**3.3. Case study: chitosan‐based biocomposites**

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

Although many authors have reported the preparation of mixtures of chitosan and calcium phosphates in the form of powders [69], membranes [70], scaffolds [71] or microspheres [72], only few publications were dedicated on developing new procedures allowing the concomi‐ tant preparation of a biocomposite material containing the two components, which is predicted to assure a more close contact between them [73–75]. For this reason, further we will exemplify the biofabrication of the compositional map of chitosan (CHT) and biomimetic apatite (BmAp) by the innovative C‐MAPLE technique. In the experiments, an excimer laser source (KrF\*, λ  = 248 nm, ζFWHM = 25 ns) operated at 10 Hz frequency repetition rate was used for the cryogenic targets evaporation. As deposition substrates were used as follows: 12 mm diameter Ti (grade 4) disks, Si wafers or glass slides. The coated samples areas deposited onto the substrate selection formed of five consecutive 12 mm Ti disks which are further denoted S1–S5; where S1 stands for the coating area having CHT as major component, S5 represents the coating area with richer content of BmAp. S2–S3–S4 series indicates blended coating areas with decreasing CHT/BmAp ratios. For comparison reasons, simple CHT and BmAp films have been also deposited on the same type of substrates [54]. After preliminary studies, deionized water was chosen as solvent to prepare solutions of 1 and 2 wt.% for CHT and BmAp, respectively. A parametric study in order to find the optimum laser energy (70, 100, 120 and 150 mJ) for which the materials are deposited unaltered was performed. The selected laser energy was as follows: 100 mJ in the case of CHT target and of 70 mJ for the BmAp one.

C‐MAPLE composite coatings have a homogeneous spongy appearance all over the substrate, which is known to be beneficial for cell adhesion as revealed by SEM micrographs (results not presented here). While CHT particulates preserve their spherical morphology, elongated filiform structures can be also noticed in the blended regions of the film (S2–S4). One can assume that these thread‐like structures may support some toughness improvements similar to the one provided by fiber reinforcing in the biocomposite materials, ensuring the desired mechanical behavior for tissue substitution [76].

The C‐MAPLE synthesized biocomposite‐based CHT thin films are amorphous, rough, with a morphology characteristic to laser deposited structures. Next, by high resolution AFM investigations, we disclose information about the nanostructuring of the film grains and their morphological evolution (**Figure 10**). One notices that a progressive increase in roughness (RRMS) occurs with the CHT concentration in the C‐MAPLE composite films [54].

For each combinatorial surface, the Ca/P molar ratio was in the range ∼1.3–1.5, lower than the stoichiometric theoretical value of hydroxyapatite (i.e., 1.67), pointing to the calcium‐deficient state of biomimetic apatite synthesized.

The Raman spectra of BmAp, HA (pure and highly crystalline) and CHT raw materials are presented comparatively in **Figure 11a** and **b** [54]. Similar vibration bands to the ones revealed by FTIR investigations (data not shown here) [54] have been also identified by micro‐Raman analyses for the CHT and BmAp source materials. However, in the case of Raman spectra the symmetric stretching bands are now the dominant ones (e.g., symmetric stretching ν1 of (PO4) 3‐ ∼956 cm‐1; symmetric stretching of aliphatic (C–H) bonds ∼2929 cm‐1). This is owned to the fact that in Raman spectroscopy, the vibration bands intensity is dependent on the polarizability induced dipole, and not on the variation of the dipole moment of the molecule, as is the case of FTIR spectroscopy. Thereby, this was to be expected, as for a given centrosym‐ metric molecule, the vibration modes which are symmetric to the inversion center of the molecule generate higher Raman intensity bands. Moreover, the electronic density between the carbon atoms in aliphatic C–H bonds that can be deformed under the action of the radiation electric field should be large. If the vibrational mode involved in the Raman scattering process is not totally symmetric, then the polarization of the photon can be partially (e.g., antisym‐ metric stretching modes) or even totally reduced.

**3.3. Case study: chitosan‐based biocomposites**

120 Composites from Renewable and Sustainable Materials

100 mJ in the case of CHT target and of 70 mJ for the BmAp one.

mechanical behavior for tissue substitution [76].

state of biomimetic apatite synthesized.

medical implants applications.

