**1. Introduction**

Bone infections, such as osteomyelitis, remain a major clinical challenge in the field of bone surgery due to their serious rate of mortality and morbidity [1]. The most common causative species of surgical site infections and medical device-associated infections are the opportunistic Gram-positive staphylococci (75% of cases), particularly *Staphylococcus aureus* and *Staphylococcus epidermidis* [2,3]. In terms of pathogenesis, osteomyelitis is complex and varied, with bacteria reaching the bone in two ways: (i) endogenously via blood or originating from another nearby or distant source of infection (hematogenous osteomyelitis); and (ii) exogenously through direct inoculation, contamination of an open trauma, or postsurgical procedures [4].

**Citation:** López-González, I.; Hernández-Heredia, A.B.; Rodríguez-López, M.I.; Auñón-Calles, D.; Boudifa, M.; Gabaldón, J.A.; Meseguer-Olmo, L. Evaluation of the In Vitro Antimicrobial Efficacy against *Staphylococcus aureus* and *epidermidis* of a Novel 3D-Printed Degradable Drug Delivery System Based on Polycaprolactone/Chitosan/ Vancomycin—Preclinical Study. *Pharmaceutics* **2023**, *15*, 1763. https://doi.org/10.3390/ pharmaceutics15061763

Academic Editor: Ana Isabel Fernandes

Received: 6 May 2023 Revised: 10 June 2023 Accepted: 14 June 2023 Published: 18 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The traditional treatment for chronic osteomyelitis includes extensive resection of the infected tissue, repair of the bone defect, and intravenous and local administration of antibiotics over long periods of time [5,6] (Figure 1A). In more specific cases such as infections associated with the implantation of prosthetic materials (hip, knee, and shoulder prosthesis) or osteosynthesis materials for fracture stabilization (plates, screws, pins, etc.), as a first step, treatment requires the simultaneous removal of the implant and intravenous and local antibiotherapy maintenance until normalization of biochemical parameters, as well as the performance of osteoarticular reconstruction surgery as required by the case [5].

**Figure 1.** Schematic representation of current antibiotic treatment strategies used to control bone infections (blue spheres: antibiotics; (**A**): intravenous route; (**B**): implantation of a local drug delivery system (DDS) fabricated by loading a scaffold with antibiotics).

Another strategy for the local treatment of chronic bone infections has been based on the administration of antibiotics by means of implantation of drug delivery systems (DDSs) at the site of infection (Figure 1B). One of the most used DDSs for the treatment of osteomyelitis have been antibiotic-loaded poly(methyl methacrylate) (PMMA) beads [7,8]. PMMA, also referred to as (acrylic) bone cement, is a non-resorbable biomaterial that works by slowly releasing antibiotics (generally gentamicin and vancomycin) over time, which can help to eradicate the bacteria causing the infection [9,10]. Despite having been used for decades in clinical practice, PMMA beads or blocks are far from being an ideal antibiotic carrier. The non-degradability of this biomaterial requires a second surgery to remove the beads 2 or 3 weeks after implantation [11].

Vancomycin (Van) is the most commonly used antibiotic in treatment of infections in arthroplasty surgery and chronic osteomyelitis of any etiology [10]. This glycopeptide antibiotic acts primarily as a cell wall synthesis inhibitor in susceptible organisms. It binds rapidly and irreversibly to the cell wall of susceptible bacteria, inhibiting the synthesis of peptidoglycan, which forms the structure of the cell wall [12,13].

Polycaprolactone (PCL) is a synthetic polymer that has been commonly used in 3Dprinting applications as a scaffolding component for bone and cartilage reconstruction [14–16]. Among its main advantages, we find its biocompatibility, biodegradability, good mechanical properties, and its low melting temperature (60 ◦C), which makes it more versatile than other synthetic polymers used for 3D printing applications [17]. However, the 3D printing temperatures used to fabricate well-defined scaffolds with controlled architecture are around 120–160 ◦C, which makes it impossible to combine them with cells, growth factors, or other bioactive molecules during the printing process [18,19]. On the other hand, PCL lacks natural motifs that provide specific binding sites for cells that facilitate tissue integration [20]. Because of this, different strategies have been developed to overcome PCL native hydrophobicity, such as surface modification using NaOH treatment or its combination with other synthetic or naturally derived materials (hydrogels) to create hybrid scaffolds with enhanced properties [21–24]. Synthetic and naturally derived hydrogels have been widely used for different biomedical applications due to their ability to encapsulate cells, drugs, growth factors, or other bioactive molecules [25,26]. Naturally derived hydrogels principally include collagen, chitosan, alginate, silk fibroin, gelatine, cellulose, hyaluronic acid, and hydrogels derived from decellularized tissues [27].

