**3.2. Case study: lignin‐based biocomposites**

C‐MAPLE technique offers the possibility to combine and immobilize two or more biomate‐ rials, dissolved in different solvents, using diverse wavelengths. Next, by physical, chemical and biological characterization of the obtained structures, one may select the best compositions

MAPLE and C‐MAPLE techniques could unlock the research frontiers in biofabrication of composite based on renewable resources with great potential for various applications.

**3. Relevant results regarding renewable materials deposited by advanced**

In this section, relevant data regarding the engineering of novel renewable materials by laser

In this chapter, we focused biomaterials based on renewable resources in form of thin films fabricated by laser‐based concepts for obtaining functionalized medical implants. The first example refers to silk fibroin and composite HA‐fibroin coatings deposited by MAPLE [63]. Silks are fibrous proteins synthesized in specialized epithelial cells of Lepidoptera larvae such

The studies of Miroiu et al. [63] showed that compared to the simple fibroin or HA thin films, obtained implants exhibit an intermediary topography, favorable to bone cells anchorage and proliferation. On the same time, it resulted that the fibroin content in films was a mixture of preponderantly random coil with crystalline forms, β‐sheet and α‐helix. The renewable silk fibroin composite evidenced by *in‐vitro* viability tests appropriate bone cell behavior nontox‐

Next, biocomposite silk fibroin‐poly(3‐hydroxybutyric‐acid‐co‐3‐hydroxyvaleric‐acid) bio‐ degradable functionalized implants were grown by MAPLE [64]. Silk fibroin and poly(3‐ hydroxybutyric‐acid‐co‐3‐hydroxyvaleric‐acid—PHVB) are both natural biopolymers with excellent biocompatibility, but different biodegradability rates and tensile strength proper‐ ties. They were combined in a composite in order to improve their properties as coatings for biomedical uses. The physical‐chemical properties of the composite coatings and princi‐ pally their degradation behavior in simulated body fluid were investigated as primary step of applicability in local controlled drug release. It was demonstrated that higher PHBV contents enhance the resistance and lead to a slower degradation rate of composite coat‐

Another MAPLE studies related to synthesis of poly(d,l‐lactide‐co‐glycolide) (PLGA) particle systems were reported by Socol et al. [65]. PLGA + polyvinyl alcohol (PVA), PLGA + PVA +  bovine serum albumin (BSA) and PLGA + PVA + chitosan nanoparticles were prepared by an

**3.1. The literature survey of renewable materials‐based composites deposited by laser**

that can be synthesized from the two biomaterials.

114 Composites from Renewable and Sustainable Materials

**laser techniques: trends and challenges**

processing and their applications are detailed.

as silkworms, spiders, scorpions, flies and mites.

icity, good spreading and normal cell morphology [63].

**techniques**

ings [64].

This subsection is intended to summarize the recent progresses and concerns involving the use of lignin in the development of new polymer biocomposite materials for high performance medical implants applications.

Until now, biocomposite coatings based on the renewable lignin were studied by electropho‐ retic deposition (EPD) only [36, 37], and the results revealed that the obtained composite exhibited enhanced stability and improved interconnected structure, with an increased coating cohesion [53]. For electrophoretic deposition (EPD) method, it was applied a constant voltage (at optimized parameters 60 V for 45 s), and it were obtained uniform composite coatings with good adhesion and mechanical properties without any phase transformation [67].

In our studies, we report MAPLE deposited a large macromolecule of undefined molecular weight‐organosolv lignin (Lig) embedded in a simple hydroxyapatite (HA) or silver (Ag) doped hydroxyapatite film matrix [53]. A pulsed KrF\* laser source (λ = 248 nm, ζFWHM = 25 ns) operating at 10 Hz was used for the Ag/HA/Lig and its counterpart without silver (HA/Lig) composite frozen targets evaporation. Pure titanium foils or silicon wafers were used as substrates. A total number of 20,000 pulses at a fluence of 0.7 Jcm‐2 were applied for the deposition of each structure while the spot size was of 25 mm2 . Then, the structures were subjected to analysis by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), X‐ray diffraction analysis (XRD), X‐ray photoelectron spectroscopy (XPS) and attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) [53].

Smooth, uniform Lig‐based thin films adherent to substrate were revealed by typical top‐view SEM images (**Figure 5**). As a first observation, rough surface of the films was reported starting from the same nano‐hydroxyapatite powder composite with Lig, when using the electropho‐ retic deposition [67] (**Figure 6**).

**Figure 5.** Top‐view SEM micrographs of the HA‐Lig (a) and Ag:HA‐Lig films (b) deposited by MAPLE on Ti sub‐ strates. Reproduced with permission from Ref. [53].

**Figure 6.** SEM micrographs of sintered Ag/HAP/Lig coating (a) before and (b) after immersion in SBF solution at 37°C. Scale bar: 5 µm. Reprinted from Ref. [67] with permission. Copyright American Chemical Society 2013.

EDS analyses revealed the presence of HA components but also evidences of traces of Ag and lignin. The deposited HA was Ca deficient, which denotes a film with increased solubility. Recorded X‐ray patterns (data not presented) were characteristic to amorphous films. Lignin presence in composite films was undoubtedly proved by both XPS and FTIR. The XPS spectra were achieved for two films: the composite Ag:HA‐Lig and the pure HA film one (**Figure 7**). The lignin dispersion in HA matrix was evidenced by the massive increase in the C‐bonded carbon signature, accompanied by a slight raise of the constituent connected with oxygen‐ bonded C or oxygen‐containing radicals. These enhancements were attributed to the lignin presence. The certain proof that lignin has been successfully transferred into the HA composite film consist in calculated experimental data regarding the stoichiometry fraction xC:yO considering the addition of 10% lignin into the HA matrix.

