**4. Experimental and results**

chains (or smaller molecular weight) diffuse faster into the polymer surface leading to an

According to Shananan et al.[72], contact angle hysteresis is related to the polymer polarity. Indeed, when a polymer gets in touch with a polar liquid (water), it orients its mobile polar groups on the surface in order to increase the interfacial water/polymer energy and there‐ fore decreasing the system surface free energy. In the other hand, when the polymer is con‐ tact with a non polar liquid, its functional groups conserve their state and will not reorient. These authors assumed the existence of two parameters behind hysteresis: the intrinsic po‐ larity of the material and the mobility of its polar groups on the surface. Nishioka et al.[73], had observed that the advancing contact angle hysteresis is under the control of surface sites

The contact angle hysteresis observed on hydrophilic and hydrated polymers is due to the polar groups' orientation on the interfaces polymer/liquid and polymer/air. This reorienta‐ tion represents the polymer reaction to every environmental change (air, liquid). The reced‐ ing contact angle (θr) depends on the contact duration with water, the environment temperature and on the glass transition temperature (Tg) of the material itself. Each material has its own glass transition temperature (Tg) allowing a defined molecular mobility suffi‐

The concepts of solid surfaces assumed that the surfaces in question were effectively rigid and immobile. Such assumptions allow one to develop certain models and mathematical re‐ lationships useful for estimating and understanding surface energies, surface stresses, and specific interactions, such as adsorption, wetting, and contact angles. It is assumed that the surfaces themselves do not change or respond in any specific way to the presence of a con‐ tacting liquid phase, thereby altering their specific surface energy [75]. Although such as‐ sumptions are (or may be) valid for truly rigid crystalline or amorphous solids, they more

In contact with condensed phases, especially liquids, surface relaxations and transitions can become quite important leading to a possible dramatically change in the interfacial charac‐ teristics of a polymer with possibly important consequences in a particular application. And since the processes are time-dependent, the changes may not be evident over the short span of a normal experiment. For critical applications in which a polymer surface will be in con‐ tact with a liquid phase, such as implant device for biomedical application, it is not only im‐ portant to know the surface characteristics (e.g., coefficient of friction, adhesion, adsorption)under normal experimental conditions but also to determine the effects of pro‐ longed (equilibrium) exposure to the liquid medium of interest. It is therefore important for biomedical as well as many other applications that the surface characteristics of a material of interest be determined under conditions that mimic as closely as possible the conditions of use and over extended periods of exposure to those conditions, in addition to the usual

more hydrophobic than those controlling the receding contact angle hysteresis.

important decrease in contact angle.

226 Advances in Biomaterials Science and Biomedical Applications

cient for an important rearrangement [74].

often than not do not apply strictly to polymeric surfaces.

**3.3. Conclusion**

characterizations.

#### **4.1. Polyelectrolyte multilayer film preparation**

Before use, glass slides were cleaned in 0.01 M SDS and then in 0.1 N HCl, both for 10 min in a boiling water bath, followed by a pure water rinse. Polyelectrolyte solutions were pre‐ pared by dissolution of the polyelectrolyte powders in 0.15 M NaCl (using ultrapure water filtered with a MilliQ system, Millipore) at a concentration of 1mg/l for PLL, PGA and HA and 5 mg/l for PEI, PSS and PAH. For all the films, the precursor layer was always PEI (pol‐ ycation), followed by the alternate adsorption of polyanions/polycations for 12 min adsorp‐ tion times and two rinses in the 0.15 M NaCl solution [76]. The glass slides held in a slide holder were dipped into the different polyelectrolyte baths for the preparation of three dif‐ ferent types of film, ending either by the polycation or polyanion: (PSS/PAH)10, (PSS/ PAH)10–PSS; (PGA/PLL)5, (PGA/PLL)5–PGA; and (HA/PLL)5, (HA/PLL)5–HA. Cleaning was made before film characterization. The films were all prepared at the same pH before being in contact with culture medium. Poly(styrene-4-sulfonate) (PSS,MW=70 kDa), Poly(allylamine hydrochloride) (PAH,MW=70 kDa) and Poly(ethyleneimine) (PEI,MW=70 kDa) are purchased from Aldrich. Poly(l-lysine) (PLL, MW=32 KDa) Poly(l-glutamic acid) (PGA, MW=72 KDa) were obtained from Sigma and Hyaluronan (HA,MW=400 kDa) from Bioiberi‐ ca. Sodium dodecyl sulfate (SDS) was purchased from Sigma and sodium chloride (NaCl, purity ~ 99%) from Aldrich, glass slides (18x18 cm2 square and 14x14 cm2 disk), respectively, were obtained from CML, France.

