2.1. Improvements in the Mechanical Properties by Incorporation of Polydopamine Coated BaSO4 Particles into PLA
The tensile stress–strain curves and mechanical properties of neat polylactide and its composites (PLA/PD-BaSO4
) were determined by tensile testing. Neat PLA, not reaching a yield point, showed typical brittle behavior, exhibiting around 6% of elongation before failure and 60 MPa of tensile strength. In contrast, PLA/PD-BaSO4
composites showed, for all PD-BaSO4
amounts studied, a clear yield point and an extended ductile behavior with a dramatic increase in elongation at break (supporting information S1
). This is accompanied with a moderate increase in the elastic modulus. As identified in our previous works with PLA/BaSO4
], the plastic deformation in composites occurs because the rigid particles act as stress concentrators and, after particles debonding from the matrix and being of a 0.7–1.9 µm size [28
], activate, at a point of applied stress, crazing and shear yielding deformation mechanisms. Since polydopamine covered particles (PD-BaSO4
) present 1.25 µm size and interactions with the matrix through the polydopamine interface, in these composites, increases in all mechanical properties (elastic modulus, strength, ductility and toughness) are achieved [29
shows the tensile mechanical properties of PLA/PD-BaSO4
composites. As can be observed, up to a 2 wt.% BaSO4
content in composites, Young’s modulus, ductility and toughness improvements can be observed. Further, if these results are to be compared with those of PLA/BaSO4
, the non PD functionalized composite counterparts, enhancements in stiffness and strength together with ductility and toughness can be noticed in the former. Please also note that a huge >2300% increase in elongation at break are determined in these novel composite formulations in regard to neat PLA, which brings about a dramatic improvement in toughness. This is attributable to specific interactions stablished between the ester groups of polylactide and the alcohol groups of polydopamine coating of the particles surface [18
] bringing about a stronger fiber/matrix interface. It is also noticeable that a 30% increase in the elongation at break is obtained in PD coated BaSO4
PLA composites in regard to the composites without polydopamine coating [3
]. Finally, beyond the 2 wt.% particle composition, a slight general decrease in mechanical properties are noticeable, suggesting the existence of particle aggregates and bundles beyond this point.
represents the break surface of the PLA/PD-BaSO4
2 wt.% composite. The SEM image corroborates the ductile and tough behavior of the PLA containing 2 wt.% barium sulfate PD-coated particles with values provided in Table 1
. A high level of dispersion of PD coated particles (indicated with white and straight arrows) within the PLA matrix can be observed. In the pictures, ductile-deformed threads of PLA matrix appear too, thinner than a micrometer in diameter. This means that the matrix has been stretched during the tensile test beyond the yield point leaving long fibers (red dot-line arrows). Therefore, radiopaque PD-BaSO4
particles were able to act as fixed points that under the external applied stress assisting the PLA matrix to develop specific ductile deformation mechanisms.
It is also remarkable that the values obtained for the 2 wt.% and 10 wt.% composites do not differ much from each other. However, radiopacity values, as expected, show a significant increase with the increasing amount of PD-BaSO4
in composites (Figures S3
) and particularly in the 10 wt.% composite. Consequently, it was concluded that among all compositions studied the 10 wt.% PLA/PD-BaSO4
composite presents the optimal properties (Figures S1 and S3
) and hence an additional analysis is conducted for this composite.
The mechanical properties of the 3D printed scaffolds are also analyzed. Details of the scaffolds geometry design can be seen in Figure S4
. In this case, mechanical properties were measured in compression to mimic the working conditions of the device. Non-reinforced polylactide (as reference) and the 10 wt.% PLA/PD-BaSO4
scaffolds with 55% of porosity have been tested.
shows the stress–strain curve’s behavior under compression of neat PLA scaffolds and those of its 10 wt.% PLA/PD-BaSO4
composite counterpart. As can be observed the 3D printed PLA and PLA/PD-BaSO4
scaffolds do show different regimes and each regime corresponds to a specific mechanism of a porous structured material, in agreement with bibliography. In the first stage (region I), the walls contribute to the resistance of the scaffolds under compressive load, which results in an elastic response region at initial loads and corresponding strains. In the second stage (II), the pores collapse by buckling of the walls (barreling effect). The third stage (III) is featured by a large increase in stress over strain which may be explained by the fact that scaffolds are now compressed to a size that the scaffold becomes denser and furthermore more strain resistant to the applied load [31
]. Despite the fact that both scaffolds have the same porosity (55%), in Figure 2
the neat PLA behaved like a more rigid structure [34
], while PLA/PD-BaSO4
shows again a flexible and softer behavior. Therefore, it can be concluded that the particles confer flexibility to the scaffolds in compression tests.
