A gradient of plasma polymerization of pyrrole was generated to model a chemoattractant surface; it was obtained by changing the polymerization time to manipulate the thickness and the deposited layer. The polymerization was generated with a power of 30W on a cylindrical vacuum reactor, the coverslip was polymerized covered by a cavity with a surface exposed of 0.7 cm every 35 min., the cells seeded were liver stellate cells (Ito); it was evaluated against a coverslip with continuous plasma polymerization of pyrrole (Ppy). The results show similar protein secretions, but cells proliferation was selective in the surface gradient regions

Godínez R

Ramírez -Fernández

Zúñiga-Aguilar E

CiteSeerX

Superficies y Vacío 27(1) 20-23, marzo de 2014 ©Sociedad Mexicana de Ciencia y Tecnología de Superficies y Materiales
 
*odinramirezfernandez@gmail.com 
20 
Plasma gradient modified scaffolds to generate a chemoattractant surface  
 
 
Ramírez-Fernández O.*, Godínez R., Zúñiga-Aguilar E. 
Departamento de Ingeniería Eléctrica Universidad Autónoma Metropolitana, Unidad Iztapalapa 
Apdo. Postal 55-534, Iztapalapa, México, D.F. 
 
López-Cobá C. A., Morales J., Olayo R. 
Departamento de Física Universidad Autónoma Metropolitana, Unidad Iztapalapa 
 Apdo. Postal 55-534, Iztapalapa, México, D.F.  
(Recibido: 28 de noviembre de 2013; Aceptado: 27 de febrero de 2014) 
 
 
A gradient of plasma polymerization of pyrrole was generated to model a chemoattractant surface; it was obtained by 
changing the polymerization time to manipulate the thickness and the deposited layer. The polymerization was generated 
with a power of 30W on a cylindrical vacuum reactor, the coverslip was polymerized covered  by a cavity  with a surface 
exposed  of 0.7 cm every 35 min., the cells seeded were liver stellate cells (Ito); it was evaluated against a coverslip with 
continuous plasma polymerization of pyrrole (Ppy). The results show  similar protein secretions, but cells proliferation 
was selective in   the surface gradient regions. 
 
Keywords: Chemotaxis; Chemoattractant; Plasma Polypyrrole; Ito cells; Cell Culture; Modified Surfaces; Biomaterials 
 
1. Introduction  
 
The aim of tissue engineering is to obtain tissue models 
that regenerate and replace a functional tissue or organ 
[1].The tendency of the in vitro culture is to generate 
physiologically functional tissue to generate models of 
complex biological tissues [2].  
The cell culture surfaces are designed to support cell 
growth and perform specific functions, as those tissues in 
vivo [3, 4].  The cell culture often requires designed 
substrates or scaffolds that allow control over cell 
proliferation and function [5]. 
At present, a set of biological, physical, and chemical 
methods are used to do surface modifications on 
biomedical devices and biomaterials [6]. These approaches 
can be used to tune a range of properties, including 
biostability and/or chemical inertness and cellular adhesion 
[7, 8]. The materials derived from the pyrrole are being 
used as an alternative to cultivate tissue. In biological 
environments the biocompatibility of the Ppy was tested 
both in vitro and in vivo [9, 10].  
Plasma polymers differ from the conventionally 
synthesized polymers with their highly complex structure, 
with branched or cross linked chains [11]. The course of 
plasma polymerization involves the generation and 
dissociation of complicated reactive species such as 
electrons, ions, radicals, atoms and molecules. Therefore, 
only limited supporting literature detailed the relationships 
between the deposition kinetics with the physical–chemical 
properties of plasma polymers [12-16]. 
The final properties of the tissue will be in function of 
their movements responding to the chemical environment 
(chemotaxis) [17]. Gradient surfaces have recently been 
established as tools allowing the screening of cell 
proliferation towards its possible optimization. Plasma 
polymerization lends itself to surface gradient formation, 
since it supports the introduction of a wide range of surface 
chemistries and forms well-adherent layers on a range of 
substrates [18-21]. The technique also allows surfaces to be 
modified independently of the underlying substrate, with 
little change to surface topography [22].  
In a modified culture surface it is possible to create a 
layer of cells with the most convenient geometry for the 
culture. In case of cocultures the different cells location 
plays a vital role because the organ functionality are 
strongly linked to the cell structure. For the particular case 
of the liver, the stellate cells synthesize extracellular matrix 
(ECM) components including collagen, which has the role 
of natural scaffolds then it is important to manipulate the 
regions where the stellate cells are growing.[10] 
A chemoattractant model was created with the gradient 
coverslip using liver stellate cells. It was evaluated against 
a coverslip with continuous Ppy. The cells morphology 
were registered by microscopy, to corroborate the cell 
proliferation on the material, pictures of the samples with 
the cells were taken  three times a week. Finally the 
metabolic functionality was measure with the protein 
secretion rate in the supernatant and was compared with the 
control material. In conclusion we obtain a Ppy surface that 
optimizes the conditions for the best cell culture. 
 