This subsection is intended to summarize the recent progresses and concerns involving the use of chitosan in the development of new biocomposite materials for high performance

Although many authors have reported the preparation of mixtures of chitosan and calcium phosphates in the form of powders [69], membranes [70], scaffolds [71] or microspheres [72], only few publications were dedicated on developing new procedures allowing the concomi‐ tant preparation of a biocomposite material containing the two components, which is predicted to assure a more close contact between them [73–75]. For this reason, further we will exemplify the biofabrication of the compositional map of chitosan (CHT) and biomimetic apatite (BmAp) by the innovative C‐MAPLE technique. In the experiments, an excimer laser source (KrF\*, λ  = 248 nm, ζFWHM = 25 ns) operated at 10 Hz frequency repetition rate was used for the cryogenic targets evaporation. As deposition substrates were used as follows: 12 mm diameter Ti (grade 4) disks, Si wafers or glass slides. The coated samples areas deposited onto the substrate selection formed of five consecutive 12 mm Ti disks which are further denoted S1–S5; where S1 stands for the coating area having CHT as major component, S5 represents the coating area with richer content of BmAp. S2–S3–S4 series indicates blended coating areas with decreasing CHT/BmAp ratios. For comparison reasons, simple CHT and BmAp films have been also deposited on the same type of substrates [54]. After preliminary studies, deionized water was chosen as solvent to prepare solutions of 1 and 2 wt.% for CHT and BmAp, respectively. A parametric study in order to find the optimum laser energy (70, 100, 120 and 150 mJ) for which the materials are deposited unaltered was performed. The selected laser energy was as follows:

C‐MAPLE composite coatings have a homogeneous spongy appearance all over the substrate, which is known to be beneficial for cell adhesion as revealed by SEM micrographs (results not presented here). While CHT particulates preserve their spherical morphology, elongated filiform structures can be also noticed in the blended regions of the film (S2–S4). One can assume that these thread‐like structures may support some toughness improvements similar to the one provided by fiber reinforcing in the biocomposite materials, ensuring the desired

The C‐MAPLE synthesized biocomposite‐based CHT thin films are amorphous, rough, with a morphology characteristic to laser deposited structures. Next, by high resolution AFM investigations, we disclose information about the nanostructuring of the film grains and their morphological evolution (**Figure 10**). One notices that a progressive increase in roughness

For each combinatorial surface, the Ca/P molar ratio was in the range ∼1.3–1.5, lower than the stoichiometric theoretical value of hydroxyapatite (i.e., 1.67), pointing to the calcium‐deficient

The Raman spectra of BmAp, HA (pure and highly crystalline) and CHT raw materials are presented comparatively in **Figure 11a** and **b** [54]. Similar vibration bands to the ones revealed by FTIR investigations (data not shown here) [54] have been also identified by micro‐Raman

(RRMS) occurs with the CHT concentration in the C‐MAPLE composite films [54].

**Figure 10.** Comparative AFM images recorded on the surface of: simple CHT and BmAp films deposited by MAPLE, and in various regions of the C‐MAPLE composite film starting from the CHT‐rich edge. The coated samples areas de‐ posited onto the substrate array composed of five consecutive 12 mm Ti disks were denoted S1 to S5; where S1 stands for the coating area having CHT as major component, S5 represents the coating area with richer content of BmAp. S2‐ S3‐S4 series indicates blended coating areas with decreasing CHT/BmAp ratios.

**Figure 11.** Comparative Raman spectra of BmAp, crystalline HA and CHT powders presented in the fingerprint (a) and functional groups (b) regions. The spectra have been normalized to the highest intensity peak (i.e., ν1(PO4) 3‐ νs. C– H bands in the case hydroxyapatite and chitosan, respectively).

**Figure 12.** 2D Raman maps obtained from (80 × 100) µm2 areas of the CHT‐BmAp composite C‐MAPLE film, based on the characteristic Raman stretching vibration bands of C–H bond in aliphatic and aromatic groups (3000–2850 cm‐1 wave numbers region) of CHT (red colored), and ν1 symmetric stretching of orthophosphate groups (965–950 cm‐1 wave numbers region) of BmAp (green colored). (a) CHT‐rich edge region (b) CHT‐BmAp central region (c) BmAp‐ richer edge region.

In perfect agreement with the FTIR results, the Raman spectra indicated that the BmAp material elicit a decreased ordering (indicated by the broader aspect of the bands) and a certain level hydration with respect to the pure and crystalline HA.

