The fused deposition modeling is the method of 3D printing, in which a continuous filament of material is heated in the nozzle to reach a semi-liquid state and then extruded on the platform (first layer) or on the top of the previously printed layers [
26]. The filament has to be thermoplastic because it allows combining subsequent layers of print and solidifying the entire object after reaching room temperature. Using bulk modification, such as copolymerization with other lactone-type monomers, PEG, monomers with functional groups, and blending with other materials, the degradation rates, hydrophilicity, mechanical properties, and surface properties of PLA-type polymers can be significantly changed [
27]. Therefore, to preserve the thermoplastic properties of the mixtures of PLA with chitosan, a maximum of 10% (
w/w) of the latter was used.
3.1. Structure of Filaments
The co-extrusion of PLA and chitosan was successful, and it was possible to obtain a filament with a diameter of 1.75 ± 0.03 mm. The results of the filament density measurements are shown in
Table 2. The density range of PLA and its derivatives, such as PLLA and PDLA, has been reported as 1.21–1.25 g/mL, 1.24–1.30 g/mL, and 1.25–1.27 g/mL for the amorphous and crystalline form of polymer, respectively [
28]. The density consistent with the literature data was obtained only for PLA and PLA-CHI
3 samples. The 3% (
w/w) share of chitosan (with a density of 0.35 g/mL) in the filament did not significantly affect its density. However, the 10% (
w/w) share of chitosan caused a decrease in the density of PLA and sPLA filament by 45% and 25%, respectively.
Values obtained for PLA and sPLA materials marked with the same letters do not differ significantly.
All samples of the sPLA filaments had a higher density than the corresponding PLA filament samples. This may be due to the higher degree of crystallinity of the lactic acid polymer in the sPLA granulate. Moreover, the presence of the high molecular weight plasticizer could provide sPLA filaments greater flexibility. A similar dependence on lowering the filament density with the increase in the contribution of the modifying additive was presented by Daver and colleagues [
29]. They created a cork-PLA filament for printing using the FDM technique, which included a 5% (
w/w) of the cork. The 3D printed composite showed slightly lower tensile mechanical properties, except elongation at break where 3D printed composite was more ductile compared to that of compression molded composites.
Figure 1 shows FT-IR spectra of PLA, PLA-CHI
3, and PLA-CHI
10 filaments. The stack spectra showed that the chitosan additive did not have a significant impact on the structure of PLA (
Figure 1A). In the range of 3100 to 2800 cm
−1 in the PLA spectrum, the characteristic stretching frequencies for –CH
3 asymmetric and –CH
3 symmetric vibrations at 2995 and 2944 cm
−1, respectively, were present (
Figure 1B). The asymmetric and symmetric bending frequencies for the same group have been identified at 1452 and 1382 cm
−1, respectively (
Figure 1C) [
30]. The peaks located at 2925 and 2852 cm
−1 showed the –CH– asymmetric and the –CH– symmetric stretching vibrations, respectively (
Figure 1B) [
31]. In
Figure 1C, two peaks at 1747 and 1360 cm
−1 corresponding to the stretching vibration of the ester carbonyl group and –CH group were also identified. The second one used to be sharper for PLA
LMW than for PLA
HMW due to the differences in chain conformation arranges in each polymer. It is one of the markers confirming that the high molecular weight PLA was used during our studies. The next marker of the PLA molecular mass is the band of carbonyl ester group appears at 1266 cm
−1, which is stronger for PLA
HMW compared to PLA
LMW due to the difference in the number of ester links [
32]. The next band at 1181cm
−1 was ascribed to –C–O– stretching bond vibration in the –C–OH– group of PLA. The specific region composed of three characteristic peaks, ascribed to –C–O– stretching vibration in –O–C=O group, was identified at 1128, 1080, and 1043 cm
−1, respectively [
33].