2. Materials and Methods 
 
2.1. Plasma Polymerization  
 
The coverslips were modified in the surface with 
polypyrrole (Aldrich 98%) by plasma glow discharge with 
the following reactor conditions: pressure was on 9x10-2 
Torr, measured with a vacuum sensor (Edwards Active 
Gauge Control), power of 30 watts, obtained from a source 
of radio frequency at 13.5 MHz (Dressler Cesar RF). 
Reaction time was 30 minutes [8]. The gradient of Ppy was  
Superficies y Vacío 27(1) 20-23, marzo de 2014 ©Sociedad Mexicana de Ciencia y Tecnología de Superficies y Materiales
 
 
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Figure 1. Schematic of coverslip gradient: the chamber has a mobile-
support that moves the coverslip through a parallel surface that help to 
control the Ppy deposited. 
 
 
 
Figure 2. FT-IR spectrum in gradient coated Ppy coverslip at different 
regions. The blue pointed line correspondence to clear coverslip, the red 
line is for the next section, the black line is for the third section, the green 
line is for the fourth section and the yellow line is for the final region. 
 
 
 
Figure 3. In A) show the gradient coverslip with different times of Ppy 
deposition, in B) show the continuous polymerization that covers the 
coverslip in a uniform way. 
 
 
 
Figure 4. After 3 day of culture the coverslips were photograph in A) 
shows the cell distribution in continuous coverslip, B),C),D),E) and F) 
shows the cells in gradient coverslip from more Ppy time exposition to 
less. 
 
generated with a power of 30W on a cylindrical reactor, 
and the substrate was moved into the cavity 0.7 cm every 
35 min. as shown in figure 1. 
 
2.2. Cell cultures 
 
The stellate cells were routinely grown in a monolayer 
culture in the presence of a Williams E medium (Gibco 
12551) supplemented with 10% fetal bovine serum (Gibco 
16000), penicillin (100 units /ml) and streptomycin (100 
mg/ml). Cells were grown at 37 ◦C in disposable plastic 
bottles (Nunc, USA), in a humidified atmosphere of 5% 
CO2 and 95% air. The medium was replaced twice a week, 
and cells were trypsinized and re-seeded every 7 days at a 
ratio of 1:3. [10].  
 
3. Experimental Design 
 
Stellate cells were seeded (5 x 105 cells per coverslip) 
directly on the gradient coverslip. It was evaluated against 
a coverslip with continuous Ppy surface polymerized at 
30w for 20 min. Each coverslip was made in triplicate, n = 
3, the dimensions of the coverslip are as follows: long 6.0 
cm, width 2.0 cm; thickness 0.05 cm. The culture medium 
was sampled daily by 7 days. The experiment was repeated 
with the continuous coverslip. 
 
3.1. Total protein rate 
 
Bicinchoninic acid (BCA) protein analysis kit (Thermo 
Scientific 23225) was used to determine the total protein in 
supernatant, which works with colorimetry, at a 
wavelength of 562 nm. The standard curve was obtained 
initially obtained using Bovine Seric Albumin (BSA) at 
various concentrations provided by the kit. Samples of 0.5 
ml were taken of the supernatant daily, they were placed in 
a 96-well plate to measure the concentration in a 
multimodal detector plates DTX 880 (Beckman Coulter) 
[10]. 
 
3.2. Distribution of living cells 
 
To identify the cells those were staining with 
immunofluorescence technique of Hoetscht. This technique 
allowed observing the nucleus of functional stellate cells 
which were marked with fluorescent material. In addition 
to show the cell distribution in the modified coverslip; also 
it serves to identify the viable cells, since it reacts only 
with the functional cell nucleus. The photographs were 
taken in random way, in different zones of the coverslip to 
probe the different cells distribution.  
 
3.3. Data analysis  
 
Data are reported as mean ±SE for at least three 
independent experiments carried out in triplicate. The 
Origin package version 8.1 was used for statistical 
significance. Comparison among groups was done by  
Superficies y Vacío 27(1) 20-23, marzo de 2014 ©Sociedad Mexicana de Ciencia y Tecnología de Superficies y Materiales
 
 
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Figure 5. Hoetscht stain on day 5 in cell culture on the coverslips with the 
gradient Ppy and the continuous surface, the cells are the blue points in 
the photographs A), B), C), D), E) and F) ; G) Continuous Ppy surface 
(100 x original, scale 100 micron). 
 
 
 
 
Figure 6. Protein secretions after 5 culture days, where day one is the 
seeded day.There is no significant difference between the continuous 
(white squares) and the gradient coverslip (black squares). p<0,05. 
 
 
 
 
 
 
means of ANOVA. Tukey's method was used for multiple 
comparisons. A P<0.05 was considered as statistically 
significant. 
 
