp, with the exception of Method 3 (longer crosslinking time) where functionalization with AuNp causes them to degrade slightly slower, although the changes are not statistically significant.

pH 9 94 99 95 *3.3. Permeability*  Method 2 (sonication) has the highest permeability coefficient, being more than twice The scaffolds' degradation by Method 3 (more crosslinking time) with respect to time and subjected to different aqueous media for 7 days are shown in Figure 3D. For all samples, there is a weight decrease, corroborating a degradation between 8 and 35%, and, as observed, it increases with increasing pH.

### the one obtained with the other scaffold processing methods (Figure 2C). When function-*3.6. FTIR-ATR*

alizing the scaffolds with AuNp and AuNp+Alg, a slight increase in permeability is observed in the four proposed scaffold elaboration methods (Figure 2D). *3.4. Porosity*  It is observed that the scaffolds of Method 3 (30 min crosslinking) are the most porous, and this property increases to more than 95% when functionalized with AuNp (Figure 2E). *3.5. Degradation*  Figure 4 shows the chemical characterization (IR spectra) of the alginate/chitosan scaffolds before and after crosslinking by Method 3 (30 min crosslinking), followed by the incorporation of AuNp and AuNp+Alg, in addition to the initial sodium alginate and chitosan spectra. For sodium alginate (Figure 4(A1)), the presence of the characteristic groups is confirmed [37]. Figure 4(A2) shows the characteristic peaks of chitosan [38]. In the case of the scaffold with alginate/chitosan before crosslinking (Figure 4(A3)), peaks that overlap due to the presence of both components can be observed (follow the dotted lines). The spectrum of the scaffold crosslinked with calcium gluconate by Method 3 (Figure 4(A4))

Degradation analysis under cell culture conditions was performed on scaffolds made

is similar to the spectrum of the scaffold before crosslinking, but an incorporation of OH and COO– due to the gluconate is present, and an increase in the intensity of the –CH peak is observed. The IR spectra of the scaffolds crosslinked and with the incorporation of AuNp and AuNp+Alg are presented in Figure 4(A5,A6), respectively; they are similar to the scaffolds before doping with AuNp, see Supplementary Materials. and COO– due to the gluconate is present, and an increase in the intensity of the –CH peak is observed. The IR spectra of the scaffolds crosslinked and with the incorporation of AuNp and AuNp+Alg are presented in Figure 4(A5,A6), respectively; they are similar to the scaffolds before doping with AuNp, see Supplementary Materials.

sonication) are the ones that degrade slower without AuNp, but when functionalized with AuNp, they degrade faster. In the case of Method 4, the opposite happens; they are the scaffolds that degrade faster without AuNp and with AuNp they degrade slower. In general, they degrade slightly faster with AuNp, with the exception of Method 3 (longer crosslinking time) where functionalization with AuNp causes them to degrade slightly slower,

The scaffolds' degradation by Method 3 (more crosslinking time) with respect to time and subjected to different aqueous media for 7 days are shown in Figure 3D. For all samples, there is a weight decrease, corroborating a degradation between 8 and 35%, and, as

Figure 4 shows the chemical characterization (IR spectra) of the alginate/chitosan scaffolds before and after crosslinking by Method 3 (30 min crosslinking), followed by the incorporation of AuNp and AuNp+Alg, in addition to the initial sodium alginate and chitosan spectra. For sodium alginate (Figure 4(A1)), the presence of the characteristic groups is confirmed [37]. Figure 4(A2) shows the characteristic peaks of chitosan [38]. In the case of the scaffold with alginate/chitosan before crosslinking (Figure 4(A3)), peaks that overlap due to the presence of both components can be observed (follow the dotted lines). The spectrum of the scaffold crosslinked with calcium gluconate by Method 3 (Figure 4(A4)) is similar to the spectrum of the scaffold before crosslinking, but an incorporation of OH

*Polymers* **2022**, *14*, x FOR PEER REVIEW 10 of 21

although the changes are not statistically significant.

observed, it increases with increasing pH.

