**4. Study of thin film growth mechanisms by laser ablation**

One should take into account that coatings are obtained by successive ejected material from the target by each laser pulse. We will start the discussion by presenting some representative results from our studies related to the processing of hydroxyapatite by laser ablation.

In **Figure 6**, the surface of a HA target before and after laser irradiation is presented. The morphology is characteristic to a material melted and then resolidified. The details presented in **Figure 6c**, at a higher magnification, are specific to HA solidification, in fractal form. All circular bumps indicated the presence of the bubbling phenomenon. Moreover, the cracks appeared on target surface are due to expansion-contraction cycles as a result of repeated heating/cooling processes.

**Figure 6.** SEM micrographs of HA target before (a) and after (b and c) laser irradiation.

Typical HA structures deposited by laser ablation at different laser fluences and studied by Scanning Electron Microscopy (SEM) are presented in **Figure 7**. It is obvious that the aspect of deposited coatings varies from acicular at low fluence to cauliflower aspect at high fluence, respectively.

In case of samples HA1 and HA2, the coatings seem to be the product of material condensation originated from plasma. The droplets, even if they are present, are of small dimensions.

These coatings were also investigated By Energy Dispersive X-ray Spectroscopy (EDS) and Xray Photoelectron Spectroscopy (XPS). EDS gives us information about the composition, on the entire thickness of the layer, while XPS provided information exclusively from surface.

Only three of the samples (HA1, HA3, and HA5) have been biologically investigated *in vitro* by cell growth. The cells used for these analyses were osteoprogenitor human cells (HOp) from bone marrow. HOp were cultured in ISCOVE (Sigma I 3390) medium, supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) glutamine, and 1% (v/v) penicillin and streptomycin. After 5 days, the samples were prepared for SEM investigation (**Figure 8**).

**Figure 7.** SEM micrographs for HA coatings deposited at (a) 1.2 J/cm2 (HA1), (b) 1.8 J/cm2 (HA2), (c) 2.7 J/cm2 (HA3), (d) 5 J/cm2 (HA4), and (e) 7.5 J/cm2 (HA5).

**Figure 6.** SEM micrographs of HA target before (a) and after (b and c) laser irradiation.

112 Applications of Laser Ablation - Thin Film Deposition, Nanomaterial Synthesis and Surface Modification

respectively.

Typical HA structures deposited by laser ablation at different laser fluences and studied by Scanning Electron Microscopy (SEM) are presented in **Figure 7**. It is obvious that the aspect of deposited coatings varies from acicular at low fluence to cauliflower aspect at high fluence,

In case of samples HA1 and HA2, the coatings seem to be the product of material condensation originated from plasma. The droplets, even if they are present, are of small dimensions.

These coatings were also investigated By Energy Dispersive X-ray Spectroscopy (EDS) and Xray Photoelectron Spectroscopy (XPS). EDS gives us information about the composition, on the entire thickness of the layer, while XPS provided information exclusively from surface. Only three of the samples (HA1, HA3, and HA5) have been biologically investigated *in vitro* by cell growth. The cells used for these analyses were osteoprogenitor human cells (HOp) from bone marrow. HOp were cultured in ISCOVE (Sigma I 3390) medium, supplemented with 10%

From **Figure 8a**, one can observe that HA1 sample was destroyed, the coating being completed and dissolved in the culture medium. We could not identify cells on the sample surface. **Figure 8b** demonstrated a partial dissolution of HA3 coating. No cell was present on the sample surface. In case of HA5, one can observe an important coverage rate of the coatings deposited at 7.5 J/cm2 in the presence of cells. The morphology of the coating was not modified, while the osteoblasts present a polygonal flattened form.

**Figure 8.** SEM micrographs for HA coatings deposited at (a) 1.2 J/cm2 (HA1), (b) 2.7 J/cm2 (HA3), and (c) 7.5 J/cm2 (HA5) after 5 days of cell growth.

A similar investigations on HA coatings were conducted by Zhu et al. They analyzed the behavior of MC3T3-E1 cells cultured on the specimens after 7 days [68].

