**3. Results and Discussion**

### *3.1. Characterization of the 3D-Printed Scaffolds*

3.1.1. Morphological Characterization of the 3D-Printed Scaffolds

Microstructural examination of the scaffolds' morphology using SEM imaging showed differences in the scaffolds' surfaces depending on the vancomycin concentration loaded. Figure 5(A1,B1) shows a micrograph of the native PCL constructs before being coated with

chitosan hydrogel. The scaffolds show a parallel distribution of the printed strands in each printed layer, and the triangular printing pattern results in scaffolds with interconnected porosity with an approximate pore size of 200 μm and an overall porosity of 59%, as calculated in a previous publication [18]. As can be seen in Figure 5(A2,B2), the chitosan coating causes a discrete thickening of the 3D-printed strands as well as at the points of contact between the strands as a result of the thin coating layer provided by CS. However, this finding intensifies progressively and proportionally when the chitosan hydrogel is loaded with increasing vancomycin concentrations (Figure 5(A3–A6,B3–B6)). The latter is observed in the micrographs corresponding to PCL/CS/20%Van scaffolds, in which almost the entire surface of the PCL scaffold was covered, making it difficult to distinguish its pores (Figure 5(A6,B6)).

**Figure 5.** SEM micrographs of the (**1**) PCL, (**2**) PCL/CS, and (**3**–**6**) PCL/CS/Van scaffolds loaded with 1%, 5%, 10%, and 20% *w*/*t* vancomycin, respectively, at different magnifications: (**A1**–**A6**) 22×, scale bar 2 mm; (**B1**–**B6**) 40×, scale bar 1 mm; (**C3**) scale bar 200 μm; (**C5**) scale bar 700 μm.

The functional characteristics of the construct developed in this study agree with the premises established by different authors for the design and fabrication of 3D scaffolds to be used for tissue engineering applications [46,47]. In fact, we have taken into account that an ideal construct or scaffold, in general, should have sufficient porosity to accommodate both differentiated and undifferentiated cells of different lineages. In addition, it was combined with a natural-derived polymer (chitosan) capable of acting as a carrier or DDS for the controlled release of drugs or biomolecules. Likewise, these reticular pore systems should have open and interconnected porosity to facilitate the diffusion of nutrients, metabolites, and cells from the external environment to the inner parts of the scaffold [20].

### 3.1.2. Wettability Assay

The wettability of solid 3D-printed PCL scaffolds was evaluated by measuring the static water contact angle of 1 μL water droplets before and after the plasma treatments. As shown in Figure 6, the static WCA of the untreated PCL scaffolds was 79.93 ± 2.36◦, demonstrating hydrophobic behavior without any surface modification. However, for plasmaactivated scaffolds, we observed water contact angles of 55.40 ± 3.16◦ and 59.62 ± 4.22◦ for Plasma 1 and Plasma 2, respectively, showing a decrease of around 20◦ that attests to a decrease in surface hydrophobicity.

**Figure 6.** (**A**) Water contact angle on 3D-printed solid (nonporous) PCL scaffolds before and after plasma treatments (Plasma 1: cold atmospheric pressure plasma jet; Plasma 2: homemade torch with piezoelectric plasma generator). Water droplets with a volume of 1 μL deposited on (**B**) nontreated PCL scaffolds and (**C**) PCL scaffolds treated using Plasma 1. Data represent mean ± SD. \* Significant differences with the water contact angle of nontreated PCL scaffold.

A change in WCA is a clear indicator of a polymer surface chemistry modification [48]. The results obtained are in total accordance with the ones obtained by Carette et al., who studied the effects of atmospheric plasma on the surface of a polylactic acid (PLA) scaffold in order to improve the adhesion of a 1% chitosan solution, obtaining a decrease of about 30◦ in WCA (from 85◦ to 55◦) after plasma activation [36].

### 3.1.3. In Vitro Degradation Kinetics

The in vitro degradation assay carried out using PCL/CS/Van scaffolds did not result in any changes in the scaffold weights (W0 = Wd) after 28 days of immersion in culture medium at 37 ◦C in a 5% CO2 atmosphere at 95% relative humidity. It is well known that CS, as well as other polysaccharides, is degraded in the body by depolymerization, oxidation, and hydrolysis (enzymatic or nonenzymatic) [49]. Lim et al. studied the degradation of CS porous beads with different degrees of acetylation (DA 10–50%) in different enzyme solutions (lysozyme, NAGase, and a mixture of both) [50]. As main findings, they determined that (i) CS beads did not degrade in the NAGase solution, (ii) CS beads with high DA degraded faster than those with low DA in lysozyme and enzymatic mixture solutions, and (iii) CS beads with DA values around 30 to 50% rapidly degraded into monomers in the enzymatic mixture in less than 30 to 60 days.

