In vitro evaluation of collagen immobilization on polytetrafluoroethylene through NH 3 plasma treatment to enhance endothelial cell adhesion and growth

Abstract

Background:

Polytetrafluoroethylene (PTFE) is poorly biocompatible due to its low surface energy and hydrophobicity, which cause weak cell attachment and proliferation and complicate its use in implants.

Objective:

NH₃ plasma was used for surface modification and binding of amine groups on the PTFE surface. Collagen was immobilized on the plasma-treated PTFE in order to enable it to support enhanced cell adhesion and growth.

Methods:

PTFE was exposed to NH₃ plasma and collagen was immobilized on the NH₃ plasma-treated surface. ATR-IR, SEM, EDXA and contact angle were conducted to determine the composition, microstructure and wettability of samples. The cytocompatibility of the samples was assessed via the growth HUVEC cells using MTT assay.

Results:

Plasma treatment resulted in an incorporation of functional groups, containing N₂ and O₂ that caused the PTFE surface to become hydrophilic with contact angle 68°. Also, a reduction in F/C ratio was observed after collagen immobilization that indicates the presence of collagen. Cells proliferated in greater numbers on the collagen immobilized-PTFE as compared to the plasma-treated one.

Conclusions:

Plasma treatment incorporates functional polar moieties on the PTFE surface, causing enhanced wettability, collagen immobilization and cell viability. Collagen-immobilized PTFE may offer a valuable solution in biomedical applications such as vessel grafts.

Keywords

Surface biological modifications collagen immobilization biocompatibility polytetrafluoroethylene artificial blood vessel hydrophilicity

1. Introduction

Cardiovascular diseases are an important cause of mortality in developed countries and patients usually require open surgical procedures such as; bypass operations in which unhealthy vascular veins are transplanted or replaced via grafts. These grafts are not transplanted from biological native organs or tissues [1–4], because regular auto-grafts are not achievable [5] or are inappropriate for the operating procedure [6], while rejection reactions of allografts are likely to occur in such cases. Thus implants are made of artificial materials, for instance polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), silk, polyurethanes (PU), polyglycolic acid (PGA) and Polylactic acid (PLA) [1,2].

PTFE, commonly referred to as ‘Teflon’, is a stable, chemically and biologically inert polymer, having properties like hydrophobicity, high oxygen permeability and the capability to withstand enzymatic and microbiological attack [7–9]. PTFE is poorly soluble and resistant to oxidants, acids and alkalis [10] and can be fabricated in various forms [11]. Consequently, PTFE has clinical applications in cardiovascular, reconstructive vascular system (include vascular grafts and heart valves), abdominal and orthopedic surgery [1,12–14]. Unfortunately, the hydrophobicity and low surface energy of PTFE cause weak cell attachment and inefficient cell spreading on the surface. To solve these problems, alteration of the surface characteristics of PTFE is needed either by chemical or physical modification or by immobilization of natural or synthetic biomolecules onto its surface [15]. Some of these methods are Extreme ultraviolet (EUV) [10], plasma discharge [11], dielectric barrier discharge [16], surface coating [11,17,18] and biomolecule grafting [19].

Plasma surface modification is a commonly used procedure to increase surface biocompatibility and protein adhesion incorporation of various functional groups without influencing any bulk properties [7,11,20–23]. Surface properties of plasma-modified polymers depend on the plasma expose time, temperature and the composition of ambient atmosphere under which the modified specimens are stored [24]. Interfacial adhesion of PTFE can be enhanced by exposing it to air, Ar, O₂, CO₂, H₂O and NH₃ plasmas [21]. Until today, plasma deposition has been used to apply different coatings with diverse chemically reactive moieties such as: amine (–NH₂), carboxyl (–COOH), hydroxyl (–OH), etc. [25]. The wettability of a PTFE surface can be altered and enhanced by using plasma [9,26]. Researchers used Ar + O₂ plasma to produce superhydrophobic PTFE surfaces with high durability [4], but the use of Ar plasma has been shown to decrease the hydrophobicity and contact angle of this polymer surface [27,28]. Dielectric barrier discharge (DBD) plasma and radio frequency (RF) plasma treatment also cause a decrease in water contact angle of a PTFE surface [29]. In addition, low-pressure H₂ and NH₃ microwave Plasma modification of PTFE powder, also resulted in enhanced wettability [30]. In another report using Ar + CO₂ plasma, it was shown that the resulting wettability of the PTFE surface depends on the plasma exposure time [31].

