FORMATION AND PROPERTIES OF POLYMER NANOLAYERS TO ENHANCE CELL GROWTH IN VITRO

Three-dimensional (3D) cultures models may more accurate representation of the in vivo environment than two-dimensional (2D) cultures while maintaining the cytoarchitecture of in situ tissue that supports cells differentiation or maturation [1, 2]. Cell adhesion is depended upon surface hydrophilicity, surface charge density, surface morpho logy, specific chemical groups present on the surface of the scaffold [3]. Given that surface chemistry is crucial for the biocompatibility of the nanolayers, specific surface modifications are used with different polymers [4, 5]. In particular, there is an increased interest in the polymeric surfaces which can change their affinity towards proteins and cells under external stimuli [6, 7] and therefore have potential applications in biology and medicine. Despite various investigations, specific and complex mechanisms govern the reactions that occur between the biomaterial and the cellular environment are still incomplete understanding. The objective of this study was to establish and comparison cells line B16F10 viability cultured onto different coatings. We used surfaces obtained by grafting APTES, dextran, albumin and their combinations to the surface of the modified glass plates.


Introduction
Three-dimensional (3D) cultures models may more accurate representation of the in vivo environment than two-dimensional (2D) cultures while maintaining the cytoarchitecture of in situ tissue that supports cells differentiation or maturation [1,2].
Cell adhesion is depended upon surface hydrophilicity, surface charge density, surface morpho logy, specific chemical groups present on the surface of the scaffold [3].Given that surface chemistry is crucial for the biocompatibility of the nanolayers, specific surface modifications are used with different polymers [4,5].
In particular, there is an increased interest in the polymeric surfaces which can change their affinity towards proteins and cells under external stimuli [6,7] and therefore have potential applications in biology and medicine.Despite various investigations, specific and complex mechanisms govern the reactions that occur between the biomaterial and the cellular environment are still incomplete understanding.
The objective of this study was to establish and comparison cells line B16F10 viability cultured onto different coatings.We used surfaces obtained by grafting APTES, dextran, albumin and their combinations to the surface of the modified glass plates.

Preparation of coatings. Glass plates (2020)
were dipped into 0.2 % (w/w) methanolic solution of (3-aminopropyl)triethoxysilane (APTES) for 24 h.After the incubation, loosely-attached silane molecules were removed with methanol in Soxhlet's apparatus.Then the plates functionalized with APTES were dipped into 1 % solution of peroxide in air dioxin for 24 h.Similarly, loosely attached peroxide was removed with dioxane in Soxhlet's apparatus for 4 h.As a result, peroxides grafted to aminated surfaces were obtained [8].
With participation of these aminogroups dialdehydedextran obtained by partial oxidation of the anhydroglucopyranoside subunits of dextran by periodate acid was covalently grafted to the surface of the modified glass plates.The oxidation of dextran was conducted for 2 h, and then the plates functionalized with amino-terminated APTES were dipped into 2 % solution of dialdehydedextran in water for a grafting time of 6 h.Similarly, loosely-attached dialdehydedextran was removed with water in Soxhlet's apparatus for 4 h.As a result, dialdehydedextran grafted to aminated surfaces were obtained.
The influence of the surface properties on the proliferation of B16-F10 cells culture and its ability to form a monolayer was investigated.Cells were allowed to attach and proliferate for 24, 48, and 72 h.After each time point, the number of plated cells was determined by counting using a hematocytometer.For each group, three samples were tested independently.Trypan blue exclusion test (0.4 %) was used to discriminate and count living cells.

Lactate dehydrogenase (LDH) assays.
Medium supernatants collected from both experimental and control cells every 24 h were tested for LDH activity using Cytotoxicity Detection kit LDH (Roche).This test is a colorimetric assay for the quantification of cell death and cell lysis based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant.The amount of enzyme activity detected in the culture supernatant correlates with the proportion of lysed cells.The assays were conducted following the manufacturer's instructions, in flat-bottomed wells of 96well plates.Following incubation, the absorbance of samples was measured at a wavelength of 490 nm as a measure of enzyme activity using an ELISA (enzyme-linked immunosorbent assay) plate reader.
Evaluation of cytotoxicity using the microtetrazolium (MTT) assay.The MTT assay was used to assess the in vitro cytotoxicity of surface-modified glasses in this study.A quantitative colorimetric MTT test was performed after 3 days of culture to characte rize cellular metabolism (vitality) and, by implication, proliferation.The MTT solution (0.5 mg/ml) was added to each well 3 h prior to the end of the experiment.After the incubation period dimethylsulfoxide was added in the dish to dissolve the purple formazan crystals that formed as a result of the restoration of MTT reagent-reductase living cells.The concentration of formazan in the wells was determined by the spectrophotometric method at a wavelength of 490 nm.The number of living cells (in percentage) was determined by the ratio of optical in which the cells were incubated for study and control mediums.
Statistical analysis.Tests were repeated three times for every type of the samples.The results were presented as the mean ± standard deviation.Differences between groups were determined by Student t-tests.