### 3.1.4. Water Absorption Assay

The water absorption of the chitosan coating of PCL/CS and PCL/CS/Van scaffolds was tested in PBS solutions. As can be seen in Figure 7, the total weight of the scaffolds increased between 34.61 ± 1.13% (PCL/CS) and 42.14 ± 1.46% (PCL/CS/20%Van) after 30 min of immersion in PBS, and remained more or less stable for at least for 48 h. The minimum absorption values were shown by the PCL/CS scaffolds (34.61 ± 1.13%), while the maximum ones were reached by the PCL/CS/20%Van scaffolds 45.32 ± 8.41%. The latter can be justified by the results obtained in the scaffolds characterization section (SEM imaging), which showed that increasing concentrations of vancomycin caused a thickening of the 3D-printed strands, having more chitosan surface to absorb PBS.

**Figure 7.** Water absorption as a function of time of PCL/CS and PCL/CS/Van scaffolds (1%, 5%, 10%, and 20%) in contact with PBS medium (pH = 7.4).

### 3.1.5. Vancomycin Release Quantification

The vancomycin release from the PCL/CS scaffolds loaded with 1%, 5%, 10%, and 20% vancomycin followed first-order release kinetics (Figure 8). Fast vancomycin release was observed during the first hour, followed by slower release during the next hours for all the scaffolds studied. As can be seen in the graph, vancomycin release finishes after 24 h and the cumulative drug release profiles seem to reach a plateau. These release profiles could be explained by the presence of two different drug fractions: (i) drug weakly bound or deposited on the CS hydrogel surface, and (ii) drug included in the CS network by chemical interactions. It is worth noting that the scaffolds loaded with 10% and 20% Van had similar drug release profiles, indicating that the solubility limit of the drug in PBS is probably between both concentrations.

These kinetics profiles are in total accordance with the results obtained by López-Iglesias et al., who studied vancomycin release from porous chitosan aerogels obtaining similar drug release profiles [51]. On the other hand, Kausar et al. developed chitosan films loaded with vancomycin (10 and 20% with respect to dry polymer mass), obtaining 100% dissolution of free vancomycin in 7 h with more than 50% of drug dissolved within the first hour [52].

**Figure 8.** In vitro cumulative drug release profiles of PCL/CS scaffolds loaded with 1%, 5%, 10%, and 20% vancomycin for 14 days (336 h).

### *3.2. Evaluation of Antimicrobial Properties*

PCL/Chitosan scaffolds loaded with different vancomycin concentrations have been shown to exhibit antimicrobial activity against both *S. epidermidis* and *S. aureus*. This is because, in all cases, the minimum inhibitory concentration for vancomycin was exceeded (2 μg/mL for *S. aureus* and 2–4 μg/mL for *S. epidermidis*) [53,54]. The inhibition halos were measured at 24 and 48 h, with no significant differences being observed between them. This may be due to the fact that no more vancomycin is released between 6 and 24 h (Figure 9).

**Figure 9.** Inhibition halos (mm) produced by PCL/CS/Van scaffolds loaded with 1%, 5%, 10%, and 20% *w*/*t* vancomycin for *S. aureus* and *S. epidermidis*. Data represent mean ± SD. No significant differences were found between the scaffolds for both microorganisms studied.

As seen in Figure 9, a halo of inhibition is shown in all cases. In the case of *S. aureus*, the highest inhibition halo is for PCL/CS/20%Van, but there are no significant differences with the PCL/CS/10%Van and PCL/CS/5%Van scaffolds. This is because the vancomycin concentrations in the media are similar to each other, 9.5 mg/mL and 8.82 mg/mL (Figure 9), respectively. In the case of *S. epidermidis*, the highest inhibition halo was obtained for the PCL/CS/10%Van scaffolds. If the PCL/CS/10%Van and PCL/CS/20%Van scaffolds are compared, there are no significant differences between them because the concentrations of vancomycin in the media are similar (Figure 9).

If we compare the inhibition halos of *S. epidermidis* and *S. aureus*, it is observed that although both bacteria are Gram-positive; vancomycin is more effective against *S. epidermidis*. No antimicrobial effect of PCL/CS was observed, this may be due to the low solubility and the limited amount of positive charges of the polysaccharide at the pH (7.4 ± 0.1) of the culture medium [55,56].

