*2.3. Characterization of the 3D-Printed Scaffolds*

### 2.3.1. Morphological Characterization of the 3D-Printed Scaffolds

The morphological characterization of the 3D-printed scaffolds was carried out using a digital camera coupled to a stereomicroscope. The surface morphology of the scaffolds was examined using scanning electron microscopy (SEM; model JEOL-6100, JEOL Ltd., Tokyo, Japan). Prior to visualization, the scaffolds were mounted on aluminum stubs with conductive paint and sputter-coated with platinum (10 mA, 120 s). SEM micrographs were

Figure 3C. This could be due to the fact that PCL is a hydrophobic polymer, which makes it difficult to adhere to hydrogels with more than 90% water content.

obtained at magnifications of ×20 and ×45 under an accelerating voltage of 15–20 kV and a working distance of 4–5 mm.

### 2.3.2. Wettability Assay

Static water contact angle (WCA) measurements were performed on a drop shape analyzer (DSA100S KRÜSS GmbH) to assess the effect of cold plasma treatment on PCL hydrophobicity. The water droplet volume was set to 1 μL and the deposit speed at 2.67 μL/s. Briefly, solid PCL scaffolds (n = 3) were treated using a cold atmospheric plasma jet (Plasma 1) and a homemade torch with a piezoelectric plasma generator (Plasma 2) (Figure 3). Three untreated scaffolds were used as control. Each measurement was performed in triplicate in three different scaffolds for better statistical significance.

### 2.3.3. In Vitro Degradation Kinetics

The in vitro degradation of the scaffolds was assessed by measuring the weight loss of the constructs at 1, 3, 7, 14, 21, and 28 days. Briefly, PCL/CS/Van scaffolds were weighed (W0, initial weight) and subsequently immersed in eppendorf tubes containing 1 mL of complete growth medium (DMEM). Then, the scaffolds were incubated at 37 ◦C in a 5% CO2 atmosphere at 95% relative humidity. At different time periods, the scaffolds were recovered from medium, sightly wiped using filter paper, and reweighed (Wd, final weight). The weight loss (WL) was calculated by applying the following equation [40]:

$$\text{WL\%} = \left[ (\text{W}\_0 - \text{W}\_\text{d}) / \text{W}\_0 \right] \times 100 \tag{1}$$

where W0 and Wd indicate the weight of the scaffold before and after the scheduled immersion time, respectively.

### 2.3.4. Water Absorption Assay

The swelling behavior of PCL/CS/Van scaffolds was tested in PBS solutions of pH 7.4. Three PCL/CS/Van scaffolds of known weight were put into stainless steel baskets in order to prevent scaffolds from floating. At different timepoints (0.5, 1, 3, 6, 24, 48 h), the scaffolds were collected and gently wiped using filter paper before being weighed. Water absorption percentage was calculated using the following equation [41]:

$$\text{Water absorption (\%)} = \frac{w\_t - w\_0}{w\_0} \times 100\tag{2}$$

where *wt* is the weight of the scaffolds at time *t*, and *w*<sup>0</sup> is the initial weight of the dry scaffolds.

### 2.3.5. Vancomycin Release Quantification

Vancomycin-loaded chitosan scaffolds were immersed into 1.5 mL eppendorf tubes containing 1 mL phosphate-buffered saline (PBS, pH 7.4) medium at 37 ◦C in darkness. At predetermined timepoints (1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 7 d, and 14 d), the medium was collected, and fresh PBS was refilled accordingly. The vancomycin concentration was measured using high-performance liquid chromatography (HPLC) (Agilent 1200 series, Agilent Technologies, Waldbronn, Germany) with diode array (DAD) and fluorescence (FLD) detector, equipped with a Vydac 218TP C18 column (250 × 4.6 mm, 5 <sup>μ</sup>m, Avantor®, Radnor Township, PA, USA). The analysis was performed under isocratic conditions (0.8 mL/min) using 89% ammonium phosphate 0.5 M and 11% acetonitrile as mobile phase. Prior to the measurements, a calibration curve of vancomycin in PBS was determined by measuring absorbance values of stock solutions of vancomycin from 100 to 0.5 μg/mL at 282 nm (linear range 12.4 to 0.5 μg/mL; R<sup>2</sup> > 0.998).

### *2.4. Evaluation of Antimicrobial Properties*

### 2.4.1. Bacterial Culture

Strains of *Staphylococcus aureus* (CECT 239) and *Staphylococcus epidermidis* (CECT 231) were obtained from the Spanish Type Culture Collection (CECT) (Valencia, Spain). To activate the *S. aureus* and *S. epidermidis* strains, they were grown in TSB medium and incubated aerobically at 35 ◦C for 24 h. The bacterial cultures were stored in TSA medium at 4 ◦C for over three months. For daily use, a working culture was prepared by transferring a single colony from TSA to 10 mL of TSB, followed by incubation at 35 ◦C for 24 h.

