**4. Cell interaction and answer to pNIPAm-based interfaces obtained by MAPLE**

Cell interaction and answer to pNIPAm substrates/surfaces are determined, besides the chemical characteristics, also by the thickness and surface morphology. The porosity and uniformity of the thin films as well as low roughness plays an important role in the cell adhesion/detachment behavior. The thickness of the films has an important role in the temperature-induced alterations of the films (changing from hydrophilic to hydrophobic properties [5–7, 28–34]. The cell culture-based studies (phase-contrast microscopy, fluorescence microscopy and MTT assays) have proved a good biocompatibility and a cell behavior correlated with the chemical composition of biomaterial substrates in the case of pNIPAm coatings obtained by MAPLE.

#### **4.1. Mammalian cells adhesion and morphology on pNIPAm coatings obtained by MAPLE**

A first observation of the effect of pNIPAm coatings onto the behavior of L929 and human mesenchymal stem cells (hMSC) is given in **Figure 6**, where cells adhesion and morphology were analyzed by phase-contrast microscopy, showing that cells spread on the coatings surfaces without shape alteration.

Smart Thermoresponsive Surfaces Based on pNIPAm Coatings and Laser Method for Biological Applications http://dx.doi.org/10.5772/66280 183

However, both protein adsorption and cell reversible attachment are correlated to pNIPAm density, grafting, or conformation onto a substrate [54]. In the case of pNIPAm coatings obtained by MAPLE, a model protein (bovine serum albumin (BSA)) was used to probe the changes in the interfacial properties coatings and two trends depending on roughness surfaces were observed. Negligible protein adsorption at 21°C was noticed for low roughness surfaces (samples 0–7 nm, 1–20 nm and 2 with roughness up to 30 nm) and increase of protein on denser and rougher pNIPAm coatings (samples 2–110 nm and 3 with roughness up

However, the changes in the pNIPAm surface properties (roughness) affected protein adsorption, with lower protein adsorption on dense and rough coatings at 37°C and higher adsorption on low roughness surfaces, which is in contradiction with the measurements reported on pNIPAm obtained by other methods [54]. This discrepancy could be explained by the swelling ratio which decreases with increasing material density, as the chains in more densely packed coatings swell less upon full hydration. When compared to the modification of the heights of the coatings under temperature influence (about 24 nm) to the protein dimensions (length 14 nm, height and width 4 nm), it can be assumed that the lowest density is insufficient for exceeding primary adsorption and the proteins are able to penetrate the rough coatings and they would adsorb to the underlying surface. Nevertheless, the thin-film irregularities or roughnesses were shown to be important factors in influencing the mobility of adsorbed

**4. Cell interaction and answer to pNIPAm-based interfaces obtained by**

Cell interaction and answer to pNIPAm substrates/surfaces are determined, besides the chemical characteristics, also by the thickness and surface morphology. The porosity and uniformity of the thin films as well as low roughness plays an important role in the cell adhesion/detachment behavior. The thickness of the films has an important role in the temperature-induced alterations of the films (changing from hydrophilic to hydrophobic properties [5–7, 28–34]. The cell culture-based studies (phase-contrast microscopy, fluorescence microscopy and MTT assays) have proved a good biocompatibility and a cell behavior correlated with the chemical composition of biomaterial substrates in the case of pNIPAm

**4.1. Mammalian cells adhesion and morphology on pNIPAm coatings obtained by MAPLE**

A first observation of the effect of pNIPAm coatings onto the behavior of L929 and human mesenchymal stem cells (hMSC) is given in **Figure 6**, where cells adhesion and morphology were analyzed by phase-contrast microscopy, showing that cells spread on the coatings

to 130 nm) (**Figure 5**).

182 Modern Technologies for Creating the Thin-film Systems and Coatings

proteins [5].

**MAPLE**

coatings obtained by MAPLE.

surfaces without shape alteration.

**Figure 6.** The attachment and morphology of the L929 cells (a) and hMSC cells (b) on pNIPAm coatings observed after 72 h of culture (using SteREO DiscoveryV20 microscope, with PlanApo S 1.0× objective, 32.5× magnification with an AxioCamICm1 camera attached to the microscope setup). L929 (mouse fibroblast) cells and human mesenchymal stem cells (hMSC) were cultured in Dulbecco's minimal essential medium (DMEM) containing 4-mM L-glutamine (Gibco) and supplemented with 10% fatal calf serum (FBS) and 50-U/ml penicillin/streptomycin (50 mg/ml) and maintained at 37°C with 5% CO2.