Results
The influence of the surface nature on the proliferative growth of B16F10 cells during 72 h was investigated (Table ).The results of our investigations show that proliferative activity of B16F10 cells was gradually increased in the experimental group with APTES (P < 0.01).While in the experimental groups with APTES/dextran cells concentration were lower compared to control.Improved the viability and proliferation of the B16F10 cells were observed after cultivation onto albumin (P < 0.001) and APTES/dextran/albumin (P < 0.001) nanolayers during 48 and 72 h compared to control and other experimental groups.
The viability and functional activity of B16F10 cells under cultivation onto different nanolayers we investigated of LDH levels (Fig. 1).
LDH concentration in all groups was sufficiently high at the beginning of the experiment.As increasing times cultivation, LDH activity was changed differently in each group.Enzyme activity significantly reduced in cells cultivated onto APTES, albumin and APTES/dextran/albumin coating at 48 and 72 h as opposed to the group with APTES/dextran coating.The higher concentration of LDH during whole time cultivation were observed in the experimental group with APTES/ albumin compared to other experimental groups.
Decreasing lactate dehydrogenase activity was observed in APTES, albumin, and APTES/dextran/ albumin coating experimental groups at 48-72 h of cultivation, which coincides with the growth of the proliferative activity of cells in these groups and high cell viability.
Biocompatibility and functional impact of modified surfaces were studied in vitro using MTT assay.In Fig. 2 the data of obtained results of MTT absorbance value for cells after cultivation onto different nanolayers are representing.The cell viability in the experimental group with APTES/dextran/albumin was the highest du ring the whole time of cultivation compared to other experimental groups.Whereas the lowest cell viability was noted in the experimental group with APTES/dextran.The absorbance values in experimental groups with albumin and APTES/albumin were also higher.The MTT assay showed 86.6-79.7 % and 69.5-73.4% of viability to B16F10 cells in this groups respectively.

Discussion
Most cells are anchorage-dependent because they grow as monolayers and require attachment to proliferate.Disposable plastic, especially polystyrene is now most commonly used for cell culture growth.But many cells prefer surfaces with high surface ene rgi es (i.e.hydrophilic surfaces).Whereas most plastics are hydrophobic and unsuitable for cell growth, they are often treated with radiation, chemicals or electric ion discharge to generate a charged, hydrophilic surface.The cha racteristics of glasses depend on organic compounds properties present in their composites its chemical composition, surface area, and textural properties (pore size, pore volume, pore structure) [10].Given that surface chemistry is crucial for the biocom- patibility of the nanolayers, the coatings should be tested on various cell types [11].But methods of investigations of effects of the biocompatible and smart bioactive surfaces on cells are still not fully developed.For these studies usually are applied cell viability tests and counted a quantity of the living cells with different morphology and also their proliferation index.Among different surface modification approaches studied, grafting of carbon nanotubes on the surface provides the growth, morphology and cell viability of bone cells (osteoblasts) [12].This positive effect on cell viability may be attributed to the fact that nanotubes create 3D cultures models that can provide additional nucleation and growth sites for cells to thrive.
In our studies, the cell viability rate and proliferation were showed an increase after cultivation of B16-F10 cell on the surface with grafted nanolayers of APTES, albumin and APTES/dextran/albumin during 72 h.While using coating glasses, a moderate cytotoxicity was observed after 72 h of incubation.The increased sensitivity of B16F10 cells (cell growth induction as well as inhibition) suggests that the effects may be dependent on nature of the polymer nanolayers.So it could be concluded that the stereochemistry of the polymers used for coating glasses greatly influence the cell growth and survival.This agrees with another study [13] and could be explained by the organization and maturation of the extracellular matrix surrounding cells by the created threedimensional system.
An additional approach for the detection of cells viability is the determination of LDH levels.LDH is a stable cytoplasmic enzyme present in all cells and is rapidly released following damage to the plasma membrane [14].Evaluation of LDH activity is crucial in the study of cell viability.From with the increasing of cultivation time, LDH activity was changed differently for each group, but, in general, its concentration in the control group and in all experimental groups was similar, which indicates to proliferative activity of cells.These results confirm by the index of MTT value: the viability of B16F10 cells cultivated onto different nanolayers after 72 h incubation number remains at a high level.

Conclusions
The different coatings were created through grafting of APTES, dextran, albumin and their combinations on glass surfaces.The effect of those nanolayers was studied on B16-F10 cell line in terms of change cell viability and morphology during 72 h of incubation.These coatings present different morphologies according to the introduced polymer nanolayers.It was found out that modification of the surface with grafted nanolayers of APTES, albumin and APTES/dextran/albumin is allows improving the viability and proliferation of B16-F10 cell.These results confirm the advantage of nanolayers surface for applications in cell biology and medicine because they create three-dimensional (3D) system and ensures optimum for the cultivation of cells in vitro.