2.2. Biocompatibility Assessment
In vitro compatibility studies were performed to determine the possible toxicity of BaSO4
particles and their resulting PLA composites using Human dermal fibroblasts (HDFs) as a basic toxicity test. Figure 3
shows the metabolic activity of Human dermal fibroblasts (HDFs) in the presence of 10, 50, 100 or 500 µg/mL of BaSO4
particles. The metabolic activity displayed in this figure was normalized at each time-point with respect to the metabolic activity of HDFs seeded in the absence of particles, which was used as a control. The presence of BaSO4
particles slightly reduced the metabolic activity of HDFs with respect to the control. However, in all the cases, the metabolic activity was higher than 80%, demonstrating that HDFs were able to maintain a normal metabolic activity in the presence of particles (see also Supplementary Materials Figure S5
). At day 1, the metabolic activity of HDFs seeded with 10, 50, 100 and 500 µg/mL of BaSO4
particles was, 95, 94, 90 and 90%, respectively, relative to that of the control. In the case of HDFs seeded with 10, 50, 100 and 500 µg/mL of PD-BaSO4
particles, the metabolic activity was 89, 86, 89 and 83% that of the control, respectively. At day 3, the metabolic activity of those cells seeded with 10 or 50 µg/mL of BaSO4
particles, as well as 10 µg/mL of PD-BaSO4
particles was not significantly different from the metabolic activity of the control. At this day, the metabolic activity in all the cases was higher than 90%, suggesting a negligible effect of the particles in the metabolic activity of HDFs. Additionally, cells observed under an inverted microscope showed normal morphology and the BaSO4
particles seemed to be internalized by cells and located around the nuclei (Supplementary Materials Figure S6
shows metabolic activity of cells seeded in PLA, PLA/BaSO4
with 10 wt.% of filler composites and control tissue culture plastic (TCP). The metabolic activity was normalized at each time-point with the metabolic activity of HDFs seeded onto the TCP, which was used as a control. Except for cells seeded on PLA after 3 days of culture (see Table S1
), no significant differences were observed in the metabolic activity of HDFs with respect to the control, indicating normal metabolic activity of cells seeded on the composites developed in this work.
The proliferation of HDFs on PLA, PLA/BaSO4
composites was evaluated via DNA quantification. As can be seen in Figure 5
, DNA content increased over culture time in all experimental and control conditions. Accordingly, significant differences were observed between DNA content observed at day 7 and that observed at day 1 for all the samples studied. For example, the calculated proliferation rates between day 1 and day 7 were 1.6, 3.2 and 2.4 for PLA, PLA/BaSO4
, respectively. The metabolic activity and proliferation results demonstrate that the materials employed in this work are not cytotoxic and can provide a cytocompatibility substrate for cells to attach and proliferate. A higher proliferation of HDFs was observed in PLA/BaSO4
composites with respect to pristine PLA samples. Is hypothesized that this higher proliferation rate may be associated to surface characteristic of the samples, such as roughness or hydrophilicity [35
Concurrently, PD coating of BaSO4 particles introduced binding sites for biologically active molecules such as proteins or drugs, increasing the use of this composite in second-generation devices for biomedical applications.