Collagen, a rope-like protein, comprises almost one thirds of all proteins in biological tissues and is an important element for the strength and toughness of tissues [32]. It is the main structural component of connective tissue and is a guide for cell growth and ell-cell interactions [33]. Collagen has numerous applications in biomedical, pharmaceutical, cosmetic and food industries, owing to its biodegradability, bioactivity, low toxicity and immunogenicity, weak antigenicity and high biocompatibility [34,35].

In the current research, NH₃ plasma was used for surface modification and binding of amine groups on the surface of PTFE. Collagen was immobilized on the surface of the modified PTFE in order to assess enhanced biocompatibility. Finally, human umbilical vein endothelial cells (HUVEC) were cultured on the PTFE surface and the surface properties were characterized by various techniques.

2. Materials and methods

2.1. Material preparation

PTFE was purchased from ^®Good Fellows Ltd., UK with a density of $2.2 g {cm}^{- 3}$ . PTFE disks were punched with a thickness of 2 mm and a diameter of 13 mm. The sample surfaces were polished and washed in an ultrasonic bath, containing high purity acetone, high purity methanol and deionized water. Samples were dried at 22°C and stored in a desiccator before use. Collagen was purchased from ^®National Cell Bank of Iran (NCBI). A collagen solution was made by mixing Vitrogen (95% collagen type I and 5% collagen type III) and deionized water in at 1:1 ratio under sterile conditions and kept in an ice bath at 4°C. HUVEC cells were also acquired from ^®NCBI and were cultured in RPMI-1640, supplemented with 10%FBS and incubated under conditions of 90% humidity, 5% oxygen and at 37°C after de-freezing. Finally, the cultured cells were rinsed and centrifuged at $180 \times g$ for 7 min.

2.2. Plasma treatment

Plasma enhanced chemical vapor deposition (PECVD) was used to modify the PTFE surface with NH₃ gas. First, the chamber air was evacuated for 30 minutes to obtain a vacuum pressure of about $- 2$ torr and the sample was then positioned in the vacuum chamber. NH₃ gas was introduced to the chamber with flow rate of $30 – 40 {cm}^{3} {min}^{- 1}$ . The pressure of the tube was kept at 0.3–0.4 torr with the help of flow meter and maintained for 5 minutes to generate a stable plasma [36]. Plasma modification was performed on 6 samples, each using a different operating power and exposure time, shown in Table 1. An untreated PTFE sample was used as a control in all of the experiments.

Table 1
Plasma treatment on samples by a different operating power and exposure time

Operating power (w) Exposure Time (s)

100 120

100 180

100 240

75 180

95 180

125 180

Operating power (w)	Exposure Time (s)
100	120
100	180
100	240
75	180
95	180
125	180

2.3. Collagen immobilization

Before collagen grafted the plasma-treated samples with the minimum contact angle, they were soaked in acetone for 20 min and rinsed with deionized water to exchange the acetone. This treatment provides a wetted surface in order to enhance collagen binding to the sample surfaces. Subsequently, the samples were placed in 2.5% glutaraldehyde to interact with the grafts and to facilitate the interaction between the PTFE surface and collagen. After washing for 1 h in deionized water to remove unbound glutaraldehyde, the samples were soaked in 1 ml of collagen/deionized water solution at 4°C to allow the formation of chains of cross-linked collagen on the PTFE surface. After immobilization, free collagen was rinsed off with nitric acid [37]. The schematic diagram of the reaction sequence of collagen attachment to plasma-treated PTFE is shown in Fig. 1.

Fig. 1.

The schematic diagram of the reaction sequence of collagen attachment to NH₃ plasma-treated PTFE surface.

2.4. Surface characterization

2.4.1. Measurement of the water contact angle

To determine the wettability of untreated PTFE and the plasma-treated samples, the contact angles between water and PTFE sample surfaces were measured by a manual contact angle measuring device and a goniometer (^®Krüss LBI02, Germany). To decrease the effect of gravity, small drops of 0.2 ml each were applied on the surface using an automated pipette. The measurements were carried out at a 22°C and 45% relative humidity (RH). The averaged value of the angles of each drop was determined from an average of 4 measurements on both sides with a standard deviation of ±2°.

2.4.2. Surface morphology and microstructure analysis

The surface morphology and elemental analysis of the untreated PTFE, plasma-treated and collagen-immobilized samples were investigated using a scanning electron microscope (SEM) and energy-dispersive X-ray analysis (EDXA) (^®VEGA/TE SCAN, USA, SEM/EDXA). In order to improve the imaging quality and prevent charging of the samples during SEM operation, the samples were first coated with a thin gold film using a sputter coating device (^®BIO-RAD-E5200, UK).

