Nitrogen-Containing Plasma Polymer Films Depos-ited by PECVD Process on Expression of Type X Collagen

Background: Nowadays, nitrogen-containing plasma poly-mers, due to their cell adhesion and influence on the response of mesenchymal stem cells (MSCs) are of great interest. The major problem of cartilage tissue engineering is that MSCs from osteoarthritis (OA) patients express high levels of type X collagen. This problem could be solved by these films. In this study, low-pressure plasma co-polymerization using binary gas mixtures of acetylene and nitrogen is investigated to deposit Nitrogen containing plasma-polymerized (L-PPA: N) coatings in radio frequency (RF: 13.56 MHz). In this research, we tried to decrease the concentration of amine groups to evaluate their effect on the expression of collagen X. To study the chemical bonding states and surface potential of the deposited films, at-tenuated total reflectance-Fourier transform infrared spectros-copy (ATR-FTIR), and zeta potential analysis was used. Final-ly, the response of mesenchymal stem cells under the influence of these films and the expression of type X collagen were exam-ined by Real-time- PCR.Materials and Methods: The preparation of Nitrogen-con-taining plasma-polymerized (L-PPA: N) coatings on surfaces were performed in the reactor operating by an RF of 13.56 MHz in a parallel plate capacitively coupled configuration, which was used for generation of plasma from a mixture of nitrogen (99.9995% purity) with acetylene as a monomer (99.6% pu-rity). The plasma deposition time, operating pressure and the gas flow ratio R (FN2/FC2H2) were kept constant at 10 min, 25 mTorr and 2, respectively. The ATR- FTIR spectra were record-ed using a Bruker Tensor 27 in the spectral range of 4000–400 cm−1with the resolution of 5 cm−1and 100scans. The meas-urement of zeta potential of the samples was made 3 hours af-ter plasma deposition using an Electrokinetic Analyzer (EKA Sur PASS–type A, Anton Paar GmbH, Graz, Austria). For this measurement, KCl 0.001M was used as titrator. Mesenchymal stem cells were seeded on L-PPA: N surface. The RNA extrac-tion was done after 4, 7 and 14 days and the expression of Col X gene was evaluated. One-way ANOVA test was applied to analyze the results statistically.Results: The Zeta potential measurement indicated that the sur-face potential of the typical L-PPA: N surface is -30.13. Since the presence of anions on surfaces caused negative zeta poten-tial, the presence of more carboxyl groups on the surface was verified. According to the spectrum, three bonds were indicat-ed: I. 3000 - 2800 cm-1 C-H stretch; II. 1800 - 1500 cm-1 C=C, C=N stretch; and III. 1500 - 1300 cm-1 CH2, CH3 bending. Band I comprises a convolution of contributions due to aliphatic CH groups. The assignment of band II is somewhat speculative, the likeliest contributions being C=C or C=N stretching modes. Finally, band III is attributed to disordered highly-ranched al-iphatic chains. The results of the Real-time q-PCR indicated that Col X gene expression significantly suppressed in 7 days. Again, in the day of 14th, the data showed a significant increase in the expression of this gene which means that the biological activity of the films is lost within about ten days.Conclusion: It was probable that the significant potential dif-ference across the cell membrane and L-PPA: N surface caused attraction between them. FTIR results showed a remarkable amount of carboxyl group and a negligible amount of amine group. It is proved that the surface containing a high amount of nitrogen moieties might lead to long-standing suppression of collagen expression. Since, cellular adhesion has a strong effect on gene expression and also on collagen expression and differentiation- functional group on L-PPA: N surface influ-ences cellular adhesion into the extracellular matrix (ECM). Accordingly, it could be concluded that the functional group has a substantial impact on gene expression and differentiation. Regarding the results of the real-time qPCR assay of the PPA:N surface, absence of suppression in the long-time period could be most probably assigned to amine groups.

Keywords:

Thin Films , Low- Pressure Radio Frequency Plasma , Cartilage , Type X Collagen