Figure 1E shows the chitosan (CHI) spectrum, which demonstrated a broad band in the range of 3600 to 2700 cm
−1, attributed to
and
vibration (3324 cm
−1), and stretching vibrations of –CH
2– group at 2874 cm
−1. The peak at 1654, cm
−1 corresponds to the amide I band. The second derivative procedure of chitosan spectrum in work of Staroszczyk and colleagues [
34] showed that the signal around 1590 cm
−1 can be related with the
vibration of the free –NH
2 group. Next two peaks at 1416 and 1375 cm
−1 come from the –CH
2– group’s vibration area, asymmetrical bending and stretching, respectively. The amide III band located at 1319 cm
−1 corresponds mainly to the stretching C–N and bending N–H vibration. The saccharide region of the spectrum includes the asymmetric stretching vibration of the C–O–C bridge at 1150 cm
−1, the skeletal vibrations involving the C–O–C stretching band at 1057 and 1023 cm
−1 and the last peak in the saccharide region of the spectrum at 892 cm
−1 corresponding to the
vibration of βthe -glycosidic bond [
35].
The addition of chitosan to the filaments slightly weakened the intensity of the bands in the range of wave numbers 1800 to 1000 cm
−1 and increased the intensity of the bands in the range of wave numbers 3000 to 2800 cm
−1. However, the differential spectra of PLA and chitosan (PLA
d, CHI
d) present in
Figure 1F–H confirmed that co-extrusion of these two components did not cause the disappearance or appearance of new bands. This means that the components of the mixture did not react with each other and formed only a physical mixture. Small differences in the intensity of the spectra and shifts of no more than 5cm
−1 indicate conformation changes in the chains of both polymers, related to their most energetically advantageous way of arranging them in a two-component filament.
Figure 2 shows FT-IR spectra of sPLA, sPLA-CHI
3, and sPLA-CHI
10 filaments. The stack spectra indicate a clear difference in the range of wave numbers 3100 to 2800 cm
−1 (
Figure 2A). In
Figure 2B, the wide band in the range of wave numbers 3500 to 3100 cm
−1 can be observed, whose intensity decreased with increasing concentration of chitosan in the filament. Such band did not appear in the PLA spectra (
Figure 1), which may indicate the stretch vibrations of the –OH groups of the plasticizer present in the sPLA granulate, for example, amorphous vinyl acetate units comes from EVA polymer [
36]. The addition of chitosan also caused the reduction in the intensity of the bands corresponding to C–H stretching vibrations of –CH
3 (2955 cm
−1) and –CH– groups (2916 cm
−1, 2850 cm
−1). The occurrence of the C=O stretching band of the carbonyl group in sPLA at 1712 cm
−1 (
Figure 2C), the wave number of approximately 40 cm
−1 lower than for the PLA may indicate the vibration in sPLA higher number of groups ester carbonyl group [
37]. The irregular band at the 1411 cm
−1 wave number may be the result of an overlap of the three bands of PLA: 1452, 1382, and 1366 cm
−1 (
Figure 1C) and absorption peak attributed to the contributions from both VA and ethylene (–CH
2–) units [
37]. The peak at 1268 and 1251 cm
−1 is assigned to –C=O bending from PLA and plasticizer, respectively (
Figure 2C) [
36]. The spectra also showed the characteristic bands ascribed to –C–O– stretching vibration in –O–C=O group at the 1200–1000 cm
−1 region (
Figure 1D). The band at 1016 cm
−1 can correspond to the symmetric stretching vibration of the C–O–C band of EVA [
37]. The addition of chitosan in filaments weakened the intensity of the bands in the whole measuring range (
Figure 2B–D). The differential spectra of sPLA and chitosan (sPLA
d, CHI
d) present in
Figure 2F–H, confirmed that co-extrusion of these two components did not cause the disappearance or appearance of new bands. Therefore, it can be concluded that sPLA, in spite of the presence of a plasticizing compound, also formed only physical mixture during co-extrusion with chitosan.
The change in the density of materials doped with chitosan has also been confirmed based on images and SEM micrographs (
Figure 3).
The PLA and sPLA filaments were homogeneous and had smooth surfaces. The 3% addition of chitosan did not cause a significant difference in the microstructure of these samples. In turn, the PLA-CHI10 filament was porous and characterized by an irregular, rough surface. Thus, its density was the smallest. A similar pattern was observed for sPLA, sPLA-CHI3, sPLA-CHI10 samples. The BSE analysis of sPLA filaments showed the presence of a homogeneously dispersed plasticizer or pigment, providing a blue color of granulate, also confirmed by FT-IR studies.