*3.6. FTIR-ATR* 

**Figure 4.** (**A**) FTIR-ATR spectra of: sodium alginate (1), chitosan (2) and alginate/chitosan scaffolds before (3) and after crosslinking (4, Method 3) and with incorporation of gold (5) or gold plus sodium alginate (6) nanoparticles. Vertical dotted and continuous lines are presented as eye guides. Representative electromicrographs of (**B**) AuNp and (**C**) AuNp+Alg obtained by TEM. (**D**) Gold nanoparticles plasmon. **Figure 4.** (**A**) FTIR-ATR spectra of: sodium alginate (1), chitosan (2) and alginate/chitosan scaffolds before (3) and after crosslinking (4, Method 3) and with incorporation of gold (5) or gold plus sodium alginate (6) nanoparticles. Vertical dotted and continuous lines are presented as eye guides. Representative electromicrographs of (**B**) AuNp and (**C**) AuNp+Alg obtained by TEM. (**D**) Gold nanoparticles plasmon.

### *3.7. Characterization of AuNp 3.7. Characterization of AuNp*

The obtained metallic nanoparticles presented an average hydrodynamic diameter of 74.5 ± 1nm and 91.1 ± 1 nm for PLGA and PLGA+Alg core-shell nanoparticles, respectively. As for the surface charge value, it was determined to be −25.4 ± 0.8 mV for PLGA-The obtained metallic nanoparticles presented an average hydrodynamic diameter of 74.5 ± 1nm and 91.1 ± 1 nm for PLGA and PLGA+Alg core-shell nanoparticles, respectively. As for the surface charge value, it was determined to be −25.4 ± 0.8 mV for PLGA-cored samples and −36.8 ± 1 mV for those modified with sodium alginate (see Supplementary Materials).

Particle morphology was determined by SEM and transmission electron microscopy (TEM). According to these techniques, AuNp presented a spheroidal structure (Figure 4B), while AuNp+Alg showed cylindrical particle characteristics (Figure 4C). The UV-Vis spectra of AuNp and AuNp+Alg solutions (before dilution) were taken (Figure 4D), presenting both a plasmon starting at ~500 nm and reaching a maximum wavelength at 571 nm and 639 nm, respectively, confirming the sizes observed by TEM. As a reference, Peña et al. showed a maximum plasmon at a wavelength of 528 nm for homogeneous spherical AuNp ~32 nm of diameter [30].

### *3.8. Cytotoxicity*

The presence of Np does not affect cell viability. The scaffolds elaborated by Methods 1, 2 and 3 were the ones that allowed the maintenance of up to 80% of viable cells (Figure 2H). The results demonstrate that the alginate/chitosan scaffolds elaborated by the different proposed methods without Np and functionalized with AuNp and with AuNp+Alg do not release toxic components that could negatively impact the viability of the cells cultured on them.

### *3.9. Scanning Electron Microscopy (SEM)*

The electromicrographs of the scaffolds show a porous appearance (Figure 5). The presence of spheroids of different diameters can be observed (Figure 5B,G–I,L). Functionalization with Np allowed the increase in the number, size and distribution of spheroids, especially for the scaffolds of Method 3 (Figure 5H,I). *Polymers* **2022**, *14*, x FOR PEER REVIEW 12 of 21

**Figure 5.** Representative electromicrographs of cross sections of the constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. Identifier: arrow (spheroids). **Figure 5.** Representative electromicrographs of cross sections of the constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. Identifier: arrow (spheroids).

### *3.10. Histological Analysis 3.10. Histological Analysis*

Histological analysis shows that there was high cell migration and penetration through the pores of the scaffolds. Consistent with the SEM, the scaffolds of Method 3 were the ones that presented the largest spheroids, reaching 200 µm in diameter, in addition to being present both in the periphery and inside the scaffold (Figure 6). All the scaffolds with Np presented a greater number of spheroids compared to the scaffolds without Np (Figure 6B,E,H,K). However, spheroids were observed in all four proposed methods. Histological analysis shows that there was high cell migration and penetration through the pores of the scaffolds. Consistent with the SEM, the scaffolds of Method 3 were the ones that presented the largest spheroids, reaching 200 µm in diameter, in addition to being present both in the periphery and inside the scaffold (Figure 6). All the scaffolds with Np presented a greater number of spheroids compared to the scaffolds without Np (Figure 6B,E,H,K). However, spheroids were observed in all four proposed methods.

