Imidazolium Salts for Candida spp. Antibiofilm High-Density Polyethylene-Based Biomaterials

The species of Candida present good capability to form fungal biofilms on polymeric surfaces and are related to several human diseases since many of the employed medical devices are designed using polymers, especially high-density polyethylene (HDPE). Herein, HDPE films containing 0; 0.125; 0.250 or 0.500 wt% of 1-hexadecyl-3-methylimidazolium chloride (C16MImCl) or its analog 1-hexadecyl-3-methylimidazolium methanesulfonate (C16MImMeS) were obtained by melt blending and posteriorly mechanically pressurized into films. This approach resulted in more flexible and less brittle films, which impeded the Candida albicans, C. parapsilosis, and C. tropicalis biofilm formation on their surfaces. The employed imidazolium salt (IS) concentrations did not present any significant cytotoxic effect, and the good cell adhesion/proliferation of human mesenchymal stem cells on the HDPE-IS films indicated good biocompatibility. These outcomes combined with the absence of microscopic lesions in pig skin after contact with HDPE-IS films demonstrated their potential as biomaterials for the development of effective medical device tools that reduce the risk of fungal infections.


Introduction
Nowadays, polymer-based medical devices such as catheters [1][2][3], prostheses [1,2], endotracheal tubes [1,2], implants [1,2], tissues for tissue engineering [1], drug delivery systems [3] and heart valves [1], are commonly used in hospitals. Candida spp. (e.g., C. tropicalis, C. albicans and C. parapsilosis) have a strong tendency to colonize these polymeric surfaces, forming fungal biofilms [4,5]. By definition, biofilms are complex communities of microorganisms, with a high degree of organization, characterized by cells that are adhered to a surface or interface and embedded in an extracellular matrix of extrapolymeric substances (polysaccharides, proteins, lipids and DNA) of microbial origin, producing a spatially organized three-dimensional structure [6]. Chemical communication between cells, called quorum sensing, allows microorganisms (bacteria and fungi) to coordinate their activity and group together in communities that provide similar benefits as those of multicellular organisms [1,7]. The process of biofilm formation occurs through adhesion

Materials and Methods
Materials. HDPE (HA 7260, Braskem S.A., Triunfo, RS, Brazil) and C 16 MImCl (CJC China Jie Chemical, Shanghai, China) were donated and purchased, respectively. C 16 MImMeS was prepared using the synthesis reported [20]. For the removal of residual water, HDPE and the ISs were vacuum dried at 60 • C for 5 h.
Melt-Blended HDPE-IS and Film Formation. HDPE was melt-blended without or with an IS (0, 0.125, 0.250, or 0.500 wt%), using a twin-screw extruder (HAAKE Rheomex PTW 16 OS, Thermo Fisher Scientific, Waltham, MA, USA). The IS was added after 1 min to the molten polymer, and the components were mixed for 6 min, maintaining the screw speed and temperature of 60 rpm and 190 • C, respectively. Processed samples were left cooling to 25 • C, air dried and milled. After vacuum drying at 60 • C for 5 h, the samples were pressed into 0.5 mm thick films using a hydraulic press (Monarch 3710, Carver, Wabash, IN, USA). Initially, the material was molten within 4 min at 190 • C, and then pressed for 30 s at 4 lbf. The obtained films were abbreviated as HDPE.IS.content (e.g., HDPE.MeS.0125 for HDPE containing 0.125 wt% of C 16 MImMeS).
Scanning Electron Microscopy (SEM). A scanning electron microscope (EVO 50, Carl Zeiss AG, Oberkochen, Germany) was used to study: (A) the morphology of the HDPE and HDPE-IS film surfaces and (B) the biofilm inhibition on these films through reported protocols [33].
Atomic Force Microscopy (AFM). The surfaces of the HDPE and HDPE-IS films were studied with the aid of a scanning probe microscope (5500, Agilent Technologies, Chandler, AZ, USA), using a reported procedure [33].
X-ray Diffraction (XRD). The crystallinity of the HDPE and HDPE-IS films was analyzed with a powder diffractometer (D500, Siemens, Munich, Germany) through a reported procedure [33].
Differential Scanning Calorimetry (DSC). A differential scanning calorimeter (Q20 V24.10 Build 122, TA Instruments, New Castle, DE, USA) was used to study the phase transitions of the HDPE and HDPE-IS films, using a reported protocol [33]. Thermogravimetric Analysis (TGA). The thermal degradation of the HDPE and HDPE-IS films was analyzed in a thermogravimetric analyzer (QA-50, TA Instruments, New Castle, DE, USA), using a reported protocol [33].