2.4.3. ATR-IR spectroscopy

Attenuated total reflection infrared spectra (ATR-IR) (^®Equinox 55, Bruker, ATR-IR) were obtained to confirm the plasma modification and to characterize the chemical composition of the surfaces of the untreated PTFE, plasma-treated and collagen-immobilized samples at wavenumbers of $500 – 4000 {cm}^{- 1}$ .

2.5. Cell proliferation and viability analysis

In vitro experiments were performed with human umbilical vein endothelial cell line (HUVEC), purchased from ^®NCBI, Pasteur Institute, Tehran, Iran. The cells were grown in RPMI-1640 (^®Gibco, USA) with 10% fetal bovine serum (^®Gibco, USA), 100 IU penicillin/ml and 100 μg streptomycin/ml. The samples were cut into pieces, rinsed with alcohol and sterilized on both sides under UVC radiations for 30 minutes before the cell culturing. The samples were placed in 24-well plates and the cells were seeded on the samples at a density of 50,000 cells/well and grown on the samples in above-mentioned culture conditions for 24 h at 37°C. The viability and proliferation of the HUVEC cells, attached to the plasma-treated PTFE were examined by the methylthiazol tetrazolium (MTT) (^®Invitrogen, Germany) assay before and after collagen immobilization. MTT solution (100 μL; at a concentration of $0.5 mg {ml}^{- 1}$ ) was added to each well to form formazan crystals. After 4 h of incubation, the medium was aspirated and isopropanol was added to dissolve the formed formazan crystals. An ELISA reader device (^®STAT FAX, USA) was used to determine the optical density of the solution at a wavelength of 570 nm. The relative cell viability in contact with the samples was calculated compared to the negative control (tissue culture palate), according to equation (1) [38]. $\begin{matrix} (1) & Relative Cell Viability % = A s / A c \times 100 \end{matrix}$ where $A s$ and $A c$ are the optical densities of sample and negative control, respectively.

2.5.1. Cell morphology assessment

SEM was utilized to study the HUVEC cell attachment and morphology on the collagen-immobilized samples. A density of 4000–5000 cells was placed on sterilized disks in 1 mL culture media, followed by culturing for 3 days. In order to perform SEM, the samples were immersed into a 2.5% glutaraldehyde solution (^®Merck, Germany) for 24 h in a refrigerator and subsequently dehydrated in graded ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100) for 5 min. The dried and fixed samples were then gold-coated using a Sputter coating device (^®BIO-RAD-E5200, UK), and viewed with an SEM.

2.6. Statistical analysis

The statistical analysis was carried out using SPSS software for Windows (^®v 17.0; IBM, NY, USA). When the statistical differences were detected, a student’s t-test was performed. Data are reported as mean ± standard deviation at a significance level of $p < 0.05$ .

3. Results and discussion

3.1. Sample characterization

The PTFE substrate was modified by NH₃ plasma discharge and the operational parameters (discharge power and time) were varied in order to deposit a varying number of amine groups on the surface of this polymer. Collagen was successfully immobilized on the surface of these NH₃ plasma-treated samples. SEM microscopy was used to evaluate the surface morphology of untreated PTFE and plasma-treated samples (Fig. 2). It was found that the surface morphology of untreated PTFE sample was flat and smooth with no significant superficial structures. This morphology was significantly altered after plasma treatment, which is attributed to the etching reactions between the PTFE surface and plasma discharge which contains active species [31].

Fig. 2.

SEM micrographs of (a) untreated PTFE, and (b) plasma-treated sample.

EDXA results were obtained in order to assess the elemental composition of the PTFE surface before and after collagen immobilization. Table 2 shows the analysis results for plasma-treated samples. Carbon is the most abundant element on the PTFE surface with a concentration of 50.39%. The next abundant element is fluorine at 39.85%, which means that the F/C ratio is 0.79 in this sample. These two elements are expected to make up the majority of the polymer surface, as they are the structural elements of PTFE. Nitrogen and oxygen were incorporated onto the PTFE surface after NH₃ plasma treatment, and the amount of nitrogen was almost twice that of oxygen. The presence of nitrogen is due to NH₃ plasma operation, which means that the plasma radiation could successfully modify the sample surface and incorporate the nitrogen onto it as well. It should be noted that the presence of oxygen may be caused by air leakage in the plasma chamber or could have arisen when the samples were exposed to air immediately after plasma treatment [39].