## *3.3. Biological Assays*

3.3.1. Cell Viability and Proliferation Assays In Vitro Cytotoxicity Assay

Cytotoxicity was assessed using an LDH activity assay after placing the PCL/CS/Van scaffolds in indirect contact with *ah*-BM-MSCs for 24 and 72 h (Figure 10). The results of LDH release demonstrated that none of the tested scaffolds were found to be cytotoxic. However, the maximum LDH release values were achieved by PCL, PCL/CS, and PCL/CS/1%Van scaffolds, showing cytotoxicity values of 11.4 ± 6.4%, 8.3 ± 4.5%, and 10.0 ± 8.0% at 24 h, and 11.1 ± 4.9%, 6.9 ± 5.0%, and 11.7 ± 6.4% at 72 h, respectively, with respect to low and high control. On another hand, PCL/CS scaffolds loaded with 5, 10, and 20% vancomycin reached cytotoxicity values of 5.7 ± 5.5%, 6.1 ± 4.7%, and 4.9 ± 5.5% at 24 h, and 4.4 ± 3.4%, 4.8 ± 4.0%, and 4.7 ± 2.9% at 72 h, respectively. No statistically significant differences were observed between the tested samples.

**Figure 10.** Cytotoxicity results using the LDH assay after placing PCL, PCL/CS, and PCL/CS/Van scaffolds in indirect contact with *ah*-BM-MSCs for 24 and 72 h. The mean cytotoxicity percentage was calculated and normalized with respect to spontaneous LDH release (low control) and maximum LDH release (high control). Bars represent standard deviations of the mean. No significant differences were observed between the tested samples.

Both polymers used in this study (PCL and CS) have been commonly used as DDSs or scaffolds components for tissue engineering applications due to their biocompatibility, biodegradability, and ease of modification [57,58]. Cytocompatibility of CS has already been studied, with results showing that it does not have an acute cytotoxic effect [59]. Our results are in line with previous publications that demonstrate the good biocompatibility of PCL and CS-based structures [33,60].

### Cellular Metabolic Activity Assay

The cellular metabolic activity of *ah*-BM-MSCs growing in the presence of PCL, PCL/CS, and PCL/CS/Van scaffolds was evaluated using the AlamarBlue® assay on days 1, 3, 7, and 14 after seeding (Figure 11). As can be seen in the graph, the metabolic activity of *ah*-BM-MSCs increased gradually from days 1 to 14 for all the scaffolds studied except for PCL/CS/20%Van, in which a slight decrease was observed after day 7. No significant differences were observed at early periods (days 1 and 3), where all the scaffolds showed a similar proliferation rate. On the other hand, significant differences were noted between PCL, PCL/CS scaffolds, and the control on day 7, and between PCL, PCL/CS/20%Van, and the control on day 14. It is worth noting that the PCL/CS/10%Van scaffolds showed higher metabolic activity with respect to the scaffolds studied.

**Figure 11.** Cellular metabolic activity of *ah*-BM-MSCs using the AlamarBlue® assay at different time periods. The metabolic activity of cells seeded on plastic (TCPs) was used as positive control. Bars represent standard deviations of the mean. \* Significant differences between the bracketed groups at the same time period.

### 3.3.2. Osteoblastic Differentiation Assays

Osteogenic differentiation was assessed on PCL, PCL/CS, and PCL/CS/10%Van scaffolds, as it provided the best results in the cytotoxicity and proliferation assays, as well as in the microbiological assays, showing a similar bactericidal effect to the scaffold loaded with 20% vancomycin.

### Alkaline Phosphatase (ALP) Activity

The Alkaline Phosphatase activity of *ah*-BM-MSCs cultured in the presence of PCL, PCL/CS, and PCL/CS/10%Van scaffolds is shown in Figure 12. From days 7 to 14, using growth medium (GM), no significant differences in ALP activity were observed between the different time periods or within the scaffolds studied. On the other hand, when using osteogenic medium (OM), the ALP activity significantly increased at 14 days, reaching the maximum values in the presence of PCL/CS/10%Van. In fact, significant differences were observed between the scaffold loaded with 10% vancomycin and the other experimental groups, indicating that PCL/CS/10%Van seems to increase the ALP activity of the cultured cells.

