The Potential of N-Rich Plasma-Polymerized Ethylene (PPE:N) Films for Regulating the Phenotype of the Nucleus Pulposus

Deposition of PPE:N

PPE:N films were deposited on biaxially-oriented polypropylene (BOPP, graciously provided by 3M Company), in the form of a 50 µm-thick isotactic polymer film [34, 35], as previously described [36]. Our atmospheric pressure plasma deposition system comprised a moveable aluminium plate electrode, on which flat sheets of BOPP (typically 22 cm x 30 cm) were uniformly coated in a 10 kHz, high voltage (~ 18 kV peak-to-peak) dielectric barrier discharge (DBD) [6]. This was accomplished by carefully-controlled linear displacement of the substrate through the ca. 1 cm wide discharge zone between the cylindrical, ceramic-coated high voltage electrode and the grounded aluminium plate, spaced 1 mm apart. Using this apparatus, films containing various nitrogen concentrations, [N], were deposited from the precursor gas mixture composed of very pure nitrogen (N₂, 10 standard liters per minute, slm) and ethylene (C₂H₄, typically some tens of standard cubic centimeters per minute, sccm).

PPE:N Films with Different Nitrogen Concentrations ([N])

Obtaining PPE:N films with different [N] values was accomplished as previously described [36, 37]. Briefly, the flow rate (F) of the ethylene gas precursor, F_C2H4, was changed from the lowest value of 5 sccm to higher values, thereby gradually reducing [N]. Thus, for F_C2H4 = 5 sccm, [N] ≈ 36%, while for the highest F_C2H4 used here, 60 sccm, [N] ≈ 17%: all other deposition conditions of course being maintained constant. Table 1 shows the characteristics of the three different PPE:N coatings we have used in the present investigation. Hereafter in this text, we shall refer to these PPE:N coatings in terms of the F_C2H4 values used in their preparation, namely 5, 10, and 60 sccm, knowing that the resulting different [N] values can lead to significantly different cell responses [36, 37]; accordingly, as shown in Table 1, coatings are designated S5, S10, and S60, in the order of increasing F_C2H4 (decreasing [N]).

The surface compositions of the thin PPE:N films were determined by X-Ray photoelectron spectroscopy (XPS) using a Kratos Axis Ultra system, with non-monochromatic Mg Kα radiation [34]; at least five samples were analysed for each condition. We will be referring to their surface-elemental concentrations, [X], in terms of the elements that comprise PPE:N, namely N, C and O; since hydrogen cannot be detected by XPS, [X] is given by:

$\left(X\right)=\frac{X}{N+C+O}100\%$

N, O, and C being determined from XPS broadscan spectra. Other methods used for the characterization of PPE:N films included attenuated total internal reflectance Fourier transform infrared spectroscopy (ATR-FTIR), contact angle goniometry, atomic force microscopy, stylus profilometry and spectroscopic ellipsometry, as described elsewhere [34].

Source of Intervertebral Discs (IVDs)

Three fetal bovine lumbar discs were obtained from the slaughterhouse Les Abattoirs Colbex-Levinoff (St-Cyrille de Wendover, QC). Ages of the fetal bovine were determined by the method of Pal et al. based on the length of the tibia [38]. The ages of the fetal bovine are about 5 months old. IVDs were classified as nondegenerated grade I according to the grading system of Thomson [39].

Cell Isolation

Cells were isolated immediately after transportation from the slaughterhouse (abattoir). The IVDs were dissected from their adjacent vertebral bodies, placed in DMEM-high glucose containing 150 ng/ml gentamycin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone (medium A). Under aseptic conditions, the IVDs were separated by dissection into regions corresponding to the AF and the NP. The NP regions were, in turn, cut into approximately 2 mm fragments and were washed twice in medium A for 15 min. Cells were enzymatically isolated from the tissue, using the method adapted from protease type XIV/collagenase protocol [9]. Briefly, the tissue was washed and then digested overnight at 37°C in washing medium (medium A, without gentamicin and fungizone) supplemented with 10% fetal bovine serum (FBS, Hyclone) and bacterial collagenase type IA (Sigma-Aldrich, Oakville, ON) at 0.03% (w/v). The resulting cell suspensions were passed through a 70-µm cell strainer (Beckton Dickinson, Oakville, ON), washed twice in DMEM-high glucose containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were counted with a hemacytometer and the viability was determined using 0.04% Trypan Blue Dye. Approximately 5 millions cells from 10 g of the NP per fetus were recovered and used.