Chitosan (CS) is a naturally derived semicrystalline polymer that is obtained by partial deacetylation of chitin under alkaline conditions [28]. It is one of the materials most widely used to prepare hydrogels due to its excellent biocompatibility, nontoxicity, and biodegradability [29,30]. However, CS hydrogels present several problems due to their unstable structures with large-sized and poorly defined pores, weak formability, and low mechanical properties, limiting their further utilization for in vivo studies [31,32]. For this reason, they have been combined with curable polymers such as polycaprolactone (PCL) and polylactic acid (PLA), which provide the scaffolds basic mechanical support [33,34].

The fabrication of hybrid materials that combine natural and synthetic polymers is a promising approach for creating new scaffolds that combine the intrinsic advantages of both materials and meet several requirements, such as being biodegradable, bioactive, having good mechanical properties, and being easy to fabricate [35]. In this work, we focus on the fabrication of a novel hybrid 3D-printed scaffold based on polycaprolactone and a chitosan hydrogel loaded with different vancomycin concentrations (1%, 5%, 10%, and 20%) as a DDS to evaluate its antimicrobial efficacy against *S. aureus* and *S. epidermidis*. In addition, we propose a novel method for improving the adhesion of hydrophobic polymers (PCL) to hydrogels using two different cold plasma treatments. The obtained scaffolds combine the natural biocompatibility, biodegradability, and antibacterial properties of CS with the excellent mechanical properties of PCL. The morphological characteristics of the scaffolds were studied by means of optical and scanning electron microscopy (SEM), showing that the CS/Van hydrogel successfully coated the PCL matrix homogeneously after the plasma treatments. The antibacterial efficacy of the scaffolds was tested against *Staphylococcus aureus* and *Staphylococcus epidermidis*, and vancomycin release was studied at different time periods by means of high-performance liquid chromatography (HPCL). Finally, we evaluated the possible systemic adverse effects of scaffolds at the cellular level by analyzing the viability, proliferation, and differentiation of a population of adult human bone marrow-derived mesenchymal stem cells (*ah*-BM-MSC).

### **2. Materials and Methods**

### *2.1. Materials*

Polycaprolactone (PCL) 3D printing filament (molecular weight: 50,000 g/mol) was purchased from 3D4makers (Haarlem, The Netherlands). Chitosan (#448869, 75–85% deacetylated, low molecular weight), acetic acid, and NaOH were obtained from Sigma-Aldrich (Saint Louis, MO, USA) and used as received. Vancomycin hydrochloride was purchased from Lab. Reig Jofre S.A. (Barcelona, Spain).

*Staphylococcus aureus* (CECT 239) and *Staphylococcus epidermidis* (CECT 231) strains were purchased from the Spanish Type Culture Collection (CECT) (Valencia, Spain). Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), and buffered peptone water were obtained from Scharlau (Barcelona, Spain).

### *2.2. Methods*

2.2.1. Fabrication of Porous Polycaprolactone/Chitosan Scaffolds Loaded with Vancomycin Design and Fabrication of 3D-Printed PCL Scaffolds with Controlled Porosity Using the Fused Deposition Modelling (FDM) Method

