**Table 2.** *Cont.*


**Table 2.** *Cont.*

(10,000–34,500)

modulus E (from 483 to 830 MPa); Decrease of degradation rate.

## *Molecules* **2020**, *25*, 4919



concentration; d visible light; e two-photon polymerization technique (2PP); f Mn of the polycondensation product; PPG—poly(propylene glycol); PTMG—poly(tretramethylene glycol); TMC – 1,3-methylene carbonate (1,3-dioxan-2-one); TEGDMA—triethylene glycol dimethacrylate; MC—5-methyl-5-cinnamoyloxymethyl-1,3-dioxan-2-one; GMA—glycidyl methacrylate; NA—data not available.

#### **4. PLA-Based Materials by Photo- and High Energy Radiation Crosslinking**

As mentioned in the previous sections, electron beam and gamma irradiations as well as UV light were predominantly used for crosslinking of PLA-based materials designed for biomedicine. However, in most articles concerning crosslinking by ionizing radiation only changes in properties of polylactide (co)polymers upon exposure to high energy radiation are discussed without indication of their specific medical application. On the other hand, some authors presented examples of daily use items prepared from gamma or electron beam irradiated PLAs.

Mitomo, who studied PLA crosslinking by electron beam irradiation for many years (the results are presented in Table 1 in Section 2) [29–33] demonstrated how irradiation improved the thermal stability of cups and plates prepared from poly(L-lactide) and how it enabled the preparation of heat-shrinkable tube which can be used as a cover for electric wire [31]. The tube was prepared by extrusion of PLLA blended with 3 wt % of TAIC at 180 ◦C and then irradiated at 50 kGy. The irradiated tube expanded two times at 180 ◦C and its shape was kept at room temperature. After that, the expanded tube shrunk up to the original size by re-heating, thus could bundle wire by heat shrinking. The result is shown in Figure 7A. In another attempt, PLLA with 3 wt % of TAIC was molded to cup and plate by the extruder and irradiated to form a crosslinked structure at 50 kGy. Boiling water was poured into unirradiated and irradiated cups. The unirradiated cup deformed and changed to milky-like transparency, but the crosslinked cup kept its original shape and transparency due to protection from the crystallization of crosslinked structure (see Figure 7B).

**Figure 7.** PLLA crosslinked by electron beam irradiation (50 kGy). ( **A**) shrinkable tube (**a**); possible use (**b**). (**B**) Appearance of cups after using for hot water: (**a**) the unirradiated product, (**b**) the product crosslinked by irradiation [31]. (Adapted with permission from Elsevier, 2005).

As polylactide is also considered as an insulating material for di fferent electronic applications, the influence of electron beam irradiation on electrical insulating properties of PLA has been studied [99]. Thus, to have better material for electric wire sheaths, a soft-resin as a plasticizer was added to polylactide but then the electric breakdown strength (EB) decreased. To keep the EB at the same level as that of neat PLA, the composite was irradiated by the electron beam at the dose 100 kGy what resulted in the PLA crosslinking.

Examples of the use of crosslinked by irradiation polylactide in the production of packaging can be found in both the scientific literature and patents [42,100–103].

Thus, new films based on PLA and montmorillonite with improved barrier and mechanical properties have been developed [42]. These were designed for use with foods being processed with electron beam technology for a shelf-life extension, phytosanitary treatment, and pathogen elimination. Only low radiation doses were applied, i.e., nanocomposite films were prepared at 1, 3, and 5 wt % of clay and exposed to target electron beam doses of 1 and 10 kGy. It was observed that PLA properties were influenced by the addition of clay and by electron beam irradiation treatment: the samples showed some surface irregularities, increases of Tg and Young modulus, and a decrease of oxygen permeability. This limited permeability was attributed to the presence of clay and crosslinks in PLA material.

The possible application of polylactide for the production of packaging made from PLA combined with another material, e.g., cardboard is reflected in some cited in this review patents [100–103]. PLA crosslinked by gamma rays or electron beam may also find the application as adhesives in conventional glue guns [104].

Contrary to attempts of PLA crosslinking by high energy radiation, PLA photo-crosslinking has been performed on the purpose of obtaining materials for biomedicine. Among them, elastic and sti ff sca ffolds were prepared as well as gels which could be applied as drug delivery systems.