**Figure 6.** Representative photomicrographs of cross sections of the constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp, and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. H&E. Identifier: arrow (spheroids). Scale bar: 200 µm, original magnification: ×100. **Figure 6.** Representative photomicrographs of cross sections of the constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp, and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. H&E. Identifier: arrow (spheroids). Scale bar: 200 µm, original magnification: ×100.

### *3.11. Immunodetection of Proliferation and Cardiomyocyte Markers 3.11. Immunodetection of Proliferation and Cardiomyocyte Markers*

Immunohistochemical analysis was performed to measure the levels of PCNA (proliferation marker, Figure 7) and tropomyosin (Figure 8). There was increased proliferation in the scaffolds prepared by Methods 3 and 4, and in particular in Method 3, functionalization with AuNp+Alg significantly increased cell proliferation compared to scaffolds without Np (Figure 7M). For the case of tropomyosin, an increase in this protein was observed mainly in the periphery of the spheroid in the scaffolds elaborated by Methods 1, 3 and 4. The scaffolds elaborated by Methods 3 and 4 showed the highest tropomyosin expression, particularly when functionalization with AuNp+Alg was present. Figure 8C shows the interaction between the scaffold and the cardiomyocytes (amplification is shown in the Supplementary Materials). Immunohistochemical analysis was performed to measure the levels of PCNA (proliferation marker, Figure 7) and tropomyosin (Figure 8). There was increased proliferation in the scaffolds prepared by Methods 3 and 4, and in particular in Method 3, functionalization with AuNp+Alg significantly increased cell proliferation compared to scaffolds without Np (Figure 7M). For the case of tropomyosin, an increase in this protein was observed mainly in the periphery of the spheroid in the scaffolds elaborated by Methods 1, 3 and 4. The scaffolds elaborated by Methods 3 and 4 showed the highest tropomyosin expression, particularly when functionalization with AuNp+Alg was present. Figure 8C shows the interaction between the scaffold and the cardiomyocytes (amplification is shown in the Supplementary Materials).

**Figure 7.** Representative photomicrographs of PCNA expression in different constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. Scale bar: 50 µm, original magnification: ×400. (**M**) Graphical representation of the quantitative analysis of PCNA expression (Int) in the different constructs. (\*) *p* < 0.001. Data are presented as the mean ± SE. **Figure 7.** Representative photomicrographs of PCNA expression in different constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. Scale bar: 50 µm, original magnification: ×400. (**M**) Graphical representation of the quantitative analysis of PCNA expression (Int) in the different constructs. (\*) *p* < 0.001. Data are presented as the mean ± SE.

**Figure 8.** Representative photomicrographs of tropomyosin expression in different constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. Scale bar: 50 µm, original magnification: ×400. (**M**) Graphical representation of the quantitative analysis of tropomyosin expression (Int) in the different constructs. (\*) *p* < 0.001. Data are presented as the mean ± SE. **Figure 8.** Representative photomicrographs of tropomyosin expression in different constructs. Method 1: (**A**) without Np, (**B**) AuNp and (**C**) AuNp+Alg. Method 2: (**D**) without Np, (**E**) AuNp and (**F**) AuNp+Alg. Method 3: (**G**) without Np, (**H**) AuNp and (**I**) AuNp+Alg. Method 4: (**J**) without Np, (**K**) AuNp and (**L**) AuNp+Alg. Scale bar: 50 µm, original magnification: ×400. (**M**) Graphical representation of the quantitative analysis of tropomyosin expression (Int) in the different constructs. (\*) *p* < 0.001. Data are presented as the mean ± SE.

Immunodetection by western blot revealed troponin I and cardiac myosin expression in all scaffold elaboration methods (Figure 9A). In addition, densitometric analysis was performed to demonstrate that for myosin, in the case of the scaffolds of Methods 2 and 4, functionalization with both AuNp and AuNp+Alg caused an increase in expression. In the case of troponin I, it can be seen that in the scaffolds prepared by Methods 2 and 3, functionalization with AuNp+Alg also promoted increased expression (Figure 9B). Immunodetection by western blot revealed troponin I and cardiac myosin expression in all scaffold elaboration methods (Figure 9A). In addition, densitometric analysis was performed to demonstrate that for myosin, in the case of the scaffolds of Methods 2 and 4, functionalization with both AuNp and AuNp+Alg caused an increase in expression. In the case of troponin I, it can be seen that in the scaffolds prepared by Methods 2 and 3, functionalization with AuNp+Alg also promoted increased expression (Figure 9B).

