*2.4. Inkjet Printing*

Recently, inkjet printing technology has been used in various industrial areas, including tissue engineering and organic or inorganic electronics. This technique can rapidly and exquisitely deposit printing materials, such as polymers, metals, nano- and micro-particles, and proteins and cells with highly controlled volume and patterning of link-droplets. Consequently, polymeric biomaterials can be LbL-assembled on substrates. Akagi et al. (2012) devised a fast printing method for sc-PLA based on LbL deposition using an inkjet printer without a redundant rinsing step [44]. Each PLLA and PDLA solution was separately printed at the same point, and stereocomplex crystals were then formed with enantiomeric homocrystallites during solvent evaporation. Figure 8 shows the schematics of the inkjet printing process for the LbL formation of sc-PLA. The first LbL-assembly method, employed by the authors for formation of the stereocomplex, involved the dissolution of PLLA and PDLA in chloroform, followed by the PLLA solution being sprayed onto a glass substrate. Then, the PDLA solution was printed over the substrate after drying the PLLA droplets (Figure 8a). One cycle indicates that PLLA and PDLA solutions were printed once on the substrate in turn. In the second LbL-assembly method, PLLA and PDLA were dissolved in chloroform, and the blended solution was printed on the substrate in the first step. Subsequently, the blended solution was printed over dried mixed droplets once again in the second step (Figure 8b). This method holds potential to provide rapid fabrication, as 1 × 10<sup>5</sup> droplets were sprayed, and the processing time was approximately

100 s in each step. As shown in Figure 9a, the X-ray diffraction (XRD) patterns of PLA composites fabricated by the second method showed peaks at 12◦, 21◦, and 24◦ of 2θ degrees. This indicates that the specimens had an orthorhombic or pseudo-orthorhombic unit cell with a 103 helical conformation, indicating an α-form crystal. The peak intensities of the sc-PLA for both XRD and FTIR results were amplified by increasing the number of printing steps with no observation of any peak indicating homopolymers. Furthermore, the thicknesses of the PLA composites increased with the increasing number of steps (220, 600, and 980 nm for 2, 5, and 10 steps, respectively). As shown in Figure 9b, the XRD patterns analyzed the effect of the number of printing cycles on the structural sc-PLA fabricated by inkjet printing. The L-D LbL-1 specimen, which was fabricated with one cycle of printing, showed two peaks at 12◦ and 17◦, indicating stereocomplex crystals and α-form crystals, respectively. In contrast, homocrystallite formation was no longer observed with increasing cycle numbers for the printed specimens. The intensity of the peaks corresponding to β-form stereocomplex crystals was higher at 2θ = 12◦, 21◦, and 24◦ with an increasing cycle number, instead of the appearance of a peak at 2θ = 17◦. Consequently, the higher cycle of inkjet printing could render sc-PLA of higher purity and increase its crystallinity. The right graph in Figure 9b shows the XRD patterns of the PLA specimens prepared using PLLA/PDLA mixed solutions. All specimens prepared by inkjet printing using a PLLA and PDLA mixed solution showed no significant increase in the peak intensity despite increasing the number of printing cycles. Furthermore, it only exhibited peaks with no homocrystallites at 12◦, 21◦, and 24◦.

**Figure 8.** Schematic illustration for sc-PLA formation using the inkjet printing based on LbL deposition. (**a**) First LbL-assembly method; alternately printing of PLLA and PDLA solution onto a substrate in sequence (2 steps = 1 cycle), (**b**) Second LbL-assembly method; simultaneous printing of PLLA/PDLA mixed solution onto a substrate at one time [44], Copyright 2012. Reproduced with permission from WILEY-VCH Verlag GmbH & Co.

**Figure 9.** (**a**) XRD patterns (left) and FTIR spectra (right) of sc-PLA fabricated by inkjet printing of the second LbL-assembly method. The steps of three specimens indicate the number of repeated times of the printing. (**b**) Influence of the number of printing cycle on crystal formation of inkjet-printed sc-PLA. XRD patterns of sc-PLA product prepared by first (left) and second (right) LbL-assembly method [44], Copyright 2012. Reproduced with permission from WILEY-VCH Verlag GmbH & Co.