Table 2

Chemical composition of PTFE samples after plasma treating

Elements	%A
Carbon	50.39
Nitrogen	6.42
Oxygen	3.34
Fluorine	39.85

The EDXA results of the collagen-immobilized samples are given in Table 3. After collagen immobilization, the concentrations of carbon, nitrogen and oxygen atoms increased while the percentage of fluorine atoms on the PTFE surface had a dramatic. With an abundance of 55.39%, carbon is still the most abundant element followed by nitrogen (18.36%) and fluorine (14.91%). The F/C atomic ratio thus decreased to 0.27, a 52% decline as compared to the plasma-treated samples before collagen immobilization. Thus, the biological modification and an increase in the amount of nitrogen and oxygen elements on the PTFE surface may be because of the C/O elements, present in the collagen structure. Similarly, an increase in the amount of PTFE-surface carbon confirms the surface modification of this polymer with collagen.

The bulk composition of the untreated, plasma-treated and collagen-immobilized PTFE samples was examined by ATR-IR (Fig. 3). In the asymmetric spectra of the untreated samples, only the characteristic bands of C–F and C–C bonds were observed, at about 1150 and $1240 {cm}^{- 1}$ wavelength, respectively. After treatment, additional absorption features were also observed in the spectra, which is indicative of the occurrence of chemical changes on the PTFE surface. For the samples treated with NH₃ plasma, a broad band appeared between $1560 – 1640 {cm}^{- 1}$ which is attributed to N–H bonds. The absorption band observed at $2900 {cm}^{- 1}$ is caused by the presence of C–H bonds. Another band at approximately $1300 {cm}^{- 1}$ is indicative of the presence of ammonium fluoride [39]. In addition, plasma treatment caused the strength of C–C and C–F absorption bands to decrease, though the width and the overall shape of the absorption bands remained intact. Similarly for the spectra of the collagen-immobilized samples, new functional groups caused the appearance of new spectral features. The band at about $1760 {cm}^{- 1}$ is associated with the $C = O$ bond in collagen molecules. A shallow wide band appeared at $3300 – 3500 {cm}^{- 1}$ , which is attributed to N–H stretching bonds, and the peak at $1020 – 1250 {cm}^{- 1}$ is indicative of the C–N bonds. The C–F and C–C bands decreased further after collagen immobilization, while the N–H peak grew as compare to that obtained for the NH₃ plasma-treated sample.

Table 3

Chemical composition of plasma-treated samples after collagen immobilization

Elements	%A
Carbon	55.39
Nitrogen	18.36
Oxygen	11.34
Fluorine	14.91

Fig. 3.

ATR-IR results of (a) untreated PTFE, (b) Plasma-treated samples (at 100 W for 180 sec), and (c) collagen-immobilized samples.

The ATR-IR test outcome of untreated PTFE demonstrates that the surface did not contain significant impurities before plasma treatment. After treatment, absorption features were present in the spectra, which is indicative of the chemical changes on the PTFE surface. As explained above, N–H, C–H and ammonium fluoride were observed in the treated samples, which proved the PTFE surface modification. Similarly, after collagen immobilization, $C = O$ , C–N and N–H bands appeared in the ATR-IR spectra that confirmed the collagen immobilization on the PTFE surface. Although the strength of C–F and C–C bonds decreased after plasma treatment and collagen immobilization, they were still the dominant features in the resultant spectra. This shows that plasma modifications could penetrate deep into the PTFE, and that the immobilized-collagen layer was not very thick.

3.2. Wettability analysis

The measurement of the contact angle between water and a surface is one of the most straightforward ways to characterize the hydrophilicity and wettability of the surface. The evaluation of the water contact angle on the untreated PTFE and the plasma-treated samples, as a function of plasma exposure time, is shown in Table 4. The water contact angle of the untreated PTFE sample was measured to be 114°, revealing the hydrophobic nature of this surface. The contact angle decreased to approximately 68° after plasma treatment at 100 W for 180 sec. However, when the PTFE samples were plasma-treated for more than 180 sec, the contact angle started to rise, reaching a value of 92°. Adding hydrophilic nitrogen/oxygen moieties on the surface was expected to cause a decrease in hydrophobicity, because these elements allow the hydrogen-bond formation between electronegative atoms, present on the treated surface and the surrounding water molecules [39,40]. The water contact-angle measurements reveal a decrease in water contact angle in all plasma-treated samples, regardless of the duration and intensity of the plasma treatment compare to the untreated sample. Oxygen moieties were incorporated into the sample from the oxygen impurities, present in the plasma chamber or exposure of sample to air or water vapor after plasma treatment [28,41]. At constant operating power and increasing exposure time, a decrease in contact angle was observed. But, as the exposure time increased, the water contact angle also showed an increase. Although such a reversal of the water contact angle is not reported in related studies, it should be noted that plasma deposition can be used to either increase or decrease the contact angle [27,42,43]. This can be the effect of the embedment of nitrogen moieties in the substrate and the destruction of the amine groups on the PTFE surface after prolonged plasma treatment. In other words, the incorporation of hydrophobic moieties can be valid up to a certain limit.