3.2. Mechanical Properties
Due to the inability to use filaments containing a 10% chitosan additive (PLA-CHI
10 and sPLA-CHI
10) in the FDM printing process, the results regarding the effect of chitosan additive on mechanical properties were determined by directly testing the filaments and not printed therefrom the 3D objects. From the tensile testing result data, mechanical property information was extracted by plotting the stress-strain curves.
Table 3 demonstrates the list of average diameters, tensile strength, elongation at break, and Young’s modulus. Co-extrusion of PLA or sPLA with chitosan allowed obtaining filaments of the same diameter. The PLA filament was characterized by tensile strength and modulus of elasticity, similar to those demonstrated by other authors [
38,
39]. The 3% (
w/w) addition of chitosan in the PLA-CHI
3 filament did not change the mechanical properties apart from the elongation at break, which was 80% lower compared to the PLA. The 10% (
w/w) addition of chitosan in PLA-CHI
10 sample significantly degraded filament properties, for which tensile stress, elongation, and modulus of elasticity were 58%, 91%, and 28% lower than for PLA, respectively. The sPLA was characterized by significantly higher elongation at break, lower tensile strength, and lower Young’s modulus compared to the PLA sample. The 3% (
w/w) addition of chitosan in the sPLA-CHI
3 sample resulted in 51% and 96% reduction of tensile strength and elongation at break, respectively. Increasing the addition of chitosan up to 10% (
w/w) in the sPLA sample further deteriorated the mechanical properties of the filaments. It also caused a decrease in the value of Young’s modulus. For the PLA and the sPLA filaments, with increasing chitosan content, a decrease in tensile strength, elongation at break, and Young’s modulus were observed. Moreover, the relationship between decreasing Young’s modulus with a decreasing density of materials has been presented by Vanleene and colleagues [
40]. Based on Young’s modulus–density and Young’s modulus–tensile strength charts available in Material Data Book [
41], all obtained sPLA filaments can be classified as elastomeric materials, while almost all obtained PLA filaments showed properties characteristic a of thermoplastic polymer. The exception was the PLA-CHI
10, which based on Young’s modulus–density chart, was classified as a natural material with properties similar to the typical wood. Wu et al. in their work recorded that tensile strength of neat PLA (44.3 MPa) decreased to 42.6 MPa following grafting with maleic anhydride (MA), and the tensile strength of PLA/chitosan composites decreased markedly with increasing chitosan content attributed to the poor dispersion of chitosan in the PLA matrix. However, the PLA/chitosan composites exhibited an increase in tensile strength at failure as the filler of the chitosan content reached saturation at content exceeding 10 wt %. This behavior was attributed to improved dispersion and covalent bonding of the chitosan in the PLA-g-MA matrix resulting from the formation of branched or cross-linked macromolecules. The tensile strength of the PLA-g-MA/chitosan composites was ca. 7–30 MPa greater than that of the PLA/chitosan composites [
24].
3.3. Thermal Properties
Differential scanning calorimetry (DSC) measures the amount of heat energy absorbed or released when a material is heated or cooled. For polymeric materials, which undergo important property changes near thermal transition, the DSC is a very useful technique to study the glass transition temperature, crystallization temperature, and melting behavior. The DSC curves of the neat PLA, neat sPLA, and their composites with chitosan are presented in
Figure 4, and determined thermal characteristics are given in
Table 4. All characteristic temperatures of PLA samples (
Tg,
Tc,
Tm) were similar to those obtained for thermal treated PLA in Carrasco and colleagues’ work [
42]. The neat PLA filament showed two small endothermic peaks. The first was the glass-transition temperature (
Tg) at 63.9 °C, and the second was melting temperature (
Tm) at 154.5 °C, respectively. The addition of chitosan into the PLA polymer led to a minor decrease in these two temperatures. This is because the chitosan increases the free volume and flexibility of polymeric chains [
43]. The addition of chitosan to the filament improved its ability to crystallize. Moreover, the crystallization temperature of the sPLA-CHI
3 and the sPLA-CHI
10 filaments were lower than for the neat sPLA sample (
Table 4), which indicates that the chitosan can act as a nucleating agent promoting crystallization.