Dynamic Mechanical Analysis (DMA). The dynamic mechanical properties of the HDPE and HDPE-IS films were studied using a reported procedure [33], and a dynamic mechanical analyzer (Q800, TA Instruments, New Castle, DE, USA).
Water Contact Angle Measurements. The water contact angles were measured with the aid of a goniometer/drop shape analyzer (DSA100, Krüss, Hamburg, Germany), using a reported procedure [33].
Biological Analysis on HDPE Films. (A) In vitro cell culture: human Mesenchymal Stem Cells (hMSC, Lonza, Italy) at the fifth passage were employed to perform biological studies, using a reported procedure for the in vitro cell culture [33]. (B) Cell attachmentmorphological analysis: A confocal microscope (TCS SP8, Leica Microsystems, Buccinasco, Milan, Italy) was employed to analyze cell-film interactions and spreading, using a fluorescent dye. In particular, HDPE films were cultured with 2 × 10 4 cells (48 h; 37 • C); later, the non-attached cells were eliminated by careful washing with phosphate buffer solution (PBS; pH = 7.4, 0.01 M, Sigma-Aldrich, Milan, Italy), while the attached cells were treated with cell tracker green 5-chloromethylfluorescein diacetate (Life Technologies, Milan, Italy) in phenol red-free medium (37 • C; 30 min). The last step before the observation by CLSM consisted of washing with PBS and incubation in complete medium (1 h). (C) Biocompatibility test-attachment and proliferation: The biocompatibility test was performed on sterilized HDPE films (ethanol (4 h) and UV light (2 h)) equilibrated in Eagle's alpha minimum essential medium (sterile-filtered, Sigma-Aldrich, Milan, Italy) overnight. Later, HDPE films with and without IS were seeded in triplicate with 1 × 10 4 hMSCs and cultured (21 days). The effect of HDPE films on cell attachment and proliferation was quantitatively estimated by the Alamar blue assay (Life Technologies, Italy) at different time points. The results were reported as % of Alamar blue reduction (% AB reduction).
Statistical Analysis. One-way Analysis of Variance was employed with the multiple Dunnett comparison test, considering a significant difference for P < 0.05. The statistical analysis data were represented as mean ± standard deviation for n = 4 (antibiofilm, and MAC) or n = 3 (biocompatibility assay).
Histopathological Evaluation in Pig Skin with HDPE Films. The pig skin preparation, penetration and histopathological evaluation were performed following the reported procedures [33]. Ethic approval number: 04/2016 of the Animal Use Ethics Committee of the Federal Catarinense Institute -Campus Concórdia, Concórdia, SC, Brazil.

Results
HDPE-based biomaterials with IS additives were prepared by melt blending, followed by pressing into films. This resulted in the preparation of HDPE films with 0; 0.125; 0.250 and 0.500 wt% of either C 16 MImCl or C 16 MImMeS. In comparison to the rigid film of HDPE, increasing the content of IS made the films more flexible and less brittle.
SEM investigations were performed to study the morphology of the HDPE-based films' surfaces. The micrographs of HDPE, HDPE.Cl.0500 and HDPE.MeS.0500 ( Figure 2) indicate that the IS incorporation did not have an expressive influence on the surface morphology of these biomaterials. This behavior is different compared to our previous work, in which the addition of C 16 MImCl and C 16 MImMeS in the PLLA matrix interfered with the morphology leading to the formation of superficial spheres and increasing the roughness of the final material [33]. This could be related to the difference in the procedures that were applied to obtain the films; solvent casting (PLLA) vs. pressure molding (HDPE), as well as the chemical interactions between the polymer and IS [35]. As such, the eventual effects of the ISs on the surface morphology of HDPE could have been eliminated during the transformation into films under heat and pressure, assuming the flat surface of the hydraulic press plates. This was further supported by the AFM images ( Figure 3), where HDPE (roughness = 13.0 nm), HDPE.Cl.0500 (roughness = 17.2 nm) and HDPE.MeS.0500 (roughness = 15.6 nm) presented smooth surfaces. The somewhat higher roughness of HDPE.Cl.0500 was most likely related to its higher crystallinity, which will be presented in Table 1. This was less pronounced for HDPE.MeS.0500.    