Processability and properties of filaments
3.1. Diameters, density, and speed analysis of filaments
The diameter, density, extrusion speed, and roughness are crucial parameters for understanding the processing of filament material, as shown in Table 2.
Table 2
Average values and standard deviation of diameter, density, temperature, speed extrusion, and roughness for each filament.
Sample | Diameter (mm) | Density (g.cm− 3) | Temperature Extrusion (oC) | Speed Extrusion (cm.s− 1) | Roughness Ra (µm) |
Pristine PLA | 1.6 ± 0.004 | 1.1 ± 0.1 | 190 | 3.9 ± 0.09 | 0.36 ± 0.07 |
PLA-5%ZnO | 1.6 ± 0.008 | 1.0 ± 0.03 | 225 | 0.58 ± 0.02 | 0.54 ± 0.11 |
PLA-5%ZnO-4%Keto | 1.7 ± 0.006 | 1.1 ± 0.2 | 230 | 0.98 ± 0.03 | 0.20 ± 0.09 |
PLA and its composites presented similar diameters and densities, with a low standard deviation demonstrating accuracy during the process [24]. This arrangement resulted in the most favorable results and improved precision during the 3D printing process. Besides, adaptations in operational conditions were necessary to guarantee the composite processing. In this context, there was a reduction of 572% in PLA-5%ZnO and 298% in PLA-5%ZnO-3%Keto in speed extrusion, indicating more challenging processing when compared to pristine PLA. This phenomenon may be attributed to the increase in thermal resistance and melting temperature (as seen in temperature extrusion) resulting from the filler incorporation. Furthermore, the low standard deviation values may indicate a satisfactory blending of the components [25]. The PLA composite with the addition of ketoprofen showed the lowest roughness among the samples, indicating a higher miscibility between the filler and the PLA in this filament, this is an important result for obtaining a good printable performance. Therefore, a key aspect of filament formulation is determining the right amount of drug for optimal extrusion and printing properties, as these affect the filament's plasticity and drug release profile. High miscibility between the drug and polymer reduces viscosity, which is essential for good rheological characteristics [26]. Dissolved drugs act as plasticizers, lowering viscosity and the glass transition temperature. However, undissolved drugs can impair mechanical and rheological properties during extrusion, making the filaments brittle and prone to breaking under stress, while larger particles can clog the nozzle and increase viscosity [27].
3.2. Fourier-transform infrared spectroscopy (FTIR)
Figure 3 shows the FTIR spectra for the ZnO particles, Ketoprofen, pristine PLA, and their composites. Ketoprofen’s spectrum showed the characteristic absorptions at 2980 referring to C-H stretch; at 1693 and 1650 cm− 1 corresponding to the acid carbonyl (C = O) group and ketonic carbonyl (C = O) group; at 1597 cm− 1 due to the presence of aromatic C = C stretch; at 1441 cm− 1 due to the presence of CH-CH3 deformation. Finally, the peaks at 966 and 716 cm− 1 are due to the presence of C-H out-of-plane deformation [28]. ZnO particles present peaks at 3381 cm− 1, which can be attributed to the bending and stretching vibrations of surface hydroxyl groups, and at 831 cm− 1 corresponding to Zn–O stretching [15, 29].
The PLA filament showed peaks at approximately 2920 cm− 1 caused by asymmetric and symmetric stretching vibrations of the –CH2 group of saturated hydrocarbons. The hyper-conjugated system generated by the α-hydrogen atom and the carbonyl group in the PLA molecule resulted in the stretching vibration of –C = O at 1748 cm− 1. In addition, PLA peaks were observed at 1452 cm− 1 (associating with CH3 bending bands); at 1181 and 1082 cm− 1 (representing C–O–C stretching vibration caused by C–O forming bonds with different atoms or functional groups to develop more complex vibrational absorptions) and 755 and 667 cm− 1 attributed to the amorphous and crystalline phases, respectively [1, 30]. These findings are similar to the FTIR spectrum of a PLA filament reported by Heydari-Majd et al. [31].