### 2.4.2. Agar Diffusion Method

In order to assess the antimicrobial properties of the chitosan coating loaded with vancomycin, experiments were conducted to measure the inhibition zones. Initially, a suspension of each microorganism (*S. aureus* and *S. epidermidis*) was prepared with a concentration of 5.0 log10 colony-forming units/mL (CFU/mL) in TSB (2X) medium. Subsequently, 100 μL aliquots of the bacterial suspension were added to 6.0 mm diameter well plates containing TSA agar.

Following a brief drying period, the samples were brought into contact with the PCL/Chitosan/Van scaffolds and incubated overnight at 37 ◦C. Finally, the inhibition halos were measured using a ruler at 24 and 48 h. All the experiments were replicated three times.

### *2.5. Biological Assays*

2.5.1. Isolation, Purification, Characterization, and Culture of Adult Human Bone Marrow-Derived Mesenchymal Stem Cells (*ah*-BM-MSCs)

Multipotent *ah*-BM-MSCs were isolated from bone marrow as previously described (additional information related to the applied methodology, cell isolation, culture, and expansion can be found in previous publications [42,43]). Briefly, bone marrow was collected from the iliac crest of three volunteer patients by direct puncture. Then, bone marrow aspirate was transferred into transfer bags containing heparin. The mononuclear cell fraction was obtained using Ficoll density gradient media and a cell-washing closed automated SEPAXTM S-100 system (Biosafe, Eysines, Switzerland). After assessing cell viability by using trypan blue staining (#T8154; Sigma-Aldrich, Saint Louis, MO, USA), cells were plated out at a density of 3.75 × 105 mL in 75 cm<sup>2</sup> culture flasks (Sarstedt, Nümbrecht, Germany) with 10 mL of basal culture growth medium (GM). The growth medium used was Dulbecco's Minimum Essential Media (DMEM) (#31885-023, Gibco, Bleiswijk, The Netherlands) complemented with 10% (*v*/*v*) inactivated fetal bovine serum (FBS) (#F7524, Sigma-Aldrich, Saint Louis, MO, USA) and routine antibiotics such as Penicillin and streptomycin (100 U mL−<sup>1</sup> penicillin and 100 μg mL−<sup>1</sup> streptomycin) (#P4333, Sigma-Aldrich, Saint Louis, MO, USA) before incubating cells under standard conditions of normoxia (21% O2) at 37 ◦C and 5% CO2. *ah*-BM-MSCs in passages 3–5 were used for in vitro experiments.

All biological experiments were in full compliance with the established regulations and the experimental protocol was reviewed 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).

### 2.5.2. Immunophenotypic Profiles of *ah*-BM-MSC Cultures

The isolated *ah*-BM-MSCs were characterized by means of flow cytometry (Beckman Dickinson & Co., Franklin Lakes, NJ, USA; Navios Software v1.2) for mesenchymal (CD90, CD73, 105) and hematopoietic (CD34, CD45) markers as previously described [42,44,45]. Before performing the in vitro assays, single-cell suspensions obtained using culture trypsinization were labeled with fluorochrome-conjugated antibodies: CD73-PE, CD90- APC, CD105-FITC, CD34-APC, and CD45-FITC (Human MSC Phenotyping Cocktail, Miltenyi Biotec, Bergisch Gladbach, Germany) in order to verify the purity of *ah*-BM-MSCs populations.

### 2.5.3. Cell Seeding Methods

For cell viability, proliferation, and osteogenic differentiation assays, *ah*-BM-MSCs were seeded at the bottom of 24 WP at a density of 10 × 103 cells cm<sup>−</sup>2, and PCL/CS/Van scaffolds were placed in 0.4 μm pore culture well inserts (Falcon®) to be in indirect contact with the culture. Cells seeded onto tissue culture-treated polystyrene wells (TCPS; Sigma-Aldrich, Corning, NY, USA) (without materials) were taken as positive control. The culture media were changed three times a week for all the experiments performed.

### 2.5.4. Cell Viability and Proliferation Assays

### In Vitro Cytotoxicity Assay

The cell viability of *ah*-BM-MSCs was evaluated using LDH cytotoxicity detection kit (#11644793001, Roche Diagnostics, Roche. Applied Science, Mannheim, Germany) after placing PCL/CS/Van scaffolds in indirect contact with cells for 24 and 72 h. Before performing the assay, it is important to determine the optimal cell concentration for the cell line used, as different cell types may contain different amounts of LDH. In general, we choose the concentration at which the difference between the low and high control is at a maximum. For our culture of *ah*-BM-MSCs, the optimal cell concentration to be used for the subsequent experiment was found to be 3 × <sup>10</sup><sup>4</sup> cells/well for 24 WP (Figure 4).