The addition of chitosan in both types of PLA samples resulted in the lowering of the melting temperature. The obtained filaments were a mixture of the amorphous and crystalline phases. After melting, the two phases mix to one phase, which after cooling, crystallizes (without de-mixing), forming one “impure” solid phase. Mucha and Królikowski [
44] found that an organic filler, such as chitosan powder, forms the amorphous inclusion in the composites. On reheating, this single phase can melt at a lower temperature because of the adsorbed “impurities” of the former amorph phase, which causes a melting point depression. The changes in the crystallization and melting temperatures associated with the presence of chitosan in the filaments would not significantly affect the printing conditions, since the largest temperature differences recorded between the sPLA and sPLA-CHI
10 samples for
Tg,
Tc,
Tm were 2.3, 4.8, and 3.5 °C, respectively.
3.4. Antimicrobial Properties
To evaluate the antimicrobial properties of the filaments the ASTM: E2149 method was used, which is designed to measure the antimicrobial activity of non-leaching (non-water soluble) antimicrobials surfaces made of plastic, rubber, silicone, and treated fabric material and is one method to test an irregularly shaped antimicrobial object, such as a thread, powder, 3D molded plastic. The results presented in
Table 5 and
Table 6 indicate that the control materials (PLA and sPLA) did not reduce the number of
E. coli and
S. aureus.
In these samples, even an increase in the number of microorganisms was noted. The obtained results seem to be correct because PLA does not show antimicrobial activity. The only effect of limiting the growth of microorganisms by pure PLA has been demonstrated in a coating experiment [
45]. However, it was related to the poor oxygen permeability of the PLA film [
38,
46]. With the increase in the concentration of chitosan in the filament, the average reduction in the number of
S. aureus bacteria increased. The highest value was achieved for PLA filament with a 10% addition of chitosan (
Table 5). A similar pattern was observed regarding
E. coli bacteria (
Table 6). It means that a greater antimicrobial effect of the filaments was obtained with respect to the Gram-positive strain. The 3D prints showed virtually the same antimicrobial properties as the filaments from which they were printed. It applies to both tested bacteria strains. This means that re-melting the PLA-chitosan material has no significant effect on the inhibition of the material’s antimicrobial activity. The antimicrobial activity was due to the presence of chitosan and depends on the degree of its deacetylation, molecular weight, concentration in solution, pH, and ionic strength of the solution [
14]. According to Goi and colleagues, there is a lack of conclusive data on whether the chitosan has higher activity on Gram-positive or on Gram-negative bacteria [
47]. On both strains, chitosan seems to act differently, though in both cases satisfactorily. The main reason for the antimicrobial activity of chitosan is the interaction of its positive charged chains with anionic components of microorganisms—lipopolysaccharides (Gram-negative bacteria) and teichoic acids (Gram-positive bacteria) that cause bacteria cell lysis. However, this mechanism takes place when the amino groups of the polymer are protonated: pH < 6 [
48]. It has been concluded that for neutral or alkaline media, the cationic nature of chitosan can no longer explain its antibacterial activity. In this case, the strong coordination capability of –NH
2 groups in the chitosan chain might be one possible mechanism [
49,
50]. Considering the test conditions and comparing them with the conditions in other, most frequently used standards [
51,
52], it can be concluded that the addition of chitosan allows obtaining at least bacteriostatic filaments. For comparison, antimicrobial activity tests performed for the
E. coli strain in Wu et al. studies showed the PLA/chitosan or PLA-g-MA/chitosan suppressed the growth of
E. coli; furthermore, all of the samples containing PLA-g-MA/CS exhibited a higher degree of bacterial suppression than the corresponding samples of PLA/CS. The antibacterial effect on bacteria was enhanced in PLA-g-MA/CS because the PLA-g-MA is stabilized in a fixed orientation by CS, resulting in bacteria death due to increasing effective concentration of CS to bacteria or contacting time elongation to bacteria [
24].