#### **4.2. Contact angle measurement and Surface Free Energy (SFE) calculation**

The measurements were performed with a Wilhelmy balance for the characterization of sol‐ ids using the 3S tensiometer and the corresponding software (GBX, France). For these ex‐ periments, the glass slides were coated with polyelectrolyte multilayer films on both sides. Before beginning the measurements, the films were washed in 18.2 MΩ Millipore water for 30–45 min in order to eliminate the NaCl traces that could modify the results. Samples were then dried at 30 °C for 2 h. The dynamic contact angle hysteresis was determined at 20°C for each film and five wetting/dewetting cycles were carried out at a 50 μm/s speed.

Three liquids were used as a probe for surface free energy calculations: diiodomethane, for‐ mamide (Sigma Chemical CO, St Louis, MO, USA) and distilled water. The final contact an‐ gle used for this calculation was the average of the 2nd to 5th cycle advancing contact angle (θa) and the surface free energies of the different films were calculated using the Van Oss (VO) approach, as usual with sessile drop method contact angles:

$$\gamma\_S = \gamma\_S \, ^d \star \, \mathcal{D} \left( \gamma\_S \, ^\* \cdot \gamma\_S \, ^\* \right)^{\vee 2}$$

This method produces the dispersive (γ<sup>S</sup> <sup>d</sup>) and the polar acid–base (γ<sup>S</sup> + , γ<sup>S</sup> - ) components. Sol‐ id and liquid SFE components and contact angle are related according to the equation be‐ low:

$$\gamma\_L \begin{pmatrix} 1 \ + \end{pmatrix} \otimes \begin{pmatrix} \end{pmatrix} = 2 \left( \langle \gamma\_S^{\;d} \cdot \gamma\_L^{\;d} \rangle^{\sharp\_{\mathcal{S}}} + \langle \gamma\_S^{\ast} \cdot \gamma\_L^{\ast} \rangle^{\sharp\_{\mathcal{S}}} + \langle \gamma\_L^{\ast} \cdot \gamma\_S^{\ast} \rangle^{\sharp\_{\mathcal{S}}} \right)$$

Were γL is the SFE of the liquid and γs the SFE of the surface.

#### **4.3. Cell adhesion, viability and morphology study**

For adhered cell counting, image analysis was performed on a Quantimet 570 (Leica, UK) fitted to an epifluorescence microscope (Axioplan, Zeiss, DE) and a black-and-white chargecoupled device (CCD) camera (LH51XX-SPU, Lhesa Electronique, FR). The scanning was carried out using a ten times lens (NA=0.3) and a filter set adapted for propidium iodide flu‐ orescence observation (BP 546/12 nm, DM 580 nm, LP 590 nm). Microscope focus and stage were motorized and software controlled.

The cell viability was determined with the MTT colorimetric assay. It was measured at 570 nm with a 96-well microplate reader (Becton Dinkinson, Lincoln Park, USA) on a spectro‐ photometer (Bio-Tek Instruments, Winooski, USA). The blank reference was taken for wells containing only the MTT solution.

The morphology of the cells was analyzed after 120 min (day 0), 2 and 7 days of culture us‐ ing a scanning electron microscopy (Philips, EDAX XL-20) and phase contrast microscopy.

#### **4.4. Results**

#### *4.4.1. Contact angle measurement*

The different contact angle values found are shown in Table 1. Experiments were performed at 20 °C at a speed of 50 μm/s. One can observe that contact angle depends on the film's na‐ ture (physico-chemical composition) which differs from a polymer to another.


**Table 1.** Dynamic contact angle

#### *4.4.2. SFE values*

Were γL is the SFE of the liquid and γs the SFE of the surface.

For adhered cell counting, image analysis was performed on a Quantimet 570 (Leica, UK) fitted to an epifluorescence microscope (Axioplan, Zeiss, DE) and a black-and-white chargecoupled device (CCD) camera (LH51XX-SPU, Lhesa Electronique, FR). The scanning was carried out using a ten times lens (NA=0.3) and a filter set adapted for propidium iodide flu‐ orescence observation (BP 546/12 nm, DM 580 nm, LP 590 nm). Microscope focus and stage

The cell viability was determined with the MTT colorimetric assay. It was measured at 570 nm with a 96-well microplate reader (Becton Dinkinson, Lincoln Park, USA) on a spectro‐ photometer (Bio-Tek Instruments, Winooski, USA). The blank reference was taken for wells

The morphology of the cells was analyzed after 120 min (day 0), 2 and 7 days of culture us‐ ing a scanning electron microscopy (Philips, EDAX XL-20) and phase contrast microscopy.