In another study, some of the HA coatings grown by PLD using a laser fluence of 3 J/cm2 were thermally treated (at 400°C for 6h) in order to improve the crystallinity.

After a first examination, the SEM images of this type of structures did not revealed significant differences (**Figure 9**).

at 7.5 J/cm2

the osteoblasts present a polygonal flattened form.

114 Applications of Laser Ablation - Thin Film Deposition, Nanomaterial Synthesis and Surface Modification

**Figure 8.** SEM micrographs for HA coatings deposited at (a) 1.2 J/cm2

behavior of MC3T3-E1 cells cultured on the specimens after 7 days [68].

thermally treated (at 400°C for 6h) in order to improve the crystallinity.

A similar investigations on HA coatings were conducted by Zhu et al. They analyzed the

In another study, some of the HA coatings grown by PLD using a laser fluence of 3 J/cm2

(HA5) after 5 days of cell growth.

in the presence of cells. The morphology of the coating was not modified, while

(HA1), (b) 2.7 J/cm2

(HA3), and (c) 7.5 J/cm2

were

**Figure 9.** SEM micrographs of HA coatings deposited by PLD (a) with (HAt) and (b) without thermal treatment (HA).

SEM micrographs, at higher magnification, showed the differences induced by the thermal treatment (**Figure 10**).

**Figure 10.** SEM micrographs of HA coatings synthesized by PLD with (left) and without (right) thermal treatment.

It can be seen that HA coatings present a more rough morphology at nano level. The droplets' shape is similar to that of snowballs, and the surface is made up of parallelepipedic structure.

The thermal treatment induced the surface structure reorganization. There are no longer irregularities, and the appearance of the droplets is smooth.

The Atomic Force Microscopy (AFM) analysis revealed differences between the two types of structures related to their roughness (**Figure 11**). The decrease in the rough value from 1.01 to 0.8 nm, at nanometric scale, proves the smoothing of the target.

**Figure 11.** AFM imaged for HA coatings deposited by PLD with (left) and without (right) thermal treatment.

EDS results showed that the value of Ca/P ratio diminishes from 2.04 (untreated sample - HA) to 1.63 (treated sample - HAt). The corroborated results demonstrated the importance of thermal treatment in obtaining crystalline hydroxyapatite, biocompatible, having a structure similar to stoichiometric HA.

HA coatings were also grown by other techniques, such as thermal spray, high velocity oxyfuel (HVOF) techniques, and plasma spraying trying to find, as in PLD depositions, the optimal conditions for good film with applications in medicine [19, 69].

A thermal treatment was also applied to Mn-CHA and OCP films obtained by PLD. In case of Mn-CHA coating, the Ca/P atomic ratio obtained by XPS and EDS investigations was 1.64– 1.66, close to the stoichiometric values.

The morphologies of the two structures, OCP and Mn-CHA, are quite different. OCP has a porous and arborescent-like structure (**Figure 12a** and **b**), and Mn-CHA has a granular and more compact (**Figure 12c** and **d**). The surface morphologies of both calcium phosphates are well matched for bone tissue growth and osteointegration.

The thermal treatment induced the surface structure reorganization. There are no longer

The Atomic Force Microscopy (AFM) analysis revealed differences between the two types of structures related to their roughness (**Figure 11**). The decrease in the rough value from 1.01 to

**Figure 11.** AFM imaged for HA coatings deposited by PLD with (left) and without (right) thermal treatment.

conditions for good film with applications in medicine [19, 69].

similar to stoichiometric HA.

1.66, close to the stoichiometric values.

EDS results showed that the value of Ca/P ratio diminishes from 2.04 (untreated sample - HA) to 1.63 (treated sample - HAt). The corroborated results demonstrated the importance of thermal treatment in obtaining crystalline hydroxyapatite, biocompatible, having a structure

HA coatings were also grown by other techniques, such as thermal spray, high velocity oxyfuel (HVOF) techniques, and plasma spraying trying to find, as in PLD depositions, the optimal

A thermal treatment was also applied to Mn-CHA and OCP films obtained by PLD. In case of Mn-CHA coating, the Ca/P atomic ratio obtained by XPS and EDS investigations was 1.64–

irregularities, and the appearance of the droplets is smooth.