Amsden and his group worked for a long time on biodegradable elastomers for biomedical applications, among them polylactide-based materials make up the majority. These materials were prepared predominantly from acrylated star PLLA-PCL but also from star PLLA-poly(trimethylene carbonate) copolymers which were crosslinked by UV light. Because the main purpose of the biodegradable elastomers synthesis was their application as implants capable of releasing biologically active compounds, most studies included the preparation of elastomeric devices containing a variety of proteins (growth factors, interferons), corticosteroids, peptides [61,64–66,88–90,93,98,105]. These devices were prepared by embedding the drug (together with accompanying/solubilizing compounds) in prepolymers in bulk or solution (containing photoinitiator) in a form and irradiation with UV light. Studies included the investigation of active compounds release.

Another group of works concerned the preparation of elastic porous sca ffolds which could be applied for the cell culture but also for the release of biological compounds [62,63,91,95–97]. Initially fabricated porous sca ffolds (including those by Amsden group) were prepared using porogens, e.g., para ffin beads or water, for the pores generation. Among these works, the preparation of porous sca ffold from 3-arm acrylated PLA/ε-CL copolymer with dual porosity (due to the introduction of two porogens, i.e., para ffin beads and water) and pore interconnectivity is situated [91] (see Figure 8**).**

As another interesting example, the preparation of the combined system, i.e., elastomeric sca ffold with the mechanical strength and a hydrophilic cell encapsulating hydrogel which formed a bi-continuous two-phase cell delivery device for the repair and/or replacement of load-bearing soft tissues can be presented [95]. Thus, an elastomer from a star-poly(ε-CL-co-<sup>d</sup>,<sup>l</sup>-lactide) triacrylate (CL:DL-LA = 0.5:0.5, M w = 4000 and 8000) and an N-methacrylate glycol chitosan (MGC) hydrogel to distribute the cells from bovine articular cartilage and enable cells growth has been prepared. Functionalized chitosan containing cell culture was mechanically mixed with functionalized PCL-PDLA copolymer and after the addition of photoinitiator, this material was cured in the appropriate form, using UV light. The obtained sca ffolds of bi-continuous morphology had mechanical properties resembling those of soft tissues. Cell culture experiments conducted with the selected sca ffold demonstrated that the chondrocytes remained viable throughout the entire manufacturing process and were able to proliferate. The authors claimed the feasibility of the sca ffolds as an injectable and in situ crosslinkable cell delivery system.

Many articles concern precisely designed porous tissue sca ffolds prepared using stereolithography. Stereolithography is an additive fabrication process that uses a liquid light-curable photopolymer and a laser to create three-dimensional (3-D) structures [106]. Thus, porous PLA scaffolds with gyroid morphology have been fabricated using stereolithography, by visible light crosslinking of PLA macromers [57]. Complex structures could be built by illuminating sequential layers of a polymerizable resin using digital pixel masks or arrays of mirrors. In stereolithography, the thickness of a solidified layer is controlled by the light irradiation dose. It was possible to form relatively large structures (up to 42 × 33 × 200 mm) at high resolutions. The size of the smallest features that can be built was determined by the size of the light pixels (32 × 32 μm in the x and y directions), the layer thickness (25 μm), and the over cure. Although cell seeding of porous structures prepared from hydrophobic polymers, such as PDLLA is difficult, the very open structure of the gyroid architecture facilitated the penetration of water into PDLLA scaffolds prepared by stereolithography and enabled the cell seeding of mouse pre-osteoblasts. The achieved results are shown in Figure 9.

**Figure 9.** Images of PDLLA network scaffolds with a gyroid architecture prepared by stereolithography: (**A**) photograph, (**B**) microcomputed tomography (μCT) visualization and (**C**) SEM image. In (**D**) a light microscopy image is shown for a scaffold seeded with mouse pre-osteoblasts after 1 d of culturing. Scale bars represent 500 μm [57]. (With permission from Elsevier, 2009).