Although the surface structure was not affected much by the presence of IS, the crystallinity of the HDPE-based biomaterials was studied by XRD ( Figure 4). Independent of the IS (C16MImCl or C16MImMeS) or the IS content (0.125, 0.250 or 0.500 wt%), the type of HDPE crystallinity was not affected by obtaining crystalline HDPE.IS materials. All materials presented the typical HDPE peaks at 21.5° and 23.9°, which correspond to the (110) and (200) planes, respectively [36]. The thermal properties of the HDPE-based biomaterials were studied by DSC and TGA, and the results are given in Table 1. In general, the incorporation of IS in the contents of 0.125, 0.250 and 0.500 wt% led to subtle modifications in the thermal properties of the HDPE.IS biomaterials. The melting and crystallization temperatures (HDPE: 132.3 °C and 116.8 °C, respectively) varied within 1 °C. Compared to neat HDPE, HDPE.Cl.0500 showed increases of 5%, 4.4% and 5.6% in the melting enthalpy, the crystallization   3 Melting enthalpy obtained using DSC. 4 Crystallization enthalpy obtained using DSC. 5 Crystallinity obtained using DSC, and Equation (1), where ∆H 0 m = 293 J/g for 100% crystalline HDPE [36], and Fp = polymer fraction. 6 Temperature at decomposition of 5 wt% obtained using TGA. 7 Temperature at decomposition of 10 wt% obtained using TGA. 8 Temperature at decomposition of 50 wt% obtained using TGA. 9 Residual weight at 550 • C obtained using TGA.
Although the surface structure was not affected much by the presence of IS, the crystallinity of the HDPE-based biomaterials was studied by XRD ( Figure 4). Independent of the IS (C 16 MImCl or C 16 MImMeS) or the IS content (0.125, 0.250 or 0.500 wt%), the type of HDPE crystallinity was not affected by obtaining crystalline HDPE.IS materials. All materials presented the typical HDPE peaks at 21.5 • and 23.9 • , which correspond to the (110) and (200) planes, respectively [36].  Although the surface structure was not affected much by the presence of IS, the crystallinity of the HDPE-based biomaterials was studied by XRD ( Figure 4). Independent of the IS (C16MImCl or C16MImMeS) or the IS content (0.125, 0.250 or 0.500 wt%), the type of HDPE crystallinity was not affected by obtaining crystalline HDPE.IS materials. All materials presented the typical HDPE peaks at 21.5° and 23.9°, which correspond to the (110) and (200) planes, respectively [36]. The thermal properties of the HDPE-based biomaterials were studied by DSC and TGA, and the results are given in Table 1. In general, the incorporation of IS in the contents of 0.125, 0.250 and 0.500 wt% led to subtle modifications in the thermal properties of the HDPE.IS biomaterials. The melting and crystallization temperatures (HDPE: 132.3 °C and 116.8 °C, respectively) varied within 1 °C. Compared to neat HDPE, HDPE.Cl.0500 showed increases of 5%, 4.4% and 5.6% in the melting enthalpy, the crystallization The thermal properties of the HDPE-based biomaterials were studied by DSC and TGA, and the results are given in Table 1. In general, the incorporation of IS in the contents of 0.125, 0.250 and 0.500 wt% led to subtle modifications in the thermal properties of the HDPE.IS biomaterials. The melting and crystallization temperatures (HDPE: 132.3 • C and 116.8 • C, respectively) varied within 1 • C. Compared to neat HDPE, HDPE.Cl.0500 showed increases of 5%, 4.4% and 5.6% in the melting enthalpy, the crystallization enthalpy and the crystallinity, respectively. The results also indicate that the ISs can be used as additives in the content range of 0.125-0.500 wt%, without modifying the thermal properties to a large extent. The same properties were studied with PLLA.IS biomaterials which showed an increase in thermal stability of 21 • C, whereas in this study the incorporation of IS into HDPE basically did not affect the thermal stability [33]. This effect may be related to the different intermolecular interactions between the ISs and the polymers. In PLLA.IS relatively strong non-covalent hydrogen bond interactions can take place whereas in HDPE.IS this is dependent on the weaker intermolecular van der Waals forces.