**Figure 4.** Determination of the optimal target cell concentration for *ah*-BM-MSCs. *ah*-BM-MSCs were titrated in 24 WP at densities of 100, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 75,000, and 100,000 cells/well. Culture medium was added for the determination of the spontaneous LDH activity (samples), and culture medium containing 1% Triton X-100 was added to determine the maximum LDH release (positive control).

Twenty-four hours after seeding *ah*-BM-MSCs on 24 WP, the culture medium was replaced using fresh assay medium containing 1% FBS to remove LDH activity released from the cells during the incubation step. Then, the PCL/CS/Van scaffolds were placed in indirect contact with cells using 0.4 μm pore culture well inserts and LDH activity was assessed at 24 and 72 h. Cells growing on tissue culture polystyrene (TCPS) plates without PCL/CS/Van scaffolds were used as low control (spontaneous LDH release) and cells cultured using assay medium containing 1% Triton X-100 solution were used as high control (maximum LDH release). At different timepoints, 100 μL aliquots of each well were transferred to an optically clear flat-bottomed 96 WP followed by the addition of 100 μL of the reaction mixture. After 30 min incubation at RT, the absorbance was read directly using a Spectramax iD3 plate reader (Molecular Devices, San Jose, CA, USA) at 490 nm.

### Cellular Metabolic Activity Assay

The cellular metabolic activity of *ah*-BM-MSCs grown in the presence of PCL/CS scaffolds loaded with different vancomycin concentrations was evaluated using the AlamarBlue® assay (#DAL1100, Invitrogen, Carlsbad, CA, USA) on days 1, 3, 7, 14 after seeding. Cells growing onto TCPS (without PCL/CS/Van scaffolds) were used as control. Briefly, at different time periods, the inserts containing PCL/CS/Van scaffolds were temporarily removed (prior to the incubation step), and fresh medium (1 mL) containing 10% (*v*/*v*) AlamarBlue® reagent was added to each well. Then, the plate was incubated for 4 h in darkness (wrapped with aluminum foil) at 37 ◦C in a 5% CO2 atmosphere with 95% of relative humidity. Finally, 150 μL aliquots of each well were transferred to a black-walled 96 WP and fluorescence was assessed at excitation and emission wavelengths of 530 and 590 nm (respectively) using a Spectramax iD3 plate reader (Molecular Devices, USA).

### 2.5.5. Osteoblastic Differentiation Assays

### Alkaline Phosphatase (ALP) Activity

The ALP activity of *ah*-BM-MSCs was assessed at 7 and 14 days using an Alkaline Phosphatase Detection Kit (#SCR004, Merck Millipore, Billerica, MA, USA) after seeding the cells in the presence of PCL, PCL/CS, and PCL/CS/10%Van scaffolds. At each time period, cells were detached using trypsin, and an aliquot of 1.5 × 104 cells (per sample) was treated by adding 100 μL p-nitrophenylphosphate (p-NPP). After 20 min incubation at RT, the catalytic reaction (p-NNP → phosphate + p-nitrophenol) affords a yellow colored byproduct that is proportional to the amount of ALP present within the reaction. The absorbance was measured using a Spectramax iD3 plate reader (Molecular Devices, USA) at a wavelength of 405 nm.

### In Vitro Mineralization Assay

In vitro mineralization was evaluated using an Osteogenesis Assay Kit (#ECM815; Millipore, Billerica, MA, USA) 21 days after seeding *ah*-BM-MSCs in the presence (indirect contact) of PCL, PCL/CS, and PCL/CS/10%Van scaffolds. Briefly, the *ah*-BM-MSCs were fixed using 10% formaldehyde, stained using alizarin red stain solution, and visualized using an optical microscope (Axio Vert.A1, ZEISS, Jena, Germany) coupled to a digital camera (Axiocam 305 color, ZEISS, Germany). Then, the samples were treated following the manufacturer's protocol to quantify matrix mineralization and the optical density (OD) was measured using a Spectramax iD3 plate reader (Molecular Devices, USA) at 405 nm.

### *2.6. Statistics*

All data were represented as mean ± SD, n = 3. The statistical significance was determined by a two-way ANOVA using GraphPrism 8.0.1 (GraphPad Software Inc., San Diego, CA, USA) for Windows. Comparison between groups was evaluated using the *t*-test, the significance level being *p* < 0.05.