The CHT vibration bands in the 1250–1000 cm‐1 wave numbers region are screened by the more intense superimposed Si–O–Si stretching band of the glass substrate (**Figure 12**). Overall, the

CHT and BmAp bands in the case of the composite C‐MAPLE films are less conspicuous and defined with respect to the ones of the source powders. The shift of ν1 symmetric stretching band of (PO4) 3‐ groups close to ∼956 cm‐1 with respect to the stoichiometric crystalline com‐ pound (i.e., ∼962–961 cm‐1) suggest a higher degree of disordering, similarly to the source BmAp material.

Subsequently, X‐Y Raman mapping measurements have been performed in three relevant regions (with areas of (80 × 100) µm2 ) of the C‐MAPLE synthesized biocomposite‐based CHT thin films deposited onto glass substrate (**Figure 12**), in order to generate detailed chemical images (in red‐green‐blue colors) of the spatial distribution of the two species (i.e., CHT and BmAp). The CHT phase (red colored) has been integrated with the stretching vibration bands of C–H bond in aliphatic and aromatic groups (3000–2850 cm‐1 wave numbers region), while the BmAp phase (green colored) has been assigned with the prominent Raman band of HA (i.e., ν1(PO4) 3‐ symmetric stretching) situated in the 965–950 cm‐1 range. The distribution of CHT seems to be homogenous in all analyzed composite film regions, whilst the BmAp phase is rather distributed in randomly dispersed aggregates.

**Figure 11.** Comparative Raman spectra of BmAp, crystalline HA and CHT powders presented in the fingerprint (a) and functional groups (b) regions. The spectra have been normalized to the highest intensity peak (i.e., ν1(PO4)

the characteristic Raman stretching vibration bands of C–H bond in aliphatic and aromatic groups (3000–2850 cm‐1 wave numbers region) of CHT (red colored), and ν1 symmetric stretching of orthophosphate groups (965–950 cm‐1 wave numbers region) of BmAp (green colored). (a) CHT‐rich edge region (b) CHT‐BmAp central region (c) BmAp‐

In perfect agreement with the FTIR results, the Raman spectra indicated that the BmAp material elicit a decreased ordering (indicated by the broader aspect of the bands) and a certain

The CHT vibration bands in the 1250–1000 cm‐1 wave numbers region are screened by the more intense superimposed Si–O–Si stretching band of the glass substrate (**Figure 12**). Overall, the

H bands in the case hydroxyapatite and chitosan, respectively).

122 Composites from Renewable and Sustainable Materials

**Figure 12.** 2D Raman maps obtained from (80 × 100) µm2

level hydration with respect to the pure and crystalline HA.

richer edge region.

3‐ νs. C–

areas of the CHT‐BmAp composite C‐MAPLE film, based on

XPS analysis confirmed the chemical composition of the C‐MAPLE synthesized biocomposite‐ based CHT thin films. XPS survey spectra collected on the surface of CHT‐BmAp samples (not presented) indicated the presence of C 1s, O 1s, N 1s, Ca 2s, Ca 2p, P 2s and P 2p photoelectron peaks [54], while **Figure 13** showed the atomic ratio N 1s/(N 1s + Ca 2p) evolution depending on the position of material deposited along the CHT‐BmAp blended sample. As predicted, the atomic ratio (N 1s/N 1s + Ca 2p) is decreasing from CHT to BmAp compounds, evidencing the composition gradient of the combinatorial films.

**Figure 13.** Typical XPS measurement along the composite CHT‐BmAp C‐MAPLE films. Reproduced with permission from Ref. [54].

The antimicrobial activity was controlled by the concentration of chitosan, the most efficient antimicrobial activity, against Gram‐positive and Gram‐negative strains, was assigned to blended S3 and S4 samples (**Figure 14**).

**Figure 14.** Graphic representation of the number of viable microbial cells adhered to the surface of CHT‐BmAp compo‐ site film after (a) 6 h (b) 24 h and (c) 48 h of incubation. Reproduced with permission from Ref. [54].

The composition gradient of the chitosan‐to‐biomimetic hydroxyapatite has been confirmed longwise the combinatorial films by FTIR and XPS analyses, validating the used C‐MAPLE technique.

C‐MAPLE technique used in this study proves to be a prospective and viable method for the fabrication of biomimetic and bioactive antimicrobial orthopedic coatings based on renewable resources which resemble the native bone extracellular matrix while creating a favorable living environment for osteogenic cells, in the same time assuring protection from microbial coloni‐ zation.