In Table 2 the storage and loss moduli and the stiffness results that were derived by DMA are summarized. Most of the HDPE.IS biomaterials showed similar storage moduli as HDPE, except for HDPE.Cl.0500, which showed lower values. For the loss moduli, the IS-containing HDPE films showed, in general, lower values although this did not follow a clear trend with increasing IS content, and HDPE.Cl.0500 presented a decrease of 24% in the value of HDPE. The lower storage and loss moduli and stiffness for HDPE.Cl.0500 could be related to its higher crystallinity as determined by DSC (Table 1). This could also explain the higher film roughness of HDPE.Cl.0500 ( Figure 3). Interestingly, the biomaterials HDPE.Cl.0250, HDPE.MeS.0125 and HDPE.MeS.0500 demonstrated a better stiffness performance than the neat HDPE; the last one showed an increase of 35% in stiffness at 40 • C. No clear trend with the increase in IS load was observed indicating the non-linearity of the results. Generally, the dynamic-mechanical properties balance depended on the IS and its content, which was optimal for HDPE.MeS.0500. These results are in agreement with those obtained for PLLA.IS, which also generally demonstrated better values when C 16 MImMeS was employed [33]. To better understand the influence of IS dispersed in the HDPE-IS films regarding wettability properties, the water contact angle technique was applied ( Figure 5). Although HDPE.Cl.0250 showed a higher water contact angle than HDPE, the other films with C 16 MImCl contents of 0.125 and 0.500 wt% only showed minor variations regarding the IS-free film. The increased hydrophobicity of HDPE.Cl.0250 suggests that C 16 MImCl was present at the surface and that its aliphatic part was preferentially oriented towards the water drop. In contrast, the HDPE films with C 16 MImMeS showed enhanced hydrophilicity according to the elevation of the IS load. The same effect was observed when 0.5 wt% of C 16 MImMeS was applied in PLLA [33]. This suggests again that the IS was present at the film surface and that the polar part (imidazolium cation ring and IS anion) was preferentially oriented towards the water drop.  Initially, the in vitro biofilm assay antibiofilm was performed to verify whether HDPE biomaterials with ISs exhibited an antibiofilm effect compared to HDPE ( Figures  S1-S3). In this test it was verified that in comparison with HDPE (without the ISs), all tested biomaterials (HDPE.Cl.0125, HDPE.Cl.0250, HDPE.Cl.0500, HDPE.MeS.0125, HDPE.MeS.0250 and HDPE.MeS.0500) showed antibiofilm activity against clinical isolates of C. tropicalis 72A, C. parapsilosis RL11 and RL20 and C. albicans CA04. Furthermore, HDPE films with C16MImCl (excluding HDPE.Cl.0250) also showed antibiofilm activity against C. tropicalis RL17.
Subsequently, the in vitro minor antibiofilm assay was performed to verify the percentage of prevention of biofilm formation on films of HDPE containing C 16 MImCl (HDPE.Cl.0125, HDPE.Cl.0250 and HDPE.Cl.0500) or C 16 MImMeS (HDPE.MeS.0125, HDPE.MeS.0250 and HDPE.MeS.0500), and the results are represented in Figures 6 and 7, respectively. Those films were differentiated from the neat HDPE against 12 isolates of C. tropicalis that are well known to form biofilms [33,37]. The results of the statistical analysis are shown in Figures S4-S9. In general, the obtained results suggest that the presence of IS reduced the growth of biofilms compared to HDPE. The biofilm inhibition varied between 0-75% and 0-64% on the HDPE films containing C 16 MImCl and C 16 MImMeS, respectively. The inhibition percentage was dependent on the tested C. tropicalis isolate, which was possibly due to genetic mutations that made some isolates more resistant to the HDPE-IS biomaterials [38]. The best percentages of impediment of biofilm formation were obtained using HDPE.Cl.0125 and HDPE.Cl.0250 with 75% for C. tropicalis 17P. When HDPE.Cl.0500 was employed, a 65% impediment was obtained for C. tropicalis 17P and 47% for C. tropicalis 17A ( Figure 6). The best percentages of impediment of biofilm formation using HDPE.MeS.0125 were 54% for C. tropicalis ATCC 750 and 41% for C. tropicalis 17P. HDPE.MeS.0250 demonstrated a 40% impediment for C. tropicalis 94P and 37% for C. tropicalis ATCC 950. In the case of HDPE.MeS.0500, a 64% impediment was obtained for C. tropicalis 17P and 46% for C. tropicalis 72P (Figure 7).    Figures  8A-D) show the formation of the biofilm with extracellular material and the cells at different stages of growth adhered to the HDPE film surface. In the cases of the films HDPE.Cl.0500 and HDPE.MeS.0500 ( Figures 8E-H), the biofilm formation was prevented as no fungal and biofilm growth of C. tropicalis 72A was observed on the surfaces of these biomaterials. The results obtained indicate that, in the same way as PLLA.IS, both ISs were effective as anti-biofilm additives for HDPE.IS [33].    Figures  8A-D) show the formation of the biofilm with extracellular material and the cells at different stages of growth adhered to the HDPE film surface. In the cases of the films HDPE.Cl.0500 and HDPE.MeS.0500 ( Figures 8E-H), the biofilm formation was prevented as no fungal and biofilm growth of C. tropicalis 72A was observed on the surfaces of these biomaterials. The results obtained indicate that, in the same way as PLLA.IS, both ISs were effective as anti-biofilm additives for HDPE.IS [33]. These results can be ascribed to the intrinsic antibiofilm property of IS, which was previously reported for C 16 MImCl in the pre-treatment of catheter surfaces [30], C 16 MImCl and C 16 MImMeS incorporated in PLA-based biomaterials [33], and imidazolium polymeric materials [27]. Now, this property was effectively transposed after their incorporation in HDPE. As HDPE alone is not an effective antibiofilm material, the ISs must be present on the biomaterial's surface for this antibiofilm property to take place. As such, the prevention of biofilm formation was the result of a surface phenomenon due to the presence of IS at the HDPE surface. Even if IS would leach into the biological medium, its antibiofilm action will only take place when it is present on the surface of the biomaterial.