The incorporation of ZnO particles significantly changed the peaks in pristine PLA, such as the narrowing (increase of the intensification) of the peaks at 2920 and 1452 cm− 1, the disappearance of the peak at 1748 cm− 1, and the sharp reduction of the peaks in the 1400 − 1000 cm− 1 range. According to the literature, these results indicated that secondary forces like hydrogen bonds were formed between the PLA polymer and ZnO particles through chemical reactions [15, 31].
The insertion of Ketoprofen on the polymeric structure caused a slight narrowing of the peaks situated at approximately 3084 and 3062 cm− 1. Changes in the vibrational frequencies of the functional groups can be considered as the result of the presence of Ketoprofen in the polymer matrix [31]. Thus, the results indicate that the presence of ZnO particles and Ketoprofen alter the arrangement of the molecular and intermolecular interactions in the filament matrix.
3.3. Thermogravimetric analysis (TGA)
Figure 4 shows the thermal profiles for the ZnO particles, Ketoprofen, pristine PLA, and their composites. Table 3 provides the results for the temperature at which the samples start to degrade (Tonset), when maximum thermal degradation is completed (Tmax), and residual percentage (R%) at 600°C.
Table 3
Weight loss (%), Onset temperature (Tonset), Maximum degradation temperature (Tmax), and Residue at 600°C (R%) of ZnO, Ketoprofen, pristine PLA, and composites.
Samples | Weight loss (%) | Tonset (°C) | Tmax (°C) | R% |
100 | 200 | 300 | 400 | 500 | 600 |
°C | °C | °C | °C | °C | °C |
ZnO | 2.5 | 12.2 | 22.8 | 31.5 | 34.7 | 36.0 | 61.4 | - | 64.0 |
Ketoprofen | 0.0 | 0.51 | 47.38 | 79.7 | 100.0 | 100.0 | 177.4 | 288.5 | 0.0 |
Pristine PLA | 0.0 | 0.0 | 0.0 | 5.0 | 97.7 | 98.4 | 268.5 | 474.7 | 1.6 |
PLA-5%ZnO | 0.0 | 0.0 | 0.41 | 17.2 | 88.1 | 89.5 | 216.9 | 421.8 | 10.5 |
PLA-5%ZnO-3%Keto | 0.0 | 0.0 | 0.89 | 20.6 | 84.7 | 87.6 | 239.7 | 421.2 | 14.4 |
In the ZnO particles’ thermal curve, any moisture was evaporated until it reached 100°C. As the temperature increased, the degradation of phenolic and flavonoid biomolecules (organic molecules) involved in the biosynthetic pathway was slow and continuous. Data above 400°C revealed great thermal stability and insignificant weight loss [32, 33]. Concerning Ketoprofen, this material began its rapid degradation at approximately 177.4°C, corresponding to the breaking of the covalent bonds in its chemical structure [34].
The thermal degradation of pristine PLA started at 268.5°C (Tonset) and reached maximum degradation at 474.7°C (Tmax). This behavior may be associated with the evaporation of water molecules from the pristine filament, followed by the degradation of the polymer chains due to the loss of the ester group [35].
With the addition of ZnO particles at 5%, the Tonset started earlier (216.9°C) than with pristine PLA (268.5°C). The decrease in thermal stability of this composite filament was probably related to the decomposition of low-molecular-weight substances (organic groups) present in the particles [36]. Ahmad and coworkers reported a similar behavior when exploring the properties of cellulose nanocrystals-reinforced PLA composite filaments [37]. Other authors observed similar trends, such as Ghalsasi et al. [38] and Suryanegara et al. [39]. From the incorporation of Ketoprofen in the composite, it can be noted increase in thermal stability of composite filament compared to PLA-ZnO composite filament. This trend can be justified by ketoprofen acting as a barrier to the rapid thermal degradation of ZnO particles and the PLA matrix, slowing their mass loss.
3.4. Differential Scanning Calorimetry (DSC)
DSC analyses were conducted to evaluate the degree of crystallinity and the glass transition and melting temperatures of the PLA and composite filaments (Fig. 5). The glass transition temperature (\({T}_{g}\)), the melting temperature (\({T}_{m}\)), the degree of crystallinity (\({X}_{c}\), %), and the melting enthalpy (\(\varDelta {H}_{m}\)) determined from the DSC results are presented in Table 4. In the literature, it is reported that pure PLA has a glass transition temperature (\({T}_{g}\)) between 55 and 65°C and a maximum melting temperature (\({T}_{m}\)) between 175 and 180°C in the purely l-isomer form. However, there is a 5°C decrease in \({T}_{m}\) for every 1% increase in d-lactate in the polymer [17]. The PLA filaments generated in this study exhibited a \({T}_{g}\) of 71°C and a \({T}_{m}\) of approximately 140°C, suggesting that there is approximately 7% d-lactide in the PLA polymer used.