Composite Coatings Based on Renewable Resources Synthesized by Advanced Laser Techniques http://dx.doi.org/10.5772/65260 117

**Figure 7.** C 1s core level high resolution XPS spectra for the pure HA (a) and Ag:HA‐Lig (b) MAPLE films. Reproduced with permission from Ref. [53].


Adapted from Ref. [67] with permission. Copyright Elsevier 2016.

from the same nano‐hydroxyapatite powder composite with Lig, when using the electropho‐

**Figure 5.** Top‐view SEM micrographs of the HA‐Lig (a) and Ag:HA‐Lig films (b) deposited by MAPLE on Ti sub‐

**Figure 6.** SEM micrographs of sintered Ag/HAP/Lig coating (a) before and (b) after immersion in SBF solution at 37°C.

EDS analyses revealed the presence of HA components but also evidences of traces of Ag and lignin. The deposited HA was Ca deficient, which denotes a film with increased solubility. Recorded X‐ray patterns (data not presented) were characteristic to amorphous films. Lignin presence in composite films was undoubtedly proved by both XPS and FTIR. The XPS spectra were achieved for two films: the composite Ag:HA‐Lig and the pure HA film one (**Figure 7**). The lignin dispersion in HA matrix was evidenced by the massive increase in the C‐bonded carbon signature, accompanied by a slight raise of the constituent connected with oxygen‐ bonded C or oxygen‐containing radicals. These enhancements were attributed to the lignin presence. The certain proof that lignin has been successfully transferred into the HA composite film consist in calculated experimental data regarding the stoichiometry fraction xC:yO

Scale bar: 5 µm. Reprinted from Ref. [67] with permission. Copyright American Chemical Society 2013.

considering the addition of 10% lignin into the HA matrix.

retic deposition [67] (**Figure 6**).

116 Composites from Renewable and Sustainable Materials

strates. Reproduced with permission from Ref. [53].

**Table 4.** Quantitative XPS analysis data for Synthetic hydroxyapatite, Ca10(PO4)6(OH)2 (HAP) and hydroxyapatite/ lignin (HAP/Lig) (0.5–10) wt.% Lig electrophoretic coatings.

According to the Ref. [67], in the electrophoretic deposition case, lignin limited the decompo‐ sition of the apatite lattice of sintered composite coatings based on Lig with (1–10) wt.% Lig. This was designated by the smaller increase in carbon content and decreased Ca/P ratio, compared to pure apatite coating and HAP/Lig coating with 0.5 wt.% Lig (**Table 4**).

In addition, FTIR investigations advocated a certain improvement of Lig‐based films mechan‐ ical properties due to lignin incorporation (**Figure 8**). In good agreement with FTIR observa‐ tions in the case of MAPLE method, it is also noticed for electrophoretic deposition that does not occur with any alteration of the initial material. This fact could be associated with the binding of hydroxyls from apatite lattice, preventing the decomposition and/or ion diffusion on substrate surface, demonstrating that Lig protects HAP/Lig coatings [68].

**Figure 8.** ATR‐FTIR spectra of Lig powder (a,b), pure HA film (c), pure HA powder (Sigma‐Aldrich) (d) and Ag:HA‐ Lig film (e,f) in the spectral regions: 1800 – 550 cm‐1 (a,c,d,e) and 3100 – 2700 cm‐1 (b,f) . Reproduced with permission from Ref. [53].

The validation of the MAPLE technique was demonstrated for deposition of such delicate renewable biomaterial, as suggested by EDS, XPS, FTIR, biological and microbial results. The MAPLE obtained biocomposites‐based Lig were found noncytotoxic, promoting the prolifer‐ ation of the adhered human mesenchymal cells (figure not shown here) [53], while the microbiological assays revealed that the coated composite assured a prolonged release of silver ions, exhibiting a high both bacterial and fungal behavior (**Figure 9**). The same comportament was noticed in the case of the other reported electrophoretic case (**Table 5**) [68], where lignin addition both boosted the antimicrobial activity and supported the normal development and growth of the adhered cells [68].

**Figure 9.** (a) Number of *Pseudomonas aeruginosa* and *C. famata* viable cells recovered from the biofilms growing on the tested specimens after 24, 48 and 72 h, respectively (b) Absorbance values at 600 nm of the *P. aeruginosa* and *C. famata* bacterial biofilm developed on the tested specimens after 24, 48 and 72 h, respectively. Adapted with permission from Ref. [53].


Reprinted from Ref. [68] with permission. Copyright Elsevier 2016.

**Figure 8.** ATR‐FTIR spectra of Lig powder (a,b), pure HA film (c), pure HA powder (Sigma‐Aldrich) (d) and Ag:HA‐ Lig film (e,f) in the spectral regions: 1800 – 550 cm‐1 (a,c,d,e) and 3100 – 2700 cm‐1 (b,f) . Reproduced with permission

The validation of the MAPLE technique was demonstrated for deposition of such delicate renewable biomaterial, as suggested by EDS, XPS, FTIR, biological and microbial results. The MAPLE obtained biocomposites‐based Lig were found noncytotoxic, promoting the prolifer‐ ation of the adhered human mesenchymal cells (figure not shown here) [53], while the microbiological assays revealed that the coated composite assured a prolonged release of silver

from Ref. [53].

118 Composites from Renewable and Sustainable Materials

**Table 5.** Reduction of viable cell number of *Staphylococcus aureus* TL after incubation with Ag/HAP/Lig coating for 0, 1 and 24 h.

Biofabrication of composites based on Lig‐renewable resources have a great potential for medical applications, supporting the osteogenic cell proliferation while assuring an antibac‐ terial behavior.