The results obtained with HDPE films with different contents of C 16 MImCl and C 16 MImMeS tested with C. tropicalis isolates showed that there is no direct relationship between the IS content used as an additive in HDPE and the percentage of prevention of formation of the biofilm (Figures 6 and 7). Considering the effectiveness of the HDPE biomaterials in relation to the IS content compared to C. tropicalis isolates, it was possible to verify that at contents of 0.125 and 0.250 wt%, C 16 MImCl was more effective in preventing biofilm formation when compared to C 16 MImMeS. At the content of 0.500 wt%, C 16 MImMeS was more effective. In the case of PLA.IS, increasing the contents of the IS C 16 MImCl and C 16 MImMeS increased the percentage of impediment of biofilm formation [33]. The absence of this trend in the case of HDPE.IS suggests that other biomaterial properties impacted their determined antibiofilm potential including hydrophilicity and roughness [39,40]. Figure 8 shows SEM micrographs of HDPE samples. After 72 h of incubation with the clinical isolate C. tropicalis 72A (biofilm builder), the micrographs of HDPE ( Figure 8A-D) show the formation of the biofilm with extracellular material and the cells at different stages of growth adhered to the HDPE film surface. In the cases of the films HDPE.Cl.0500 and HDPE.MeS.0500 ( Figure 8E-H), the biofilm formation was prevented as no fungal and biofilm growth of C. tropicalis 72A was observed on the surfaces of these biomaterials. The results obtained indicate that, in the same way as PLLA.IS, both ISs were effective as anti-biofilm additives for HDPE.IS [33]. The biocompatibility of a material is the principal parameter that governs the decision about the possibility to apply it in implants for human bodies. Such a biomaterial, when used in tissue engineering, should be non-toxic and biocompatible, without causing an intolerable degree of damage to that body [41]. In general, the in vitro cell-material interaction study is frequently used as an initial preliminary analysis of cell biocompatibility [42]. Herein, human mesenchymal stem cells (hMSC), generally used to evaluate the regeneration of mineralized extracellular matrix (ECM) in bone defects [43][44][45][46], were used for the in vitro testing of the biocompatibility of the HDPE-based materials with IS [43]. In particular, the effect of HDPE.IS biomaterials on the hMSC's behavior was evaluated by cell adhesion, which is the first step involved in the biocompatibility process (Figures 9 and 10A,B). Indeed, this cell attachment is the main stage to assess the influence of material surfaces on the hMSC behavior in the first hours of culture time. Both qualitative and quantitative analyses were performed with the aim to obtain information about the cell adhesion process. The morphological analysis ( Figure 9) demonstrated a change in morphology with increasing IS contents. hMSC seeded on HDPE (without IS) showed a thin and elongated structure typical of fibroblast cells. Differently, the HDPE.IS biomaterials show a correlation between the presence of IS and the hMSC morphology. The biocompatibility of a material is the principal parameter that governs the decision about the possibility to apply it in implants for human bodies. Such a biomaterial, when used in tissue engineering, should be non-toxic and biocompatible, without causing an intolerable degree of damage to that body [41]. In general, the in vitro cell-material interaction study is frequently used as an initial preliminary analysis of cell biocompatibility [42]. Herein, human mesenchymal stem cells (hMSC), generally used to evaluate the regeneration of mineralized extracellular matrix (ECM) in bone defects [43][44][45][46], were used for the in vitro testing of the biocompatibility of the HDPE-based materials with IS [43]. In particular, the effect of HDPE.IS biomaterials on the hMSC's behavior was evaluated by cell adhesion, which is the first step involved in the biocompatibility process (Figures 9 and 10A,B). Indeed, this cell attachment is the main stage to assess the influence of material surfaces on the hMSC behavior in the first hours of culture time. Both qualitative and quantitative analyses were performed with the aim to obtain information about the cell adhesion process. The morphological analysis ( Figure 