Photolithography was also applied for the network synthesized by a different approach [107]. PDLA network was prepared by a thiol-yne photo click reaction where alkyne functionalized star-shaped and linear PLAs were coupled with tetrafunctional thiols. Crosslinking was performed by UV irradiation of prepared polymer films. Amorphous crosslinked polymers were stable when hydrolyzed—no significant weight loss was observed during the first 10 weeks (around 4%). Films prepared by the casting of solutions containing functionalized PLA, tetrathiol and photoinitiator were also crosslinked using the direct laser writing (DLW) technique which enabled the preparation of structured patterns. Patterned samples were prepared by moving the thin film of the photopolymerizable material within the focal plane using a computer-controlled XY translation stage. Photopolymerization selectively took place in the exposed areas leaving the non-exposed material unreacted. This unexposed material was subsequently etched away using acetone as a solvent. The results are shown in Figure 10. Patterned

films were used for the cell culture and independent experiments concerning cell viability indicated that studied materials based on crosslinked PLA were not toxic. The presented study showed that advanced photolithographic techniques allowed the microfabrication of well-defined micrometer-scale structures for cell patterning.

**Figure 10.** Topography images of crossing lines generated by direct laser writing using a formulation comprising the macromonomer: (**a**) linear-YNE and (**b**) star-YNE PLAs, both with a stoichiometric amount of the thiol (stoichiometry alkyne/thiol 1:2) and 3 wt % of photoinitiator. Images were obtained using a confocal microscope [107]. (Adapted with permission from Elsevier, 2017).

Micro-stereolithography has been also used for PLA composites. For example, a well-defined three-dimensional 3D pore network has been prepared starting from composite PDLLA/nanosized hydroxyapatite (HAP) [108]. The authors dispersed nano-HAP powder in a photo-curable PDLLA macromer in N-methyl pyrrolidone (as not reactive diluent) and after the addition of photoinitiator and some additives (inhibitor and dye improving the depth of light penetration), the composition was used to fabricate porous structure in a standard stereolithography apparatus. Subsequent layers were cured a dozen times by UV irradiation. As a result, a Schwarz pore network containing 5 wt % of nano-HAP has been fabricated what is illustrated in Figure 11. The ceramic component remained well dispersed in the polymeric matrix and HAP particles on the pore surface could allow the interaction between the bone-forming nano-HAP and cells. Investigation of mechanical properties showed that with increasing nano-HAP content the elasticity modulus of the composite PDLLA/nano-HAP network materials increased.

**Figure 11.** SEM images of porous structures with Schwarz primitive pore network architecture prepared by stereolithography from PDLLA and nano-HAP composite resins containing 5 wt % nano-HAP. Scale bars 200 μm [108]. (Adapted with permission from Elsevier, 2013).

Some authors applied a two-photon polymerization technique (2PP) as the type of stereolithography [109] for the preparation of UV crosslinked PLA-based materials designed for tissue scaffolds [70,71,73–75]. 2PP is a computer-aided microfabrication method by which it is possible to produce biomimetic synthetic scaffolds with high precision and reproducibility. This process uses simultaneous absorption of two photons of near-infrared (780 nm) or green (515 nm) laser light. For example, photoactive material was prepared by dissolving star-shaped methacrylate-functionalized poly(<sup>d</sup>,<sup>l</sup>-lactide) (Mn = 2600) in dichloromethane and mixing it with photoinitiator [71]. This material was next used for the fabrication of 3D structures (shown in Figure 12a) by the 2PP technique. It was

demonstrated that the fabricated PLA-based scaffolds were a beneficial microenvironment for the osteogenic differentiation of mesenchymal stem cells in vitro and the potential of prepared scaffolds as implants in cranial defects was proved by tests in vivo upon their implantation into the cranial bone defect in mice. Figure 12 illustrates prepared PLA scaffolds and their behavior as implants in mice.

**Figure 12.** (**a**) Micrographs of a 2PP-fabricated PLA scaffold, (**b**) fluorescence of PLA scaffolds after implantation into mice; MSC—mesenchymal stem cell [71]. (Adapted from Future Medicine, 2016).

The above-presented selected examples of the formation of complicated 3D structures by different stereolithography techniques concern mainly the fabrication of porous scaffolds which, as authors claim, could be used as implants for bone regeneration. However, it seems that these methods may be also useful in the production of precise elements for some other applications, as, e.g., electronic devices.