**Figure 7.** AFM image of pNIPAm coating obtained for a fluence of 400 mJcm−2 (a) and (b) fluorescence microscopy image of L929 cells stained with Hoechst (Sigma) nucleus—blue and fluorescein phalloidin (ThermoFisher) green-actin fibers on pNIPAm samples and on controls (c).

The above observations were confirmed by the fluorescence microscopy analysis. The initial cell attachment, spreading and morphological features of fibroblasts cells were assessed and correlated to surface characteristics.

As fibroblasts are ubiquitous in the body and are the first to populate prostheses surface (implants), the morphology of the cells (seeded at a density of 5000 cells/100 µl) for 24 h can give a first information on how and if pNIPAm coating obtained by MAPLE can support cell adherence and if the confluence on highly dense coatings is possible.

As shown by florescence images in **Figure 7**, L929 cells adhere and spread uniformly onto the laser-engineered pNIPAm surfaces and start projecting long dendrites along the surface area. These results are confirmed by scanning electron microscopy (SEM) analysis of cells cultured onto the pNIPAm coatings for 24 h (**Figure 8**).

**Figure 8.** SEM images of the cells seeded on pNIPAm materials for 24 h: overview on the cells distributions over large area (a) and a close-up on the shape of the adhered L929 cells (b). For preparation of the samples for SEM analysis, the samples were washed with PBS and fixed with 2.5% glutaraldehyde in PBS for 20 min and subjected for ethanol gradient dehydration.

Nevertheless, the spreading of the cells and adherence can be inhibited by either surface chemistry or surface morphology [5]. The viability of the fibroblast cells after 3 days of culture on the pNIPAM coatings was calculated showing similar proliferation rate for the coatings obtained in the range of 200–600 mJcm−2. A cytotoxic effect and a decrease in proliferation rate in the case of the coatings obtained for higher fluences [5] were observed.

### **4.2. Cell harvesting based on temperature change**

A recent study by Rusen et al. [5] on the cell detachment upon temperature reduction showed that the roughness of the thin films represents a significant parameter in tuning the detachment time. The quantitative analysis on the fibroblast cell harvesting upon temperature reduction showed that if cells detached from rougher films within 10–20 min, the detachment time on smooth surfaces increased with more than 25%.

The sequence of images presenting an example of the cell detachment from a pNIPAm thin film obtained by MAPLE is shown in **Figure 9**. The cells presented flat, spread, normal morphology at the beginning of the observations (**Figure 9a**), when the temperature was 37°C. However, by lowering the temperature the cells morphology started to change, becoming less spread and rounder (**Figure 9b**–**d**). When compared with previously reported data on cell detachment from pNIPAm coatings obtained by MAPLE [5], the detachment time is increased due to the low roughness (20 nm) pNIPAm surfaces used in this study. Moreover, the cells collected from pNIPAm coating obtained by MAPLE did not change their shape or viability. This represent a strong indication that a nondestructive reversible cell detachment takes place when using pNIPAm bio-interfaces obtained by MAPLE [5].

Smart Thermoresponsive Surfaces Based on pNIPAm Coatings and Laser Method for Biological Applications http://dx.doi.org/10.5772/66280 185

**Figure 9.** Cell detachment sequence images at 0 min (a), 5 min (b), 10 min (c), 15 min (d), 20 min (e) from pNIPAm coatings obtained by MAPLE at 250 mJ cm−2. (f) Fluorescence microscopy image of L929 cells grew on other pNIPAm surfaces, indicating no damage to either the structural or functional aspects of the cells recovered from pNIPAm samples.

### **4.3. Multifunctional pNIPAm-based surfaces with biocidal and releasing properties obtained by MAPLE**

**Figure 8.** SEM images of the cells seeded on pNIPAm materials for 24 h: overview on the cells distributions over large area (a) and a close-up on the shape of the adhered L929 cells (b). For preparation of the samples for SEM analysis, the samples were washed with PBS and fixed with 2.5% glutaraldehyde in PBS for 20 min and subjected for ethanol gradi-

Nevertheless, the spreading of the cells and adherence can be inhibited by either surface chemistry or surface morphology [5]. The viability of the fibroblast cells after 3 days of culture on the pNIPAM coatings was calculated showing similar proliferation rate for the coatings obtained in the range of 200–600 mJcm−2. A cytotoxic effect and a decrease in proliferation rate