**Figure 9.** (**A**) Representative Western blot figure of myosin and troponin I in the different constructs: without AuNp and with AuNp and AuNp+Alg. Graphical representation of the quantitative analysis of (**B**) myosin and (**C**) troponin I expression**.** (\*) *p* < 0.001. Data are presented as mean ± SE. **Figure 9.** (**A**) Representative Western blot figure of myosin and troponin I in the different constructs: without AuNp and with AuNp and AuNp+Alg. Graphical representation of the quantitative analysis of (**B**) myosin and (**C**) troponin I expression. (\*) *p* < 0.001. Data are presented as mean ± SE.

### **4. Discussion**

**4. Discussion**  AMI generates a high incidence of deaths worldwide; thus, it is necessary to develop AMI generates a high incidence of deaths worldwide; thus, it is necessary to develop new therapeutic strategies to regenerate cardiac tissue that often loses its function [39].

new therapeutic strategies to regenerate cardiac tissue that often loses its function [39]. In this work, four different methods for the elaboration of sodium alginate–chitosan scaffolds are proposed. Crosslinking between polymers is important because it provides the scaffolds with increased stability, higher mechanical strength and hydrolysis resistance [40]. In addition, crosslinking agents promote chemical interactions between exposed functional groups. Although various crosslinking agents have been proposed to improve the biological and mechanical properties of these hydrogels [13,41], in this work the use of calcium gluconate as a crosslinking agent was proposed, favoring cell adhesion and cardiomyocyte growth. In addition, it was found that the fabrication method has an important impact on the structure and properties of the scaffolds and that in general the In this work, four different methods for the elaboration of sodium alginate–chitosan scaffolds are proposed. Crosslinking between polymers is important because it provides the scaffolds with increased stability, higher mechanical strength and hydrolysis resistance [40]. In addition, crosslinking agents promote chemical interactions between exposed functional groups. Although various crosslinking agents have been proposed to improve the biological and mechanical properties of these hydrogels [13,41], in this work the use of calcium gluconate as a crosslinking agent was proposed, favoring cell adhesion and cardiomyocyte growth. In addition, it was found that the fabrication method has an important impact on the structure and properties of the scaffolds and that in general the proposed methods favor the cardiomyocytes growth and contractility.

proposed methods favor the cardiomyocytes growth and contractility. Scaffolds that have low electrical conductivity can be modified with gold, carbon, selenium or silver Np to increase their conductivity and improve their biocompatibility [29,42]. In particular, AuNp considerably increases scaffold conductivity, favors cell organization, enhances cell contractile activity and promotes cardiomyocyte maturation, Scaffolds that have low electrical conductivity can be modified with gold, carbon, selenium or silver Np to increase their conductivity and improve their biocompatibility [29,42]. In particular, AuNp considerably increases scaffold conductivity, favors cell organization, enhances cell contractile activity and promotes cardiomyocyte maturation, proliferation and migration [21,27,31,33,43].

proliferation and migration [21,27,31,33,43]. Nanoparticles tend to be more stable and interact better with biological systems when they are coated with materials of biological origin such as alginate [44,45] or albumin [46,47], among others. In this work, we proposed to use sodium alginate as a stabilizing agent and we evaluated the physicochemical and biological properties of the scaffolds Nanoparticles tend to be more stable and interact better with biological systems when they are coated with materials of biological origin such as alginate [44,45] or albumin [46,47], among others. In this work, we proposed to use sodium alginate as a stabilizing agent and we evaluated the physicochemical and biological properties of the scaffolds and the metallic nanoparticles that were developed.

and the metallic nanoparticles that were developed. It has been reported that a high swelling percentage is favorable for cell growth, adhesion and vascularization [48]. Our results show that the scaffolds that were prepared had swelling percentages above 3000%, a percentage higher or similar to what is reported in other alginate/chitosan scaffolds, in addition to corroborating their pH dependence It has been reported that a high swelling percentage is favorable for cell growth, adhesion and vascularization [48]. Our results show that the scaffolds that were prepared had swelling percentages above 3000%, a percentage higher or similar to what is reported in other alginate/chitosan scaffolds, in addition to corroborating their pH dependence [14,49]. Baei et al. [27] reported that the addition of AuNp in chitosan scaffolds decreases the

hydrophilic functional groups exposed, which translates into lower swelling percentages. Contrary to what they report, in our alginate/chitosan scaffolds, when functionalized with AuNp, the swelling percentage increased, probably due to the fabrication and crosslinking method that was proposed.

