1. Introduction
Lactic acid (2-hydroxy propionic acid) is a chiral molecule consisting of two enantiomers. As a consequence, different stereoisomeric forms of poly(lactic acid) (PLA) are available, including poly(
l-lactic acid) (PLLA), poly(
d-lactic acid) (PDLA), poly(
d,
l-lactic acid) (PDLLA), and poly(
l-lactic-co-
d,
l-lactic acid) (PLDLLA) (
Figure 1) [
1]. High molecular weight polymer batches are synthesized on an industrial scale through ring-opening polymerization of lactide, the cyclic dimer of lactic acid [
2]. For this reason, commercial PLA is often referred to as poly(lactide). PLA is generally considered an environmentally friendly polymer because lactic acid is produced by microbial fermentation, typically in the L-form, and the resulting macromolecule, in turn, degrades back to lactic acid [
3].
PLLA is a semicrystalline, thermoplastic polymer that undergoes a glass transition above room temperature (glass transition temperature, T
g ≅ 55–70 °C) [
4]. As a consequence, and considering its hydrophobic nature, PLLA behaves as a brittle material when employed under physiological conditions (T = 37 °C, pH = 7.4). The crystallinity degree of PDLLA is decreased by increasing the D-isomer content until a complete amorphous morphology is obtained for molar percentages higher than 10–20%. As a consequence, PDLLA typically shows a lower T
g in the range of 50–60 °C, higher flexibility, and faster biodegradation than PLLA [
5]. PLA is degraded in the human body mainly through nonspecific hydrolytic scission of ester bonds into oligomers and monomeric acids, which are metabolized through the tricarboxylic acid cycle and then excreted from the body in the form of carbon dioxide and water [
6]. While the complete in vivo resorption of PLLA devices takes years [
7,
8], amorphous PDLLA can be completely resorbed by 12 months after implantation [
9].
Thanks to their biocompatibility, suitable mechanical properties, and processing versatility, semicrystalline PLLA and PDLLA have been widely investigated in the biomedical field resulting in a range of biodegradable products currently available in the market for clinical use, such as suture reinforcements and anchors, meniscal darts, devices for osteosynthesis, and orthopedic fixation [
10,
11]. In addition, the relatively low T
g of PLLA is exploited for intravascular stent implantation procedures based on balloon expansion [
12]. A considerable amount of literature has also been published on amorphous PDLLA to exploit its faster biodegradation for the controlled release of bioactive molecules [
13].
PLLA and PDLLA are widely employed in additive manufacturing (AM) techniques involving processing polymers as a melt or solution [
14], such as material extrusion (MEX) based on melt or solution processing, powder bed fusion, and binder jetting [
15]. MEX of PLA was first described in 2001 in an article focused on a pneumatic system for polymeric grains melt processing [
16]. After that, a number of articles have demonstrated the possibility of applying technologies commercially, referred to as fused deposition modeling or fused filament fabrication, to process PLLA filaments also loaded with natural fibers or inorganic fillers [
17,
18]. For instance, the successful fabrication and processing of PLA filaments loaded with hydroxyapatite and chitosan [
19] or cellulose nanofibrils [
20] were recently demonstrated. Several PLA products in the form of filament tailored to MEX are currently available on the market and supplied with optimized processing protocols. However, in most cases, the composition and stereoisomeric form of the supplied PLA batch is not specified. This is a critical aspect considering the great interest of the scientific community in AM of PLA for bone regeneration [
21,
22] and breast reconstruction [
23], as well as for the production of custom-made oral dosage pharmaceutical forms or implantable drug-releasing systems [
24]. Indeed, besides the obvious relationship between polymeric material composition and its biocompatibility, polymer crystallinity degree and molecular weight significantly affect the biodegradation rate of the resulting scaffold, as well as other properties, such as the mechanical behaviour [
25,
26].
The aim of this study was the design, fabrication, and characterization of customized PLA samples tailored to biomedical applications by means of a novel AM apparatus suitable for the employment of different techniques, i.e., solution- and melt-based MEX approaches, possibly integrated with electrospinning. As described in a recent article [
27], this AM machine was already employed to fabricate tissue engineering scaffolds made of microbial polyester-based blends by means of computer-aided wet-spinning, a processing technique involving the extrusion and layer-by-layer deposition of a polymeric solution or suspension directly into a coagulation bath [
28]. The present article deals with testing the melt-extrusion configuration of the machine through a computer-aided design (CAD) and manufacturing (CAM) process, enabling the fabrication of anatomically-shaped and clinically-sized physical models made of a commercial PLA (Sigma Aldrich), as well as relevant porous scaffolds for tissue regeneration. Additively manufactured PLA samples with a dog-bone shape tailored to tensile mechanical test, as well as a tuneable porous structure, were also fabricated and characterized by scanning electron microscopy (SEM) and uniaxial mechanical test. The fabricated samples were also characterized by proton nuclear magnetic resonance (
1H-NMR), size exclusion chromatography (SEC), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) to investigate the effect of melt processing on polymer molecular structure, as well as the stereoisomeric form of the employed PLA.