The different contact angle values found are shown in Table 1. Experiments were performed at 20 °C at a speed of 50 μm/s. One can observe that contact angle depends on the film's na‐

Glass 43 ± 2 23 ± 3 43 ± 3.1

(HA/PLL) 10 12 ± 2 00 00

(HA/PLL)5 81.9 ± 1.8 49.6 ± 2 43.5 ± 3

(HA/PLL)5-HA 87.8 ± 1.2 00 45 ± 2.9

(PGA/PLL)5 55.2 ± 3 14.7 ± 2.5 39.1 ± 1.2

(PGA/PLL)5-PGA 44.1 ± 3.1 00 40.7 ± 1.6

(PSS/PAH)10 49.2 ± 1.8 23.6 ± 3 00

(PSS/PAH)10-PSS 53 ± 1.9 12.1 ± 3.3 00

**Water Formamide Diiodomethane**

ture (physico-chemical composition) which differs from a polymer to another.

**4.3. Cell adhesion, viability and morphology study**

228 Advances in Biomaterials Science and Biomedical Applications

were motorized and software controlled.

containing only the MTT solution.

*4.4.1. Contact angle measurement*

**Table 1.** Dynamic contact angle

**4.4. Results**

SFE and its component's values are summarized in Table 2. (HA/PLL) films have the lowest SFE value and (PSS/PAH) films have the highest value. The outermost layer of the film does not have a great influence.


**Table 2.** Surface Free Energy (SFE) and its components for the different films used. The SFE of PSS/PAH is higher compared to the other films.

#### *4.4.3. Cell adhesion*

Figure 8 shows the percentage of fibroblasts that have adhered after 2 h in culture. The high‐ est adhesion is found with (PGA/PLL)5 film (95%) and the lowest on (HA/PLL)5 film (49%).

**Figure 8.** Fibroblast adhesion rate after 2 h in culture onto different films. The percentage represents the number of the adhered cells compared to the initial number of seeded cells.

#### *4.4.4. Cell viability and proliferation rate*

Cell viability was evaluated on the different types of film at different time intervals (0, 2 and 7 days) with the MTT assay (Figure 9A). The (PGA/PLL)5–PGA films exhibited a good proliferation rate (Figure 9B) and the (PSS/PAH)10 films were the most favorable to cell proliferation.

#### *4.4.5. Cell morphology*

Good adhesion is observed on (PGA/PLL)5 film (Figure 10A) whereas bad adhesion was found on (HA/PLL)5-HA film (Figure 10B). Typical morphology at day 2 on a (PGA/PLL)5– PGA film is presented in Figure 10C. After seven days in culture, the difference in morphol‐ ogy for the cells that had adhered to the different films was even more striking. Cells in con‐ tact with (HA/PLL)5–HA exhibit necroses (Figure 11A) whereas the cells exhibit elongated and spread morphologies on the highly proliferative (PSS/PAH)10 films (Figure 11B).

cell proliferation.

*4.4.5. Cell morphology*

66

the adhered cells compared to the initial number of seeded cells.

*4.4.4. Cell viability and proliferation rate*

Adhesion rate (%)

230 Advances in Biomaterials Science and Biomedical Applications

49

95

65

**Figure 8.** Fibroblast adhesion rate after 2 h in culture onto different films. The percentage represents the number of

Cell viability was evaluated on the different types of film at different time intervals (0, 2 and 7 days) with the MTT assay (Figure 9A). The (PGA/PLL)5–PGA films exhibited a good proliferation rate (Figure 9B) and the (PSS/PAH)10 films were the most favorable to

Good adhesion is observed on (PGA/PLL)5 film (Figure 10A) whereas bad adhesion was found on (HA/PLL)5-HA film (Figure 10B). Typical morphology at day 2 on a (PGA/PLL)5– PGA film is presented in Figure 10C. After seven days in culture, the difference in morphol‐ ogy for the cells that had adhered to the different films was even more striking. Cells in con‐ tact with (HA/PLL)5–HA exhibit necroses (Figure 11A) whereas the cells exhibit elongated

and spread morphologies on the highly proliferative (PSS/PAH)10 films (Figure 11B).