Cell Culture

NP cells were cultured on each of the three different PPE:N coatings on BOPP in DMEM-high glucose supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. At the beginning about 1.0 million cells were layered on S5 surfaces tightly covering 100 x 20 mm culture dishes. Commercial polystyrene (PS) tissue culture dishes (Sarstedt, Montreal, QC) were used as controls (hereafter designated “PS control”). These commercial culture dishes are known to be plasma-treated for improved hydrophilicity and cell-adhesion, and our XPS measurements show that they possess a surface oxygen concentration, [O], of 18%. In all cases, the medium was changed every 2 days. The cells were then harvested at day 3 and day 14 to study the short- and long-term effect, respectively, of surfaces on gene expression.

Total RNA Isolation

Total RNA was extracted from NP cells by a modification of the method of Chomcynski and Sacchi [40] using TRIzol reagent (Invitrogen, Burlington ON). The aqueous phase was precipitated in 1 volume of isopropanol. The resulting RNA pellet was air-dried, resuspended in 50 µl of diethylpyrocarbonate-treated water, and 10 µl was assayed for RNA concentration and purity by measuring A₂₆₀/A₂₈₀.

Reverse Transcription (RT) and Polymerase Chain Reaction (PCR)

The RT reactions were performed using 1 µg total RNA isolated from the NP cells in a total volume of 20 µ1, containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 50 µM each of dATP, dGTP, dCTP and dTTP, and 200 units of Superscript II - RNase H reverse transcriptase (Invitrogen).

PCR was performed in a total volume of 25 µl containing: 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.4 mM of dATP, dGTP, dCTP, dTTP, 0.8 µM of each primer, 1 µl of RT mixture and 2.5 units of Taq DNA polymerase (Invitrogen) as previously described [40]. The 25 cycles of PCR (except GPC3 with 35 cycles) included denaturation (94°C, 1 min), annealing (56°C, 45 sec) and extension (72°C, 40sec). The PCR condition was applicable to all genes. After agarose (2%) gel electrophoresis, PCR products were visualized by ethidium bromide staining and analyzed using a Bio-Rad VersaDoc image analysis system, equipped with a cooled 12 bit CCD camera (Bio-Rad, Mississauga ON). GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as housekeeping gene. To confirm the absence of chromosomal DNA contamination of RNA samples, PCR was also performed with RNA aliquots. The primers were obtained from InVitrogen. Their sequences, shown in Table 2, were chosen because they are specific for bovine genes and amplify a unique product.

Statistical Analysis

Data are presented as the mean ± standard error of 3 experiments. Statistical differences were calculated using Student t-test, with p < 0.05 as the level of significance.