Table 4
Average contact angle in different exposure time and constant operating power

Operating power (w) Exposure time (s) Contact angle (degree) Schematic

100 120 74

100 180 68

100 240 92

Operating power (w)	Exposure time (s)	Contact angle (degree)
100	120	74
100	180	68
100	240	92

A similar behavior was observed for the experiment in which the exposure time was constant, at increasing operating power. Our results show a rise in wettability, followed by a decreasing trend. Therefore, the hydrophilicity is shown to depend on the plasma treatment time as well as the plasma intensity (Table 5).

Table 5

Average contact angle in different operating power and constant exposure time

Operating power (w)	Exposure time (s)	Contact angle (degree)	Schematic
75	180	76
95	180	70
125	180	97

3.3. Biocompatibility performance and cell morphology observations

To investigate the improved biocompatibility and cells interaction on samples, the MTT assay and SEM analysis were carried out. Figure 4 shows the HUVEC cell viability after 24 h of incubation with samples. The results demonstrate a promoted cell viability and proliferation functions without the onset of cell necrosis on the collagen-immobilized PTFE surfaces. Collagen immobilization increased cell viability to 99%, as compared to the plasma-treated samples, in which only 67% of the cells could. Therefore, the collagen-immobilized samples can be considered to be non-cytotoxic and highly cell-compatible.

Fig. 4.

The HVEC cell viability after 24 h of incubation with the samples.

SEM observations in Fig. 5 demonstrate the morphology of HVEC cells on the collagen-immobilized sample after 3 days of culture. Cells were flattened and well-spread across the surface and were adhered to the substrate with cellular micro-extensions (e.g., filopodia) that is indicative of live cells. Collagen immobilization can therefore be used as a convenient modification for artificial vessels and medical applications.

Fig. 5.

SEM micrograph of HVEC cells on collagen-immobilized sample after 3 days of culture.

4. Conclusions

This work presents a mechanism for increasing biocompatibility of PTFE polymer by using NH₃ plasma treatment and subsequent collagen immobilization. The resulting contact angle was directly correlated to the exposure time and plasma power. While, the optimal parameters of plasma were the operating power of 100 W and the exposure time of 180 sec for polymer-surface treatment, which resulted in the achievement of minimum contact angle of 68°. Plasma treatment resulted in an incorporation of functional groups, containing N₂ and O₂ that caused the PTFE surface to become hydrophilic. Also, an increase in the amount of N₂ and O₂ and a reduction in F/C ratio were observed after collagen immobilization that indicates the presence of collagen and occurrence of defluorination phenomenon. In vitro experiments demonstrated that collagen immobilization can increase cell viability to 99% and that cells can homogeneously spread over the modified surface, having flattened shape and filopodia. Therefore, hydrophilic PTFE samples can prove to be suitable in a variety of medical applications.

Footnotes

Acknowledgements

The authors gratefully acknowledge the scientific help and assistance of Pasteur Institute of Iran (IPI) and Department of Biomedical Engineering, Islamic Azad University, Yazd Branch.

Conflict of interest

The authors have no conflict of interest to report.

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In vitro evaluation of collagen immobilization on polytetrafluoroethylene through NH 3 plasma treatment to enhance endothelial cell adhesion and growth

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

1. Introduction

2. Materials and methods

2.1. Material preparation

2.2. Plasma treatment

Table 1 Plasma treatment on samples by a different operating power and exposure time Operating power (w) Exposure Time (s) 100 120 100 180 100 240 75 180 95 180 125 180

2.4.1. Measurement of the water contact angle

2.4.2. Surface morphology and microstructure analysis

2.4.3. ATR-IR spectroscopy

2.5. Cell proliferation and viability analysis

2.5.1. Cell morphology assessment

2.6. Statistical analysis

3. Results and discussion

3.1. Sample characterization

Table 4 Average contact angle in different exposure time and constant operating power Operating power (w) Exposure time (s) Contact angle (degree) Schematic 100 120 74 100 180 68 100 240 92

Footnotes

Acknowledgements

Conflict of interest

References

Table 1
Plasma treatment on samples by a different operating power and exposure time

Operating power (w) Exposure Time (s)

100 120

100 180

100 240

75 180

95 180

125 180

Table 4
Average contact angle in different exposure time and constant operating power

Operating power (w) Exposure time (s) Contact angle (degree) Schematic

100 120 74

100 180 68

100 240 92