75 76

60

76

B

**Figure 9.** A. Cell viability (MTT test) on each film type followed over a seven day period at: day 0 (D0), day 2 (D2), and day 7 (D7), B. Proliferation rate on the different films as estimated by the ratio (D7/D0)

**Figure 10.** SEM images of cells adhering to different polyelectrolyte multilayer films. (A) (PGA/PLL)5(x800) on the first day, (B) HA/PLL)5–HA (x800) on the first day, (C) (PGA/PLL)5–PGA film observed on the second day (x2725).

**Figure 11.** SEM images of cell morphology after seven days of culture. (A) (HA/PLL)5–HA (x800) film, a (PSS/PAH)10 film observed at different magnifications (B) (x1398): some areas are at confluency.

#### *4.4.6. Correlation between cell adhesion and films SFE*

**Figure 10.** SEM images of cells adhering to different polyelectrolyte multilayer films. (A) (PGA/PLL)5(x800) on the first

day, (B) HA/PLL)5–HA (x800) on the first day, (C) (PGA/PLL)5–PGA film observed on the second day (x2725).

232 Advances in Biomaterials Science and Biomedical Applications

No correlation was found between the wettability parameters or the SFE parameters and the fibroblast proliferation ratio. However, the adhesion rate at 2 h was correlated to both SFE basic component and the SFE acid component (Figure 12). For the adhesion rate, the SFE ba‐ sic component is optimum at 15 mN/m (Figure 12A) whereas the acid one is optimum at about 5 mN/m (Figure 12B).

**Figure 12.** SEM images of cell morphology after seven days of culture. (A) (HA/PLL)5–HA (x800) film, a (PSS/PAH)10 film observed at different magnifications (B) (x1398): some areas are at confluency. Figure 12B. Correlation between cell adhesion rate and Basic SFE component. An optimum is found for 15 mN/m with good polynomial correlation (R2=0.93)

B

#### **4.5. General conclusion**

sic component is optimum at 15 mN/m (Figure 12A) whereas the acid one is optimum at

A

0 2 4 6 8 10 12 14 Acid SFE component (mN/m)

> y = -0.2194x2 + 6.4466x + 46.02 R² = 0.93

y = -0.9072x2 + 9.1169x + 65.471 R² = 0.8009

B

**Figure 12.** SEM images of cell morphology after seven days of culture. (A) (HA/PLL)5–HA (x800) film, a (PSS/PAH)10 film observed at different magnifications (B) (x1398): some areas are at confluency. Figure 12B. Correlation between cell adhesion rate and Basic SFE component. An optimum is found for 15 mN/m with good polynomial correlation

0 5 10 15 20 25 Basic SFE component (mN/m)

about 5 mN/m (Figure 12B).

0

B

0

(R2=0.93)

20

40

60

80

Adhesion rate (%)

100

120

20

40

60

80

Adhesion rate (%)

100

120

234 Advances in Biomaterials Science and Biomedical Applications

A

Cell adhesion is a paramount parameter for the biomaterial tissue. These biomaterials, by their surface properties (chemical composition, topography, roughness, surface energy) hold the key of the control of the cell adhesion, proliferation and orientation. Thus, the concept of biocompatibility is seen imposed, it is primarily focused on the interface, sites of the interac‐ tions between cells and biomaterials.

The influence of different polyelectrolyte multilayer films (PEM) on gingival fibroblast cell response was studied. Roughness and hydrophobicity/hydrophilicity of the PEM were char‐ acterized by contact angle measurement. Polar (acid-basic) components of the surface free energy (SFE) were determined. Surface advancing and receding angles were measured and hysteresis was determined. Cell adhesion, viability and morphology were analyzed.

This work pointed out that cell adherence is a complex process modulated by numerous pa‐ rameters. Usually, in cell adherence studies and particularly in biomaterial approaches, sur‐ face physico-chemical properties are analysed (chemistry, roughness, motility, wettability…).

In our work we tackled the subject of the cellular behavior in contact with a biomaterial by the characterization of the surface of this material. We were interested in physical (topogra‐ phy) and chemical (composition) properties of various polyelectrolyte multilayer films de‐ posited on glass slides, with different charge densities scale and thickness. We have evaluated the wettability of theses biomaterials by measuring the contact angle hysteresis using the Wilhelmy balance tensiometry to study their physico-chemical characteristics in order to understand the effects of surface roughness and chemistry on the fibroblasts behav‐ ior. Epifluorescence microscopy, SEM, phase contrast microscopy and MTT test were used to study cell adhesion, proliferation and morphology in order to correlate the film's proper‐ ties and the cultivated cells response.

Surface hydrophobicity and roughness were found to be unfavourable for both adhesion and proliferation. Adhesion and proliferation were found not to be correlated.