9) demonstrated a change in morphology with increasing IS contents. hMSC seeded on HDPE (without IS) showed a thin and elongated structure typical of fibroblast cells. Differently, the HDPE.IS biomaterials show a correlation between the presence of IS and the hMSC morphology. HDPE-based biomaterials with the ISs C 16 MImCl and C 16 MImMeS induced the stem cells to assume a polygonal structure, typical of osteoblast cells. This behavior was more pronounced for the biomaterials with higher IS contents ( Figure 9C,D,F,G), which is highly favorable for bone repair processes. HDPE, HDPE.Cl and HDPE.MeS showed excellent values in the quantitative cell adhesion analysis which demonstrated good surface properties, promoting the extension of filopodia from the body cell and ensuring a stable cell attachment in the first 48 h of incubation ( Figure 10A). For the C 16 MImCl-based biomaterials, the cell adhesion increased with an increasing IS content, showing a higher cell adhesion percentage for HDPE.Cl.0500 in comparison to HDPE. All HDPE.MeS samples showed values comparable to those obtained with HDPE. The confocal micrographs demonstrate that HDPE.Cl.0500 and HDPE.MeS.0500 improved the spreading of hMSC at the cell-material interface. Indeed, the cells are polygonal in shape, which is different from the elongated morphology observed for the substrates with lower IS amounts. These results were also obtained with PLLA.IS substrates in previous work as reported [33]. After the initial cell adhesion ( Figure 10A), which is important for the next biocompatibility step, the cell proliferation after longer exposure times was studied (4, 7, 10, 14 and 21 days). This enables evaluating its continued cell development after initial adaptation to the biomaterial. In general, the HDPE materials, without and with IS, showed lower proliferation percentages than the control after 7 days ( Figure 10B). Nevertheless, this was compensated for in all materials after 21 days; the cells became acquainted over time with their new environment. HDPE, HDPE.Cl.0250, HDPE.Cl.0500 and HDPE.MeS.0250 exceeded the proliferation percentages of the control after 10 days. The best proliferation results were achieved with the biomaterials containing 0.250 wt% of IS. After the initial cell adhesion ( Figure 10A), which is important for the next biocompatibility step, the cell proliferation after longer exposure times was studied (4, 7, 10, 14 and 21 days). This enables evaluating its continued cell development after initial adaptation to the biomaterial. In general, the HDPE materials, without and with IS, showed lower proliferation percentages than the control after 7 days ( Figure 10B). Nevertheless, this was compensated for in all materials after 21 days; the cells became acquainted over time with their new environment. HDPE, HDPE.Cl.0250, HDPE.Cl.0500 and HDPE.MeS.0250 exceeded the proliferation percentages of the control after 10 days. The best proliferation results were achieved with the biomaterials containing 0.250 wt% of IS.
Finally, the results of the histopathological evaluation of skin of pig ear incubated with the HDPE films containing the ISs ( Figure S10) showed no microscopic lesions.
showed lower proliferation percentages than the control after 7 days ( Figure 10B). Nevertheless, this was compensated for in all materials after 21 days; the cells became acquainted over time with their new environment. HDPE, HDPE.Cl.0250, HDPE.Cl.0500 and HDPE.MeS.0250 exceeded the proliferation percentages of the control after 10 days. The best proliferation results were achieved with the biomaterials containing 0.250 wt% of IS. Finally, the results of the histopathological evaluation of skin of pig ear incubated with the HDPE films containing the ISs ( Figure S10) showed no microscopic lesions.

Conclusions
In conclusion, the melt-blending of HDPE with the IS additives C 16 MImCl and C 16 MImMeS, and subsequent pressure molding, provided promising biomaterial films. Altogether, the ability of HDPE.IS to act effectively against the biofilm formation of Candida species, being biocompatible with hMSC, affording good cell adhesion and proliferation and being highly favorable for bone repair processes may open alternatives for the development of innovative medical devices.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.