Compared to the pure PLA filament, the PLA + 5%ZnO filament and the PLA + 5%ZnO + 3%KETO filament practically maintained the \({T}_{g}\) value, this phenomenon having already been observed in previous studies containing the addition of ZnO filler [17, 40]. Furthermore, as the DSC curves did not show the characteristic melting peak of ketoprofen (94°C) [41], we can state that the drug had a complete conversion to the amorphous state and complete solubilization in the matrix [42, 43].
Table 4
DSC thermal parameters, such as melting temperature (Tm), enthalpy (ΔH), and crystallinity (Xc) of PLA, PLA-ZnO5%, and PLA-ZnO5%-KETO3% filaments.
Samples | ΔHm (J.g− 1) | Xc (%) | 𝑇𝑔 (℃) | Tm1(℃) | Tm2(℃) |
Pure PLA | 2.55 | 2.74 | 68.57 | 108.98 | 141.34 |
PLA-5%ZnO | 2.28 | 2.58 | 70.51 | 110.68 | 142.69 |
PLA-5%ZnO-3%KETO | 2.19 | 2.17 | 68.81 | 108.49 | 140.79 |
Regarding the values found for \({T}_{m}\), it can be observed that the addition of 5% ZnO caused a slight increase in \({T}_{m}\), on the other hand, the addition of ketoprofen in the PLA + 5%ZnO sample caused a decrease in \({T}_{m}\) which could be explained by the Keto mix in the matrix. The ZnO and Keto content did not have a significant influence on the thermal properties of the biocomposite filaments, which shows that they have little influence on the intermolecular interactions or the flexibility of the PLA polymer chain [15].
As for crystallinity, the degrees of crystallinity of the biocomposite filaments with PLA increased compared to those of pure PLA. This result indicated that ZnO increased the crystalline portion of the matrix and, consequently, acted as a nucleating agent for the PLA chains. This phenomenon has been previously reported in the literature [15, 17].
3.5 Morphology of filaments
Figure 6 shows the fractured filament morphology obtained by SEM. Pristine PLA presented a smooth and homogeneous surface compared to composites [30]. Besides, with the addition of ZnO content, it could be noticed an increase in white spots and a higher roughness surface, as seen in Table 2 [30]. This increase in surface roughness (Ra) is associated with structural modification caused by the insertion of ZnO in the polymer matrix, which can influence the selective protein adsorption process onto the biomaterial [44]. In contrast, PLA-5%ZnO-Keto revealed a reduction in roughness and an increase of distributed pores with interlayer and inner-layer voids (represented by white arrows)[45].
Properties of scaffolds
3.6 Morphology of scaffolds
Figure 7 displays the SEM images of scaffold morphology with different magnitudes. An irregular surface can be seen on all scaffolds. The pure micrograph of PLA shows a robust structure with an intact cell wall. After incorporating ZnO (5 wt.%) into the polymer matrix, a deformed structure with broken walls was observed. However, when ZnO (5 wt.%) and Keto (3 wt. 3%) were inserted, the scaffold acquired a more robust structure, with an irregular shape and well-defined porosity, presenting less roughness (Fig. 8). Similar behavior was reported by Zarei et al. [46] when evaluating the morphology and 3D printing properties of PLA/Ti6Al4V biocomposite scaffolds, further reporting that the major challenge of the biocomposite manufacturing process is to ensure uniform distribution of the load in the matrix.
3.7. Wettability (WCA) and surface roughness
The water contact angle (WCA) measurement is a crucial and widely accepted approach for examining the wettability of polymer surfaces [47]. An important term of this analysis is the “apparent contact angle,” which refers to the average angle observed along the entire three-phase contact line of a water droplet. This angle is determined after the rapid removal of the needle from the droplet deposited on the material surface. Figure 8 shows the water contact angle and surface roughness of pristine PLA, PLA-ZnO5%, and PLA-ZnO5%-Keto3% scaffolds composites and their standard deviation.