The scaffolds were designed by using REGEMAT 3D Designer software (v1.4.4) and 3Dprinted using REGEMAT 3D Bio V1® bioprinter (REG4Life, REGEMAT 3D, Granada, Spain) equipped with a glass bed and a thermoplastic extruder with a 0.4 mm diameter nozzle. The scaffolds were designed with the following parameters: scaffold size 1.50 × 20 × 20 mm (height, width, length), pore size 200 μm, layer height 0.25 mm, perimeters 0, solid bottom/top layers 0, infill pattern triangular; and manufactured using a medical-grade PCL filament printed at 160 ◦C with an infill speed of 11 mm/s. As described in a previous work, an 8 mm biopsy punch was used to prepare defined and reproducible disk-shaped scaffolds of 8 × 1.50 mm (diameter, height) from each printed scaffold (Figure 2A) [18]. In addition, nonporous (solid) PCL scaffolds of 12 × 12 × 1.50 mm (width, length, height) were printed in order to avoid porosity interfering with some of the experimental results (Figure 2B). Porous disk-shaped scaffolds were characterized and used for all biological and microbiological experiments and solid scaffolds were only used to evaluate the effect of cold plasma treatment on the adhesion of CS/Van hydrogel to the PCL matrix (see Section 2.3.2), as it was noticed that the porosity interfered in the experimental results.

**Figure 2.** Micrographs of (**A**) porous and (**B**) solid PCL scaffolds printed using REGEMAT 3D Bio V1® bioprinter.

### Vancomycin-Loaded Chitosan Hydrogel Preparation

The chitosan hydrogels were prepared by dissolving low molecular weight chitosan at a concentration of 4% (*w*/*v*) in demineralized water containing 1.5% (*v*/*v*) acetic acid. The solutions were mechanically stirred for 24 h (250 rpm) and vancomycin was added to the chitosan hydrogels in a content of 1%, 5%, 10%, and 20% (*w*/*w* of chitosan). After 24 h, the solutions were centrifuged (3000 rpm, 1 h) to remove air bubbles.

### Hybrid Scaffold Preparation

The 3D-printed PCL scaffolds were dip-coated in the chitosan hydrogel and left to dry overnight. Then, the scaffolds were neutralized in a 1 M NaOH solution for 2 h and rinsed three times with distilled water to remove residual acids. The whole process was performed in a laminar flow cabinet to avoid sample contamination.

Preliminary tests showed that the chitosan coating remained firmly adhered to the porous PCL scaffolds, as it was trapped between the 3D-printed strands. However, that was not the case for solid PCL scaffolds, which repelled the chitosan coating as seen in

**Figure 3.** Plasma treatments performed on the PCL scaffolds. (**A**) Cold atmospheric plasma jet; (**B**) homemade torch with a piezoelectric plasma generator. Optical microscopy images of (**C**) Solid PCL scaffold (untreated) coated using CS hydrogel (scale bar 400 μm); (**D**) solid PCL scaffold coated

using CS hydrogel after being exposed to cold plasma for 30 s (scale bar 400 μm).

Plasma treatments have recently been used to lower the hydrophobicity of polymer surfaces by forming reactive oxygen species (ROS) [36]. These newly formed reactive species generate hydrophilic groups (hydroxyl or carboxylic) on the polymer surface that can interact with the hydrogels [37]. Even if plasma treatment was not necessary for porous scaffolds, two different plasma technologies were tested on the surfaces of solid PCL scaffolds: (i) a cold atmospheric pressure plasma jet developed in GREMI-CNRS consisting of a plasma gun that is a dielectric barrier discharge (DBD) plasma and helium fed and powered using a microsecond voltage pulse [38] (Figure 3A); and (ii) a homemade torch with a piezoelectric plasma generator (CeraPlas®) developed by TDK Electronics GmbH & Co [39] (Figure 3B). This technology is based on a piezo ceramic component and a driving circuit that allows for generating cold atmospheric plasma. After being exposed for 30 s to plasma treatments with 5 mm gap, the PCL scaffolds were dip-coated in the chitosan hydrogels and left to dry overnight. As can be seen in Figure 3D, the CS hydrogel was not repelled by the scaffold, showing a homogeneous distribution.

In order to determine the amount of chitosan adhered to the scaffolds, the weight of the PCL scaffolds was compared to the weight of PCL/CS/Van scaffolds after the coating process. After weighing the scaffolds (n = 10) in an analytical balance, an increment from 33.34 ± 0.34 mg (native PCL) to 41.16 ± 1.01 mg (PCL/CS/Van scaffolds) was observed. No significant differences were obtained between the weight of PCL/CS scaffolds at different vancomycin concentrations.