2. Materials and Methods
2.1. Materials
PLA (3D Printing filament; diameter: 1.75 mm; density: 1.24 g/cm3; tensile modulus: 65.5 MPa; vicat point: 55–60 °C; melting point: 144 °C), chloroform (CHCl3) for HPLC and deuterated chloroform (CDCl3) were bought from Sigma Aldrich (Milan, Italy) and used as received.
2.2. Additive Manufacturing
Samples were fabricated by means of a multifunctional AM machine prototype (Fabrica Machinale S.r.l, Pisa, Italy) designed to process polymeric materials through either melt- or solution-based MEX by changing the operating head (
Figure 2). As previously described in detail, the machine is equipped with six carbon bars terminating with magnetic spheres that are connected to the extrusion head, enabling its easy manual substitution.
Samples fabrication was carried out by employing optimized processing parameters in terms of infill angle, infill density, layer height (dz), infill speed, extruder T, and bed T. Anatomical and dog-bone shape samples were fabricated by processing customized digital models and G-code files. After printing, the samples were collected and stored in a desiccator before characterization.
2.3. Scanning Electron Microscopy (SEM)
Samples were analyzed by means of SEM (JEOL LSM 5600LV, Tokyo, Japan) after platinum sputter coating. Micrographs of sample top-view and cross-section were acquired at different magnifications. The arithmetical mean roughness (Ra) of the polymer matrix surface was estimated by processing top-view micrographs with Image J software (SurfCharJ_1q plugin).
2.4. Proton Nuclear Magnetic Resonance (1H-NMR)
1H-NMR spectroscopy was performed on a JEOL YH400 MHz spectrometer (JEOL Ltd., Arkishima, Tokyo, Japan). The spectra were recorded on a 1.6% w/v polymer solution in CDCl3 at 25 °C.
2.5. Size Exclusion Chromatography (SEC)
SEC analysis was carried out employing a liquid chromatograph PU-2089Plus (Jasco, Milan, Italy) with two columns PL gel 5_1 Mixed-D and a refractive index detector PI-2031 (Jasco, Lecco, Italy). Samples were dissolved in CHCl3 (0.5% w/v) and eluted in CHCl3 at a flow rate of 1 mL min−1. Number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity index (PI) were obtained. The instrument was calibrated by using polystyrene standard samples (range 0.5–300 kDa).
2.6. Thermogravimetric Analysis (TGA)
TGA was carried out using a Q500 instrument (TA Instruments, Milan, Italy) in the temperature range of 30–700 °C, at a heating rate of 10 °C min−1, and under a nitrogen flow of 60 mL min−1. The temperature corresponding to the maximum value of the peak on TGA first derivative thermograms (Tmax), as well as the temperature at which the sample starts to degrade (Tonset), were recorded.
2.7. Differential Scanning Calorimetry (DSC)
DSC analysis was carried out using a Mettler DSC-822 instrument (Mettler Toledo, Milan, Italy) by employing a heating and cooling rate of 10 °C min
−1 in the temperature range of 25 to 200 °C, under a nitrogen flow rate of 80 mL min
−1. The glass transition temperature (T
g) was determined at the inflection point, and the melting temperature (
Tm) as the minimum of the endothermic peak in the first and the second heating cycles. The weight fraction of the
d-lactide repeating unit in the macromolecular chain was calculated according to the following equation [
29]:
where
w is the fraction of meso-lactide repeating unit (i.e., an
l-lactic acid and an
d-lactic acid sequence) in the macromolecular structure.
2.8. Mechanical Characterization
Uniaxial tensile properties of dog-bone-shaped samples were evaluated at room temperature with an Instron 5564 instrument (Norwood, Instron, MA) by following ASTM standards D 1708-93 [
30] and D 882-91 [
31]. Five replicates for each kind of printed sample (38 × 15 mm
2 overall size, 5 × 22 mm
2 in the gage area; average thickness smaller than 1 mm and measured with a micrometer) were stretched to the breaking point at room temperature and ambient humidity, under a constant crosshead displacement of 4 mm min
−1. Tensile stress-strain curves, elastic modulus, and stress and strain at break were obtained from software recording data (Merlin).
2.9. Statistical Analysis
Data are reported as mean ± standard deviation. Data were processed by two-way analysis of variance (ANOVA) and Tukey test for post hoc analysis. Differences were considered significant for a p-value < 0.05.