Permeability is a property that is directly related to the pore interconnectivity degree, as it controls the nutrient flow through the scaffold to the cells migrating inside it, thus achieving efficient cell growth [50]. Tresoldi et al. [51] reported permeabilities of <sup>9</sup> <sup>×</sup> <sup>10</sup>−<sup>15</sup> <sup>m</sup><sup>2</sup> in alginate–gelatin scaffolds, while in studies performed by Rai et al. [52], permeabilities of 2 <sup>×</sup> <sup>10</sup>−<sup>15</sup> <sup>m</sup><sup>2</sup> were obtained for fibrous poly(glycerol sebacate)-poly(εcaprolactone) scaffolds. In the case of this work, a 1 <sup>×</sup> <sup>10</sup>−<sup>8</sup> <sup>m</sup><sup>2</sup> permeability was obtained in all the proposed fabrication methods, which favors the flow of nutrients and oxygen to the interior of the scaffolds, for greater cell proliferation.

It was demonstrated that our scaffolds were highly porous, which is a feature that directly influences cell penetration and nutrient and oxygen transport [53], with adequate pore size to favor cell infiltration and colonization. The scaffolds elaborated by Method 3 (longer crosslinking time) were the most porous, and functionalizing them with AuNp was found to increase their porosity, to increase their swelling percentage and to decrease their degradation time, which favors the formation of cardiac tissue.

The degradation rate depends on the regeneration rate of the tissue to be replaced. In this work, the degradation percentage was analyzed by placing the scaffolds in culture medium supplemented with fetal bovine serum, which had not been previously reported, to know their behavior under culture conditions. Similar to what has been reported in other works, functionalization with AuNp decreased the degradation rate [54], which in our case favored cardiomyocyte cell growth.

The physicochemical characteristics of proposed gold Np (size and zeta potential) were similar to those reported with potential applications in the biomedical field, specifically in tissue engineering [18–21]. Stable Np do not agglomerate and help improve the electrical conductivity of the scaffold. Our AuNp+Alg had a Z potential (−36 mV) similar to the AuNp+Alg (−30 mV) elaborated by Shen, K, et al. [45], which were stable and helped to generate percentages of cell viability around 95%.

The difference in morphological characteristics between AuNp and AuNp+Alg could be explained by the presence of sodium alginate surrounding the PLGA nuclei, which acts as a structural growth director in a favored specific direction, as has also been reported by Pal et al. [55].

Nanoparticles used in tissue engineering have various sizes. Small Np (20 nm) [56] and large Np (156 nm) [19] have been reported to increase cell viability by up to 60%, suggesting that the size of the Np does not influence their biocompatibility. In our case, the AuNp+Alg (91 nm) were the ones that increased cardiac cell proliferation (Figure 6).

This work demonstrated that natural biomaterials such as alginate and chitosan are not cytotoxic and allow for cardiomyocyte growth as reported in other study groups [57]. The final architecture of our constructs allowed for the adhesion, growth and maintenance of cardiac cell integrity; the cultures remained healthy for 7 days; and the cells were able to enter the scaffolds, grow between the pores and form spheroids. In addition, histological and molecular analysis allowed us to observe the expression of the characteristic proteins of cardiac tissue, which increased with the incorporation of the AuNp+Alg that was proposed (Figures 7 and 8), which is in agreement with the work of Dvir et al., where functionalized scaffolds were shown to increase the expression of cardiac markers [58]. In our case, it was probably due to the biological properties reported for alginate [6].

In addition, an increase in cardiac cell differentiation has been reported when aggregated into spheroids, particularly if these aggregates can bind to a substrate [59], a situation that occurred in this study system.