2.3. Adsorption/Release Test in 3D Printed Scaffolds
Following confirmation of cytocompatibility, the potential for antibiotic delivery with 3D printed scaffolds of PLA/PD-BaSO4
was evaluated in vitro, with the aim of preventing an infection due to the insertion surgery (open wound) and the consequent rejection of the device [37
]. To this end, levofloxacin was incorporated into the material via PD-BaSO4
particle functionalization as a local drug delivery system for avoiding the oral administration common in this kind of surgeries. Levofloxacin is used for fighting and preventing osteomyelitis, since it is a fluoroquinolone with anti-staphylococcal activity in osteoarticular tissues [38
Here, in order to compare the levofloxacin loading efficiency of the developed composites a polydopamine coated neat PLA sample (termed PD-PLA) is introduced. The release was performed at pH 5 to simulate the state of infection and at body temperature of 37 °C.
shows a non-detectable release of PLA, this is because the scaffolds have been previously washed, therefore there was a very low level of drug in PLA scaffolds, reflecting also that PLA does not adsorb the antibiotic and consequently does not release it either. Furthermore, in the case of PD-PLA a burst release was observed during the first 24 h where almost 80% of levofloxacin eluted. The remaining amount of drug (20%) was eluted in 4 days. This type of release profile is adequate in the context of an infection, since at the beginning a high rate of drug release is desirable followed by a slower drug release. When the PLA/PD-BaSO4
was analyzed, a more moderate burst release was observed where almost 60% of levofloxacin eluted and later, during the next 3 days the 30% of drug was eluted while the last two days shows a very slow release with an 8% release (see Figure S6
). In the end the PLA only releases 0.43 µg/mL due to the drug that is trapped in the holes. While PD-PLA was the one with the highest amount of drug released, 3.12 µg/mL, the PLA/PD-BaSO4
composite drug released a 2.4 µg/mL.
These release results in global show that PD-PLA and PLA/PD-BaSO4
display potential antimicrobial properties. Comparing these two materials, the release profile of PLA/PD-BaSO4
composites was more interesting as it generated a second elution stage with greater release, 40% versus 20% in PD-PLA. It is remarked finally that these scaffolds were immersed in trizma buffer (pH 10) prior to analysis so the results of Figure 6
correspond to bound levofloxacin after adsorption.
2.4. Antimicrobial Activity of 3D PLA/PD-BaSO4 Scaffolds with Levofloxacin
To analyze the antibiotic efficacy of the 3D printed scaffolds the Agar Disk Diffusion tests against Staphylococcus aureus (S. aureus)
were carried out. S. aureus
was chosen because in bone infections is one of the most important pathogens due to its ability to adhere and form biofilms when in contact with tissue [38
]. Figure 7
shows the Agar disk diffusion test corresponding to 3D printed scaffolds of PLA/PD-BaSO4
with levofloxacin, PLA/PD-BaSO4
scaffold as a negative control without levofloxacin and the disk of levofloxacin (5 µg) as a positive control. Cicuéndez et al. calculated that the minimum inhibitory concentration (MIC) of levofloxacin has a value of 0.06 μg/mL [39
]. As observed in the release assay (Figure 6
), these scaffolds release larger amounts of the drug.
Analyzing the Agar Disk Diffusion tests is observed that PLA/PD-BaSO4
scaffolds with levofloxacin effectively inhibit bacterial growth, being the inhibition zone diameters of 38 ± 4 mm (Figure 7
a), while the positive control (5 µg of Levofloxacin) exhibited a diameter average 28 ± 0 mm (Figure 7
c). No inhibition zone could be observed for the sample without levofloxacin, PLA/PD-BaSO4
(negative control, Figure 7
b). It should be noted here that the scaffolds were washed after the drug adsorption, therefore the effective inhibition of bacterial growth is attributed to the amount of levofloxacin bound to polydopamine coating of the particles and not to an unspecified content of drug that could remain in the scaffold holds. This would still lead to a higher inhibition zone.
From Agar diffusion tests can be concluded that the polydopamine coating method of BaSO4 particles used for drug tethering within the PLA composites is effective and facilitates the release of the drug inhibiting the S. aureus growth. The described method is simple and allows the addition of the antibiotic after a 3D printing process of scaffolds. This fact has two advantages since the drug is not processed with the scaffold avoiding either the contact with organic solvents nor high processing temperatures that could degrade the molecules, and, on the other hand, the adsorption of the medicament by a scaffold or implant can be done when clinically necessary, in situ.