A recent study by Rusen et al. [5] on the cell detachment upon temperature reduction showed that the roughness of the thin films represents a significant parameter in tuning the detachment time. The quantitative analysis on the fibroblast cell harvesting upon temperature reduction showed that if cells detached from rougher films within 10–20 min, the detachment time on

The sequence of images presenting an example of the cell detachment from a pNIPAm thin film obtained by MAPLE is shown in **Figure 9**. The cells presented flat, spread, normal morphology at the beginning of the observations (**Figure 9a**), when the temperature was 37°C. However, by lowering the temperature the cells morphology started to change, becoming less spread and rounder (**Figure 9b**–**d**). When compared with previously reported data on cell detachment from pNIPAm coatings obtained by MAPLE [5], the detachment time is increased due to the low roughness (20 nm) pNIPAm surfaces used in this study. Moreover, the cells collected from pNIPAm coating obtained by MAPLE did not change their shape or viability. This represent a strong indication that a nondestructive reversible cell detachment takes place

in the case of the coatings obtained for higher fluences [5] were observed.

**4.2. Cell harvesting based on temperature change**

184 Modern Technologies for Creating the Thin-film Systems and Coatings

smooth surfaces increased with more than 25%.

when using pNIPAm bio-interfaces obtained by MAPLE [5].

ent dehydration.

Based on the same principle, resonant infrared matrix-assisted pulsed-laser evaporation (RIR-MAPLE) was used to co-deposit two components OPE/pNIPAm films with precise ratio control [39]. The sequential co-deposition mode allows depositions of a multifunctional coating comprising OPE and PNIPAm, used to kill bacteria and then release the accumulated dead bacteria from the film surface [39]. If laser fluence was previously used to change surface morphology [5], in this case, both surface morphology and wettability were adjusted by changing the OPE/PNIPAAm volume ratio in emulsion target used in the sequential deposition mode in RIR-MAPLE.

The biocidal activity and bacterial-releasing ability of the multifunctional films implied the use of both Gram-negative and Gram-positive bacteria strains such as *Escherichia coli* K12, respectively, *Staphylococcus epidermidis*. An inverse relationship between the biocidal activity and capability for bacterial release based on OPE content was observed. In this way, by increasing the quantity of OPE within the coatings, the resultant films showed increased biocidal activity but lower bacterial release capability. By optimizing the OPE/PNIPAAm ratio, coatings with enhanced biocidal activity against attached bacteria as well as good-removal capacity for the debris and bacteria when rinsing the films at a temperature below the LCST [39] were observed.

### **5. Conclusions and future prospects**

In this chapter, the new and rapidly emerging importance of smart-coating engineering was introduced, with focused attention on smart thermoresponsive pNIPAm interfaces obtained by matrix-assisted laser evaporation-based method. In this chapter, the thermoresponsivecoatings characteristics obtained by MAPLE-based method were emphasized along with some of the deposition parameters and used for biological assays *in vitro* implying BSA model proteins, mammalian cells and microorganisms.

MAPLE as technique for obtaining smart polymeric coatings with specific characteristics envisaging biological application provides the advantage of tailoring not only the thickness of the pNIPAm layer, which is an important parameter in the cell attachment, but also the morphology of the deposited thin films for influencing protein and cells detachment and its increased stability in the fluid medium. Although significant progress has been achieved in the field of smart coatings based on stimuli-responsive materials, the materials and methods discussed within this chapter still have limitations in practical applications that need to be overcome in the future.

Although the majority of the previous works in this field have used insoluble pNIPAm-based coatings, future research should be more directed toward biomimetic bio-interfaces, with integrated analysis platforms able to address the complexity of bio-environments accordingly to the desired application.

Considering the abovementioned advantages of the MAPLE method on tuning not only the surface characteristics and properties but also the chemical composition and film functionality, this approach could provide a new strategy to engineer multifunctional films for biological studies, regenerative medicine, tissue engineering and industrial applications.

### **Conflict of interest**

The authors declare no competing interests.

### **Acknowledgements**

The research leading to these results has received funding from the Romanian National Authority for Scientific Research (CNCS – UEFISCDI), under the projects PN-II-PT-PCCA-2013-4-1643, PNII- PT-PCCA-2013-4-199, PN-II-RU-TE-2014-4-2434, Romanian Academy Project 1/2015-2016 of the Institute of Biochemistry, University of Bucharest-Biology Doctoral School and Nucleus program—contract 4N/2016.