Characteristics of PPE:N Coatings

In an earlier article, we reported in considerable details the methodology for depositing PPE:N coatings on BOPP and on other (polymeric or glass) substrates, as well as methods used for characterizing the resulting thin film materials, and the biological responses, mostly adhesion or non-adhesion of various cell lineages of interest to these authors [36]. In order not to unduly repeat those earlier-published data, we have limited ourselves here to describing how [N] varies with F_C2H4. As shown earlier [36], [N] decreases in a non-linear monotonic manner from the maximum value, [N] ≈ 36% for the lowest value of F_C2H4 (5 sccm; S5), to [N] ≈ 17% for the highest F_C2H4 value used (60 sccm; S60) (Table 1). In this article, we limit our characterization of different PPE:N substrates to their elemental composition, in particular their total [N]; however, more realistically, the substrates’ effect on adhering cells is mediated by the concentrations of various chemical functionalities at the surface, for example amines, imines, nitriles, amides, acids and alcohols (bound oxygen is always incorporated in plasma polymer films due to the reaction of residual surface radicals with ambient air). Unfortunately, plasma polymers are difficult to characterize, on account of their random highly cross-linked structure; the resulting peak broadening, therefore, greatly complicates quantitative, even qualitative, analysis by most spectroscopic methods (high resolution XPS, FTIR, solid state NMR, etc.), and this inherently limits our knowledge of the specific surface chemistries of PPE:N films at the present time. However, as reported earlier [34], we do know (from chemical derivatization experiments, coupled with XPS, and from FTIR measurements) that primary amines account for 5 to 10 % of [N], and that nitriles (-C≡N) also constitute an important surface functionality; these results are in keeping with surface-analytical experiments on these same materials now in progress, using Near-Edge X-Ray Absorption Fine-Structure (NEXAFS), to be reported elsewhere. Primary amines are positively charged, and their particular efficacy is believed to result from the fact that they attract negatively charged proteins and other bio-molecules from the culture medium; alternately, proteins secreted from the cells’ own ECM preferentially form covalent bonds with these favored reactive amine sites.

Cells from the Fetal Bovine Lumbar Spine

Fig. (1) shows the microscopic aspect of NP cells in culture. When cultured on PS control, NP cells were able to grow and reach confluence after 14 days (Fig. 1A); at low [N] (sample S60), cell growth was not affected (Fig. 1B) while cell proliferation was decreased on S5 surfaces (Fig. 1D). Quantitatively, cell number increased by 2.3 times on the PS control over the 14 days of culture while this increase was of 2.2, 2.0, and 1.6 times on S60, S10, and S5 surfaces, respectively, suggesting a dose-dependent effect of N on NP cell proliferation.

Expression of Glypican-3 (GPC3)

The results of RT-PCR analyses of GCP3 gene expression, normalized to GAPDH expression, are shown in Fig. (2). The expression of GPC3 decreased with time on PS controls. Its expression was significantly reduced in the NP cells cultured on the S60 surface after short (3 days) and long (14 days) culture time. Interestingly, there was less downregulated of GPC3 expression when cells were cultured for 14 days on the S5 layer.

Expression of Vimentin (VIM)

Since VIM has been shown to be expressed at significant levels in the NP [26], we next explored whether this message could be regulated by PPE:N surfaces in fetal bovine NP cells. Unlike the GPC3 message (Fig. 3), the expression of VIM was maintained throughout the culture when cells were cultured on PS controls (Fig. 3).

Expression of Pleiotrophin Heparin Binding Factor (PTN)

Since PTN was recently discovered to be expressed in more than 10-fold higher concentration in the NP than in the AF [26], we next explored its expression on the different culture surfaces. Results are shown on Fig. (4). The expression of PTN on the PS controls did not change significantly from day 3 to day 14, but decreased when cultured on all three PPE:N coatings on day 3 and day 14. The weakest expression of PTN was observed on day 3 when NP cells were cultured on S60.

Expression of Matrix Gla Protein (MGP)

The results of RT-PCR analyses of gene expression of MGP are shown in Fig. (5). The expression of MGP on PS control was higher on day 3 compared to day 14. Its expression was decreased when NP cells were cultured on all three PPE:N surfaces on day 3 and day 14. Similarly to PTN, its expression decreased with decreasing [N].

Expression of Cartilage Oligomeric Matrix Protein (COMP)

The results of RT-PCR analyses of COMP gene expression are shown in Fig. (6). Its expression was consistently detectable in NP cells cultured on PS controls from day 3, following which its expression decreased progressively and significantly with time. A decrease was observed when cells were cultured on S5, S10 and S60 surfaces on day 3, but the different PPE:N surfaces had no significant effect on COMP expression on day 14.

Expression of Aggrecan, Collagen Type I, and Collagen Type II

We finally looked at the effect of PPE:N surfaces on genes of the extracellular matrix of NP cells from fetal bovine lumbar discs. Aggrecan (AGG), collagen type I alpha 2 (COL1A2), and collagen type II alpha 1 (COL2A1) gene expression stayed stable throughout the culture time. There was also no effect of the PPE:N surfaces on their expression (results not shown).