116 Applications of Laser Ablation - Thin Film Deposition, Nanomaterial Synthesis and Surface Modification

0.8 nm, at nanometric scale, proves the smoothing of the target.

**Figure 12.** (a) Scanning and (b) Transmission Electron Microscopy (TEM) images of OCP coatings; (c) SEM and (d) TEM images of Mn-CHA coatings (reproduced with permission from Ref. [25]).

In case of OCP coatings, XPS measurements showed the total dissolution and disappearance of coating after 7 days of immersion in simulated body fluid (SBF) (**Figure 13a**). SEM investigations confirmed this advanced dissolution. As for Mn-CHA, the SEM and XPS investigations demonstrated that the coatings preserve the basic composition even the intensity of Ca and P peaks decreased (**Figure 13b**). After 7 days of immersion in SBF, the surface becomes slightly smoother.

Some interesting results were obtained by laser ablation of Mg:OCP and Sr:OCP compounds using the MAPLE technique [31]. The X-ray Diffraction (XRD) patterns revealed that all MAPLE coatings are constituted of OCP. This remark is sustained by the presence of the strong low angle reflection 2*θ* of 4.7° and the series of reflections in the range of 30–34°. Comparing these results with the previous one related to OCP deposition by PLD [70], one can remark that the gentle deposition conditions of MAPLE offer a higher degree of OCP crystallinity with respect to PLD [31]. The homogeneous distribution of magnesium and strontium on the thin film surface was evidenced by EDS analysis (**Figure 14**).

**Figure 13.** XPS spectra of (a) OCP and (b) Mn-CHA before and after degradation tests (reproduced with permission from Ref. [25]).

**Figure 14.** EDS maps recorded for (a) Mg:OCP and (b) Sr:OCP coatings (reproduced with permission from Ref. [31]).

To evaluate the proliferation and morphology of MG63 cells, one can also perform phalloidin staining on OCP, Mg:OCP, and Sr:OCP coatings deposited by MAPLE. The surface topography and chemical composition can influence cell behavior. Looking the images of MAPLE coating stain with phalloidin for 14 days, no visible differences were observed (**Figure 15**, left). Similar results are visible from SEM images of the same structures (**Figure 15**, right). Deeper studies showed that Mg:OCP and Sr:OCP coatings improved the proliferation and differentiation of MG63 cells.

**Figure 15.** Phalloidin staining (left) and SEM images (right) of MG63 cells after 14 days of culture grown on (a) OCP, (b) Mg:OCP, and (c) Sr:OCP (reproduced with permission from Ref. [31]).

**Figure 13.** XPS spectra of (a) OCP and (b) Mn-CHA before and after degradation tests (reproduced with permission

118 Applications of Laser Ablation - Thin Film Deposition, Nanomaterial Synthesis and Surface Modification

**Figure 14.** EDS maps recorded for (a) Mg:OCP and (b) Sr:OCP coatings (reproduced with permission from Ref. [31]).

To evaluate the proliferation and morphology of MG63 cells, one can also perform phalloidin staining on OCP, Mg:OCP, and Sr:OCP coatings deposited by MAPLE. The surface topography and chemical composition can influence cell behavior. Looking the images of MAPLE coating

from Ref. [25]).

The biological analysis conducted on Mg:OCP and Sr:OCP coatings revealed no visible cytotoxic or inflammatory effects on osteoblast-like cells in all experimental times (**Table 1**).


**Table 1.** Control values of proliferation and differentiation for MG63 osteoblast cells at 3, 7, and 14 days culture.

Mroz et al. also evaluated the performances of HA and OCP coatings deposited by PLD. The biological assays revealed that both layers are biocompatible with respect to human osteoblast cells, offering favorable conditions for their proliferation [71].