**Figure 12.** Alkaline phosphatase activity of *ah*-BM-MSCs after being cultured for 7 and 14 days in the presence of PCL, PCL/CS, and PCL/CS/10%Van scaffolds using growth medium (GM) and osteogenic medium (OM). Cells seeded on plastic (TCPs) were taken as positive control. Results are shown as a function of optical density (OD405nm) units. Data represent mean ± SD. Significant differences were found between PCL/CS/10%Van and the other samples at 14 days using OM; \* *p* < 0.001 and \*\* *p* < 0.0001.

However, the PCL/CS scaffold did not show an increase in ALP activity, so this finding cannot be attributed to the presence of the chitosan coating. Other authors have followed novel strategies to increase the osteogenic activity of chitosan, consisting of its combination with hydroxyapatite (HA) via the fabrication of carboxymethyl chitosan–HA nanofibers [61] or HA/resveratrol/chitosan composite microspheres [62]. This kind of HA composite can provide necessary calcium sources that can consequently induce cell differentiation, deposition of extracellular matrix, and mineralization [63].

### In Vitro Mineralization Assay

The osteogenic differentiation of *ah*-BM-MSCs was determined by quantifying the presence of calcium deposits after placing the PCL/CS/Van scaffolds in indirect contact with *ah*-BM-MSCs for 21 days. As shown in Figure 13A, no major visual differences were observed between the samples, although the control appears to have a slightly higher reddish coloration. Quantitative examination (Figure 13B) showed that the alizarin red staining concentration was higher in the control than in the cells exposed to the scaffolds, even if no significant differences were observed between the samples. This could be due to a cellular adaptation phase to varying environmental changes caused by the presence of the scaffolds, which could have slightly delayed or affected mineralization.

**Figure 13.** Alizarin red staining of *ah*-BM-MSCs after being cultured for 21 days in the presence of PCL, PCL/CS, and PCL/CS/10%Van scaffolds. (**A**) Alizarin red staining showing mineralization (original magnification ×10); (**B**) quantitative determination of alizarin red staining. Cells seeded on plastic (TCPs) were taken as positive control. Quantitative data are presented as mean ± SD. No significant differences were observed between groups.

As demonstrated by both osteoblastic differentiation assays, the PCL, PCL/CS, and PCL/CS/10%Van scaffolds did not affect *ah*-BM-MSCs differentiation capacity, indicating that both the scaffold components and the vancomycin dose used did not cause adverse effects or alterations at the cellular level.

### **4. Conclusions**

Biocompatible 3D-printed hybrid PCL/CS/Van scaffolds were developed as drug delivery systems with antimicrobial efficacy against *S. aureus* and *S. epidermidis*. Two cold plasma treatments were presented as novel methods to improve the adhesion of hydrophobic polymers to hydrogels, resulting in a decrease in PCL water contact angle of

20◦. All the scaffolds studied (1%, 5%, 10%, and 20%Van) had antimicrobial activity against both *S. aureus* and *S. epidermidis*, showing higher inhibition halos for PCL/CS/10%Van and PCL/CS/20%Van scaffolds. In vitro assays demonstrated that the PCL/CS/Van scaffolds were found to be biocompatible and bioactive as demonstrated by no cytotoxicity or functional alteration of the cultured *ah*-BM-MSCs. In conclusion, the developed DDS could be useful in achieving a controlled and effective local release of vancomycin against *S. aureus* and *S. epidermidis*, considered as the main causes of bone infections, but further preclinical studies in vivo using animal models are needed to confirm these results.

**Author Contributions:** Conceptualization, I.L.-G. and L.M.-O.; methodology, I.L.-G., A.B.H.-H., M.B., J.A.G. and L.M.-O.; software, I.L.-G., M.B. and D.A.-C.; investigation and validation, I.L.-G., M.I.R.-L., M.B., J.A.G. and L.M.-O.; formal analysis, I.L.-G., A.B.H.-H. and L.M.-O.; data curation, I.L.-G., D.A.-C., M.I.R.-L. and L.M.-O.; writing—original draft preparation, I.L.-G., D.A.-C., M.I.R.-L., M.B. and L.M.-O.; writing—review and editing, I.L.-G., A.B.H.-H., D.A.-C., M.I.R.-L., J.A.G. and L.M.-O.; visualization, I.L.-G. and L.M.-O.; supervision, L.M.-O.; project administration, I.L.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of UCAM-Universidad Católica de Murcia (authorization no. CE051904) UCAM ethics committee (CE nº 052114/05.28.2022).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are disclosed in the main text.

**Acknowledgments:** The authors of this study would like to thank the Centre Régional d'Innovation et de Transfert de Technologie—Matériaux Innovation (CRITT-MI) for support in this study.

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

### **References**


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