Akashi et al. (2014) reported an advanced method that models drugs such as an 8-mer peptide, ovalbumin (OVA), where proteins could be loaded onto the sc-PLA substrate based on inkjet printing technology [45]. The sc-PLA composites could encapsulate drugs through alternate overprinting of PLLA, PDLA, and drugs on one substrate (Figure 10a). PLLA and PDLA were dissolved in chloroform at a concentration of 0.5 mg/mL. Each polymer solution was alternately printed onto the same substrate. First, the PLLA solution was printed onto a glass substrate and dried at room temperature, and then the PDLA solution was printed onto the substrate. Finally, 0.1 mg/mL of the drug, like OVA-protein, OVA-NPs, or peptide was printed onto the printed sc-PLA composite. A single cycle for the entire inkjet-printing process comprised these three steps. This process was conducted for up to a maximum of 10 cycles. As shown in Figure 10b, 25% of the peptide in the sc-PLA carrier was released in the initial 1 h, and 50% of the peptide was released after 1 day. Then, 90% of the peptide was released for 5 days via diffusion. In contrast, 40% of ovalbumin in the sc-PLA carrier was released for 1 day, and the remainder of the loaded protein failed to be released. This was assumed to be due to the aggregation of ovalbumin proteins in the sc-PLA matrix. In contrast, the sc-PLA with OVA-NPs group, which was printed by ovalbumin encapsulated nanoparticles (OVA-NPs), released 30% of the protein for the initial 1 day, and subsequently the remainder of the contents could be sustainably released during the following 30 days. It was considered that denaturation and aggregation of protein could occur to a lesser extent in the sc-PLA composite, because encapsulation of ovalbumin in the nanoparticle could offer resistance to protein denaturation and stability to the carrier. As shown in Figure 10c, the in vitro release profile of sc-PLA with ovalbumin encapsulated nanoparticles and homopolymer (PLLA and PDLA) with ovalbumin

encapsulated nanoparticles was analyzed. All groups exhibited burst release kinetics of the protein for 24 h, and their release rates gradually decreased. The sc-PLA composite group showed a lower cumulative release of ovalbumin than the homopolymers after 24 h (28 vs. 35 vs. 41% for sc-PLA-OVA-NPs, PLLA-OVA-NPs, and PDLA-OVA-NPs, respectively). Furthermore, the sc-PLA-OVA-NPs had a lower release rate with sustainable release compared to the other groups. It was supposed that sc-PLA had superior hydrolysis resistance and carrier stability owing to its higher crystallinity compared to homopolymers.

**Figure 10.** (**a**) Schematic illustration for sc-PLA with drugs composites fabricated by inkjet printing based on LbL-assembly. (**b**) Drug release profiles of the sc-PLA with drug (peptide or OVA) composites in phosphate-buffered saline (PBS). The data were plotted as mean values ± SD (*n* = 3). (**c**) OVA release behaviors from sc-PLA-OVA-NPs (named PLAs-OVA-NPs) and PLLA or PDLA-OVA-NPs in PBS. Results were plotted as mean values ± SD (*n* = 3) [45], Copyright 2014. Reproduced with permission from the American Chemical Society.

Recently, Akashi et al. (2017) reported that sc-PLA composites could be conjugated to benzyl alcohol and 3,4-diacetoxycinnamic acid (DACA) of bio-based aromatic compounds at the hydroxyl groups of both terminals using inkjet printing [46]. This DACA conjugation enhanced the thermal stability of sc-PLA by increasing the thermal decomposition temperature by 10% to above 90 ◦C. Consequently, inkjet printing can be considered as an innovative technology for fabricating sc-PLA scaffolds rapidly and easily based on LbL deposition and assembly. Furthermore, it has the advantage of versatile control of the shape, thickness, and amount of printed scaffolds composed of sc-PLA. Therefore, this technology has the potential to bring innovation to some fields, such as tissue engineering and biomaterials, if it can be converged with 3D printing for freely customized fabrication of scaffolds and substrates.