Observing the results, the PLA-ZnO-5% composite presented the highest water contact angle when compared to the other scaffolds, but none of them can be considered hydrophobic surfaces [48]. Pristine PLA presented a WCA of 77.5º, consistent with the ranges (60 to 80 degrees) reported in previous literature [49]. These measurements can be associated with the methyl groups in the PLA chemical structure [50]. However, surface wettability depends on various parameters such as materials, building orientation, and infill density[51], and these factors may cause changes in the contact angle.
Considering the addition of ZnO in the PLA matrix, there is an observed increase of 6% in the WCA. Insoo Kim et al., 2019 also reported a similar behavior when studied PLA/ZnO bionanocomposites films. Pristine PLA films showed a WCA of 60.7º, while with the increase of ZnO in PLA matrix, the WCA rosed to 92.6º. They attributed this change to variations in surface roughness and chemical affinity of the ZnO bionanocomposties films.
The incorporation of Ketoprofen into the PLA structure led to a reduction in the water contact angle (16% compared to the PLA/ZnO composite), enhancing the material's hydrophilicity. Due to the presence of acid groups in their molecular structures, NSAIDs demonstrate a high level of hydrophilicity, resulting in significant water solubility [52]. This property can explain the notable phenomenon of contact angle reduction in the PLA/ZnO/Keto composite. While this can be advantageous, as hydrophilic surfaces generally favor cell proliferation, allowing for the reorganization of fibronectins [53], it is crucial to note that this phenomenon is also linked to cell spreading, proliferation, and differentiation [54]. However, additional analyses are necessary to determine the biocompatibility of these materials.
Furthermore, as it was mentioned, the addition of ketoprofen decreased the roughness of the filaments and scaffolds. Figure 7 showed that the increase in roughness is related to the increase in the contact angle, this effect is commonly known as the Lotus Effect [55]. Hydrophilicity is also crucial for medical applications. According to Barberi (2021), hydrophilic surfaces can promote interactions with surface proteins, thereby favoring adsorption on wettable surfaces [56].
3.9. Compression Strength
Concerning the bioengineering field, studying the compression resistance and the elastic modulus of materials is crucial, especially considering that bones typically experience this type of stress. Therefore, they present an impact on mechanical stability, durability, load bearing capacity, promotion of osteogenesis, and the direct comfort of patients with implants and scaffolds [57]. Figure 9 shows the compressive strength of pristine PLA and their composites.
Observing the graph, it is evident that there are higher values of compressive strength compared to the major literature [58]. Pushpendra Yadav et al., 2020, studied the influence of different infill densities in 3D printed specimens and found the same order of magnitude for a cubic PLA sample with 80% infill density and attributed the great result to the strong bonding between the rasters and layers. Besides, when the material was being printed, there was a short travel distance of the nozzle, which helped in maintaining the high and uniform temperature of layers. Consequently, there was an improvement in the load bearing capacity when compared to less filled layers [59].
Furthermore, compared to pristine PLA, the composites presented a slight improvement in strength, with the following values: 144.57 ± 16.4 MPa, 160.83 ± 0.95 MPa and 155.96 ± 2.32 MPa for PLA, PLA/5%ZnO and PLA/5%ZnO/3%Keto respectively. It was noted an increase of 11% from pristine PLA to PLA reinforced with ZnO. The increase in compressive strength when ZnO was added, must be related to the formation of strong interfacial bonding between PLA and Zn [60].
3.10. Antibacterial activity
The tested material did not show bactericidal activity when using the strains Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 10145). No halos were observed around the discs of the evaluated material.
3.11. Viability test
Assessment of cell viability and biocompatibility of the material showed that it did not cause cell death in the tested strain. The disc diffusion test showed that all membranes didn't have halo formation, indicating absent toxicity (Fig. 10). The formazan test results indicated no significant difference in the control (Fig. 11).
Based on the results, these materials are probably inert, presenting only action related to the material that will be introduced into the base membrane. The fact that we did not find significant differences between exposures may indicate the neutrality of this membrane, where there is an indication of biocompatibility without any change in toxicity parameters.