4. Results and discussion  
 
The FT-IR spectrum for the control coverslip at 30 watts 
30 minutes was presented in previous works, and the 
values show the vibration of primary amines at 3350cm-1, 
the aliphatic carbons at 2964cm-1, the nitrile groups at 
2200cm-1, at 1624cm-1carbonyl groups [8].  
Figure 2 shows the Ppy composition for the different 
sections of the gradient Ppy coverslip that change along the 
film thickness direction of the gradient. As show in the 
peak for the silicates (principal component of glass) at 850 
cm-1 [23]; where the pointed blue line is for the clear glass 
and the yellow line is for the largest reaction time. The 
silicate peak absorbance, decrease in function of exposure 
time. The better culture regions show a largest aliphatic 
and primary amines peaks which are presents in the 
second, third and fourth increase exposure regions that 
correspondence to red, black and green lines. We suppose 
that those chemical groups increase the cellular adhesion 
and eventually the proliferation. 
Figure 3 shows the results of the polymerization in the 
surface of the coverslips, been the principal indicator of the 
Ppy thin film, the brown color; this film cover all the 
structure in a way that depends of the time exposure. When 
the regions and the exposure time were controlled the 
coverslip shows a concentration gradient showed on the 
thickness of the Ppy. 
In previous experiments the film thicknesses were 
characterized in base of the exposure time of the coverslips 
and they were correlated with the cell viability and their 
physiology, in base of these results we obtain a coverslip 
where we control the cell proliferation by regions with 
different Ppy concentrations. Because the stellate cell plays 
a scaffold role in the liver coculture and the extracellular 
matrix don’t have a specific protein to measure, we decide 
to measure the total protein secreted to the supernatant.  
The comparison of stellate cell cultures, using the 
coverslips with Ppy and the gradient coat was done, 5x105 
cells by coverslip were used; passage 5; with 15 ml of 
culture medium for 5 days in a CO2 incubator. 
Figure 4 shows, the cell culture on continuous coverslip 
(A) with a uniform cell distribution, and the cell culture on 
the gradient coverslip from the bigger to the lower Ppy 
deposition (B to F); the proliferation increases in the 
central zones, the typical cell shape in the scaffold is 
marked in a black circle. 
To probe the cellular distribution in the gradient surface 
at day 5, the cells were marked with Hoetscht stain to 
identify the cell nucleus. Figure 5 shows the photographs 
of the stained cells with the fluorescent marker (A to F), it 
shows the cellular distribution on the gradient scaffold 
from less to greater concentration of Ppy, the dyed cells are 
shown in cyan color. G Shows the cellular distribution on 
the continuous Ppy coverslip, there are clusters of dyed 
Superficies y Vacío 27(1) 20-23, marzo de 2014 ©Sociedad Mexicana de Ciencia y Tecnología de Superficies y Materiales
 
 
23 
cells in the entire surface. 
In the gradient coverslip the cells prefer the central places 
to proliferate and it decrease in function of either lower or 
larger concentration of Ppy, since the F sample  shows only 
2 cells in the caption, the central regions have a similar 
concentration of Ppy as the continuous coverslip. 
The cell adhesion, commonly known as; a direct 
reflection of the amount of Ppy functionalities on the 
surface of materials, did not correspond to the higher 
deposition time or to the thicker plasma polymerized thin 
film. We didn't observed morphological differences 
between the coated surfaces. 
Figure 6 show the total protein rate in the supernatant of 
the cell culture medium. In the figure, the black squares 
line, represents the change of protein secretion in the 
medium after 5 days of culture on the gradient coverslip. 
The white squares line shows the total protein secretion in 
the continuous coverslip. As it can be seen, there is no 
significant difference in the two experiments. 
Also, we analyzed the whole supernatant protein 
secreted, the results show similar values in the secreted 
protein when we used the Ppy gradient in comparison to 
the continuous surface, we obtained maximums of 14 
µg/ml with no significant differences. That suggests that 
the cell physiology is strongly linked to the cell 
distribution. 
 
5. Conclusion 
 
In this work is describing a novel method to fabricate 
plasma polymer gradients by using a tilted mask to control 
the concentration of plasma in the substrate. By forming a 
gradient layer of Ppy film, we created a chemical gradient 
surface suitable to examine the effect of cell proliferation 
and spreading on stellate cells protein secretion. The cells 
colony and morphology correlated with the continuous 
coverslip as show in the nucleus cell markers photographs. 
Cell protein response was similar in both experiments that 
show a comfortable surface for the cells. In conclusion we 
obtain a Ppy surface that models the conditions of 
chemoattractant surface to obtain a controlled cell culture. 
 
Acknowledgements 
 
The authors would like to thanks to Universidad 
AutonomaMetropolitana (UAM), ConsejoNacional de 
Ciencia y Tecnologia (CONACYT) (project 155239) and 
Instituto de Ciencia Y Tecnologia Del D.F. (ICyT-DF) 
(PIUTE 10-63276/2010) y (PICSA 11-14/2011), for their 
collaboration to bring to perform this work. 
 
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Plasma gradient modified scaffolds to generate a chemoattractant surface

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Plasma gradient modified scaffolds to generate a chemoattractant surface

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