Similar proliferation was observed in all scaffolds, but the proposed new functionalization with AuNp+Alg considerably increased cell proliferation.

It is interesting to note that in addition to functionalization, the proposed processing methods also have an impact on the expression of cardiac markers, confirming that scaffold topology plays a fundamental role in cell distribution and development. Several factors influence bioengineered tissue generation: on the one hand, the scaffolds and their structure, and on the other hand, the integrity of the cells to be cultured and the appropriate culture conditions. Taken together, the results suggest that these cardiac tissue constructs may be a novel implant material to study cardiac patch–cell interactions and can be used to study their biocompatibility, degradation and vascularization in an in vivo model.

### **5. Conclusions**

The data as a whole demonstrate that the proposed alginate/chitosan scaffolds elaborated by the working group favor the growth of cardiomyocytes and spheroid formation and that functionalization with AuNp and AuNp+Alg not only increases cell proliferation but also promotes the increase in cardiac proteins such as troponin I, myosin and tropomyosin.

The presence of a carbohydrate-based biopolymer such as sodium alginate in AuNp+Alg proved to be an effective means to modify the structural and physicochemical properties of AuNp. Furthermore, it was demonstrated that AuNp+Alg scaffolds can be used as a therapeutic alternative in cardiac tissue engineering.

The proposed scaffolds were found to be useful for cardiomyocyte growth, which presents the possibility of applying them to the generation of a patch that can be used as a therapeutic alternative in AMI.

### **6. Patents**

Soporte de hidrogel de alginato y quitosano para crecimiento de tejidos. Beltran NE, Francisco E., Vaquero D, Arroyo I. Instituto Mexicano de la propiedad industrial, MX/a/2020/012621. 24/11/2020.

**Supplementary Materials:** The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/polym14163233/s1. Figure S1: Custom device constructed to measure scaffold's permeability. Figure S2: Weight and thickness for the four scaffold preparation methods; File S1: S\_database.

**Author Contributions:** Conceptualization, N.E.B.-V. and E.P.-M.; methodology, N.E.B.-V., E.P.-M., C.S.-G., M.G.-L., J.-C.R., I.A.-M., S.H.-Y. and J.C.-T.; validation, N.E.B.-V., E.P.-M., M.G.-L., J.-C.R., I.A.-M. and J.C.-T.; formal analysis, N.E.B.-V., E.P.-M., J.-C.R., I.A.-M. and J.C.-T.; investigation, N.E.B.- V., E.P.-M. and J.-C.R.; resources, N.E.B.-V. and C.S.-G.; data curation, N.E.B.-V., E.P.-M., C.S.-G., M.G.-L., J.-C.R., I.A.-M., S.H.-Y. and J.C.-T.; writing—original draft preparation, N.E.B.-V., E.P.-M., J.-C.R. and I.A.-M.; writing—review and editing, N.E.B.-V., E.P.-M., C.S.-G., M.G.-L., J.-C.R., I.A.-M., S.H.-Y. and J.C.-T.; visualization, N.E.B.-V., E.P.-M., C.S.-G., M.G.-L., J.-C.R., I.A.-M., S.H.-Y. and J.C.-T.; supervision, N.E.B.-V., C.S.-G., M.G.-L. and J.C.-T.; project administration, N.E.B.-V.; funding acquisition, N.E.B.-V., C.S.-G. and M.G.-L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received support from Secretaria de Educación, Ciencia, Tecnología e Innovación (SECTEI), project SECTEI/211/10357c19; and also from the Hospital Infantil de México Federico Gómez, protocol HIM/2020/059 SSA.1688.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are contained within the article or are available as Supplementary Materials.

**Acknowledgments:** The authors thank Daniela Ángeles, Cinthya González and Yenifer Alba for their support in the elaboration and characterization of the scaffolds, Daniela Angeles for Figure 1 design and implementation, Daniela Vaquero for her support in developing the nanoparticles, Anahis Cruz-Ledesma and Mayra Montecillo-Aguado for processing the tissues and Carlos C. Patiño for cell culture and sample processing. We are also grateful for the support received from the Hospital Infantil de México Federico Gómez, protocol HIM/2020/059 SSA.1688.

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